108 results on '"Hultquist JF"'
Search Results
2. Corrections to Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection. (Immunity 44, 46-58, January 19, 2016).
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Heaton, NS, Heaton, NS, Moshkina, N, Fenouil, R, Gardner, TJ, Aguirre, S, Shah, PS, Zhao, N, Manganaro, L, Hultquist, JF, Noel, J, Sachs, D, Hamilton, J, Leon, PE, Chawdury, A, Tripathi, S, Melegari, C, Campisi, L, Hai, R, Metreveli, G, Gamarnik, AV, García-Sastre, A, Greenbaum, B, Simon, V, Fernandez-Sesma, A, Krogan, NJ, Mulder, LCF, van Bakel, H, Tortorella, D, Taunton, J, Palese, P, Marazzi, I, Heaton, NS, Heaton, NS, Moshkina, N, Fenouil, R, Gardner, TJ, Aguirre, S, Shah, PS, Zhao, N, Manganaro, L, Hultquist, JF, Noel, J, Sachs, D, Hamilton, J, Leon, PE, Chawdury, A, Tripathi, S, Melegari, C, Campisi, L, Hai, R, Metreveli, G, Gamarnik, AV, García-Sastre, A, Greenbaum, B, Simon, V, Fernandez-Sesma, A, Krogan, NJ, Mulder, LCF, van Bakel, H, Tortorella, D, Taunton, J, Palese, P, and Marazzi, I
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- 2016
3. Lineage-Specific Viral Hijacking of Non-canonical E3Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity
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Kane, JR, Stanley, DJ, Hultquist, JF, Johnson, JR, Mietrach, N, Binning, JM, Jónsson, SR, Barelier, S, Newton, BW, Johnson, TL, Franks-Skiba, KE, Li, M, Brown, WL, Gunnarsson, HI, Adalbjornsdóttir, A, Fraser, JS, Harris, RS, Andrésdóttir, V, Gross, JD, and Krogan, NJ
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viruses ,virus diseases ,biochemical phenomena, metabolism, and nutrition - Abstract
© 2015 The Authors. HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity. How viral proteins evolve functions without compromising ancestral ones is not well understood. Kane etal. now report that the lentiviral protein Vif has evolved to hijack host factors in a lineage-specific manner while conserving an ancestral viral-host complex promoting viral infection.
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- 2014
4. Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.
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Cisneros WJ, Soliman SHA, Walter M, Simons LM, Cornish D, De Fabritiis S, Halle AW, Kim EY, Wolinsky SM, Lorenzo-Redondo R, Shilatifard A, and Hultquist JF
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- Humans, Virus Activation drug effects, Virus Replication, Gene Expression Regulation, Viral, HIV-1 physiology, HIV-1 genetics, Virus Latency physiology, Virus Latency drug effects, Positive Transcriptional Elongation Factor B metabolism, HIV Infections virology, HIV Infections metabolism, HIV Infections drug therapy, CD4-Positive T-Lymphocytes virology, CD4-Positive T-Lymphocytes metabolism
- Abstract
The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation., Competing Interests: J.F.H. has received research support, paid to Northwestern University, from Gilead Sciences, and is a paid consultant for Merck. All other authors declare no competing interests., (Copyright: © 2024 Cisneros et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2024
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5. Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification.
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Class J, Simons LM, Lorenzo-Redondo R, Achi JG, Cooper L, Dangi T, Penaloza-MacMaster P, Ozer EA, Lutz SE, Rong L, Hultquist JF, and Richner JM
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- Animals, Mice, Furin metabolism, Furin genetics, Virulence, Humans, Mutation, Evolution, Molecular, Female, Disease Models, Animal, SARS-CoV-2 genetics, SARS-CoV-2 pathogenicity, SARS-CoV-2 physiology, COVID-19 virology, Lung virology, Lung pathology, Central Nervous System virology, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus metabolism, Viral Tropism
- Abstract
Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2024
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6. Distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.
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Markov NS, Ren Z, Senkow KJ, Grant RA, Gao CA, Malsin ES, Sichizya L, Kihshen H, Helmin KA, Jovisic M, Arnold JM, Pérez-Leonor XG, Abdala-Valencia H, Swaminathan S, Nwaezeapu J, Kang M, Rasmussen L, Ozer EA, Lorenzo-Redondo R, Hultquist JF, Simons LM, Rios-Guzman E, Misharin AV, Wunderink RG, Budinger GRS, Singer BD, and Morales-Nebreda L
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- Humans, Male, Female, Middle Aged, Aged, Bronchoalveolar Lavage Fluid immunology, Adult, Signal Transduction immunology, Spike Glycoprotein, Coronavirus immunology, Interferons metabolism, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, T-Lymphocytes immunology, Pulmonary Alveoli immunology, Pulmonary Alveoli pathology, COVID-19 immunology, SARS-CoV-2 immunology, NF-kappa B metabolism
- Abstract
The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
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- 2024
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7. Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.
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Quan J, Fan Q, Simons LM, Smukowski SN, Pegg C, Longnecker R, Savas JN, Hultquist JF, and Smith GA
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- Humans, Zyxin metabolism, Zyxin genetics, Animals, Cell Line, Herpesvirus 1, Human genetics, Herpesvirus 1, Human physiology, Herpesvirus 1, Suid genetics, Herpesvirus 1, Suid physiology, Host-Pathogen Interactions, Alphaherpesvirinae genetics, Alphaherpesvirinae metabolism, CRISPR-Cas Systems, Epithelial Cells virology, Epithelial Cells metabolism, Biotin metabolism
- Abstract
Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo . To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection., Competing Interests: J.F.H. has received research support, paid to Northwestern University, from Gilead Sciences and is a paid consultant for Merck. G.A.S. has received research support, paid to Northwestern University, from Dechra Pharmaceuticals, is a paid consultant for EG427, and is a founder and president of Thyreos Inc. All other authors declare no competing interests.
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- 2024
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8. Publisher Correction: Interferon subverts an AHR-JUN axis to promote CXCL13 + T cells in lupus.
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Law C, Wacleche VS, Cao Y, Pillai A, Sowerby J, Hancock B, Horisberger A, Bracero S, Skidanova V, Li Z, Adejoorin I, Dillon E, Benque IJ, Nunez DP, Simmons DP, Keegan J, Chen L, Baker T, Brohawn PZ, Al-Mossawi H, Hao LY, Jones B, Rao N, Qu Y, Alves SE, Jonsson AH, Shaw KS, Vleugels RA, Massarotti E, Costenbader KH, Brenner MB, Lederer JA, Hultquist JF, Choi J, and Rao DA
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- 2024
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9. Global siRNA Screen Reveals Critical Human Host Factors of SARS-CoV-2 Multicycle Replication.
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Yin X, Pu Y, Yuan S, Pache L, Churas C, Weston S, Riva L, Simons LM, Cisneros WJ, Clausen T, De Jesus PD, Kim HN, Fuentes D, Whitelock J, Esko J, Lord M, Mena I, García-Sastre A, Hultquist JF, Frieman MB, Ideker T, Pratt D, Martin-Sancho L, and Chanda SK
- Abstract
Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo . These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals., Competing Interests: COMPETING INTERESTS STATEMENT J.F.H. has received research support, paid to Northwestern University, from Gilead Sciences, and is a paid consultant for Merck. The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. All other authors declare no competing interests.
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- 2024
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10. Interferon subverts an AHR-JUN axis to promote CXCL13 + T cells in lupus.
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Law C, Wacleche VS, Cao Y, Pillai A, Sowerby J, Hancock B, Horisberger A, Bracero S, Skidanova V, Li Z, Adejoorin I, Dillon E, Benque IJ, Nunez DP, Simmons DP, Keegan J, Chen L, Baker T, Brohawn PZ, Al-Mossawi H, Hao LY, Jones B, Rao N, Qu Y, Alves SE, Jonsson AH, Shaw KS, Vleugels RA, Massarotti E, Costenbader KH, Brenner MB, Lederer JA, Hultquist JF, Choi J, and Rao DA
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- Female, Humans, Male, Cell Differentiation, Epigenomics, Gene Expression Profiling, Interleukin-22 immunology, Interleukin-22 metabolism, T-Lymphocytes, Helper-Inducer immunology, T-Lymphocytes, Helper-Inducer metabolism, Basic Helix-Loop-Helix Transcription Factors metabolism, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, Chemokine CXCL13 metabolism, Interferon Type I immunology, Interferon Type I metabolism, Lupus Erythematosus, Systemic immunology, Lupus Erythematosus, Systemic metabolism, Lupus Erythematosus, Systemic genetics, Proto-Oncogene Proteins c-jun metabolism, Receptors, Aryl Hydrocarbon metabolism
- Abstract
Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions
1,2 . Expansion of T follicular helper (TFH ) and T peripheral helper (TPH ) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4 . Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs.5,6 ), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+ /ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH /TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7 , opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH /TFH cells on a polarization axis opposite from T helper 22 (TH 22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2024
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11. Evolution of the Umbilical Cord Blood Proteome Across Gestational Development.
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Mithal LB, Lancki N, Ling-Hu T, Goo YA, Otero S, Rhodes NJ, Cho BK, Grobman WA, Hultquist JF, Scholtens D, Mestan KG, and Seed PC
- Abstract
Neonatal health is dependent on early risk stratification, diagnosis, and timely management of potentially devastating conditions, particularly in the setting of prematurity. Many of these conditions are poorly predicted in real-time by clinical data and current diagnostics. Umbilical cord blood may represent a novel source of molecular signatures that provides a window into the state of the fetus at birth. In this study, we comprehensively characterized the cord blood proteome of infants born between 24 to 42 weeks using untargeted mass spectrometry and functional enrichment analysis. We determined that the cord blood proteome at birth varies significantly across gestational development. Proteins that function in structural development and growth (e.g., extracellular matrix organization, lipid particle remodeling, and blood vessel development) are more abundant earlier in gestation. In later gestations, proteins with increased abundance are in immune response and inflammatory pathways, including complements and calcium-binding proteins. Furthermore, these data contribute to the knowledge of the physiologic state of neonates across gestational age, which is crucial to understand as we strive to best support postnatal development in preterm infants, determine mechanisms of pathology causing adverse health outcomes, and develop cord blood biomarkers to help tailor our diagnosis and therapeutics for critical neonatal conditions., Competing Interests: JFH has received research support, paid to Northwestern University, from Gilead Sciences, and is a paid consultant for Merck. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2024
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12. COVID-19 outbreak and genomic investigation in an inpatient behavioral health unit.
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Rios-Guzman E, Stancovici AG, Simons LM, Barajas G, Glenn K, Weber RT, Ozer EA, Lorenzo-Redondo R, Hultquist JF, and Bolon MK
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Background: Inpatient behavioral health units (BHUs) had unique challenges in implementing interventions to mitigate coronavirus disease 2019 (COVID-19) transmission, in part due to socialization in BHU settings. The objective of this study was to identify the transmission routes and the efficacy of the mitigation strategies employed during a COVID-19 outbreak in an inpatient BHU during the Omicron surge from December 2021 to January 2022., Methods: An outbreak investigation was performed after identifying 2 COVID-19-positive BHU inpatients on December 16 and 20, 2021. Mitigation measures involved weekly point prevalence testing for all inpatients, healthcare workers (HCWs), and staff, followed by infection prevention mitigation measures and molecular surveillance. Whole-genome sequencing on a subset of COVID-19-positive individuals was performed to identify the outbreak source. Finally, an outbreak control sustainability plan was formulated for future BHU outbreak resurgences., Results: We identified 35 HCWs and 8 inpatients who tested positive in the BHU between December 16, 2021, and January 17, 2022. We generated severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomes from 15 HCWs and all inpatients. Phylogenetic analyses revealed 3 distinct but genetically related clusters: (1) an HCW and inpatient outbreak likely initiated by staff, (2) an HCW and inpatient outbreak likely initiated by an inpatient visitor, and (3) an HCW-only cluster initiated by staff., Conclusions: Distinct transmission clusters are consistent with multiple, independent SARS-CoV-2 introductions with further inpatient transmission occurring in communal settings. The implemented outbreak control plan comprised of enhanced personal protective equipment requirements, limited socialization, and molecular surveillance likely minimized disruptions to patient care as a model for future pandemics., Competing Interests: All other authors declare no conflicts of interest., (© The Author(s) 2024.)
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- 2024
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13. Deviations in RSV epidemiological patterns and population structures in the United States following the COVID-19 pandemic.
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Rios-Guzman E, Simons LM, Dean TJ, Agnes F, Pawlowski A, Alisoltanidehkordi A, Nam HH, Ison MG, Ozer EA, Lorenzo-Redondo R, and Hultquist JF
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- Infant, Humans, United States epidemiology, Aged, Retrospective Studies, Pandemics, Respiratory Syncytial Virus Infections, COVID-19 epidemiology, Respiratory Syncytial Virus, Human genetics
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Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with the greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially in the United States (US) following the implementation of COVID-19-related non-pharmaceutical interventions but later rebounded with abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study from 2009 to 2023 in Chicago, Illinois, US, we examined RSV epidemiology, clinical severity, and genetic diversity. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings post-2020 and that hospitalized adults infected with RSV-A were at higher risk of intensive care admission than those with RSV-B. While population structures of RSV-A remained unchanged, RSV-B exhibited a genetic shift into geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development., (© 2024. The Author(s).)
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- 2024
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14. CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.
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Rathore U, Haas P, Easwar Kumar V, Hiatt J, Haas KM, Bouhaddou M, Swaney DL, Stevenson E, Zuliani-Alvarez L, McGregor MJ, Turner-Groth A, Ochieng' Olwal C, Bediako Y, Braberg H, Soucheray M, Ott M, Eckhardt M, Hultquist JF, Marson A, Kaake RM, and Krogan NJ
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- Humans, CD4-Positive T-Lymphocytes, CRISPR-Cas Systems, Ubiquitins metabolism, Virus Replication, CCAAT-Enhancer-Binding Proteins metabolism, HIV Infections, TNF Receptor-Associated Factor 2 metabolism, Ubiquitin-Protein Ligases metabolism, Virus Latency, HIV physiology
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During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies., Importance: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.
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- 2024
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15. Activity and inhibition of the SARS-CoV-2 Omicron nsp13 R392C variant using RNA duplex unwinding assays.
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Inniss NL, Rzhetskaya M, Ling-Hu T, Lorenzo-Redondo R, Bachta KE, Satchell KJF, and Hultquist JF
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- Humans, RNA, Double-Stranded genetics, RNA, Double-Stranded metabolism, Mutation genetics, RNA, Viral genetics, RNA Helicases antagonists & inhibitors, RNA Helicases genetics, RNA Helicases metabolism, Virus Replication drug effects, Virus Replication genetics, COVID-19 virology, SARS-CoV-2 drug effects, SARS-CoV-2 genetics, Antiviral Agents pharmacology, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins antagonists & inhibitors, Viral Nonstructural Proteins metabolism, Methyltransferases
- Abstract
SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13
R392C variant, which is predominant in currently circulating lineages. Using in vitro gel- and fluorescence-based assays, we found that both nsp13 and nsp13R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Judd F. Hultquist, Karla J. F. Satchell reports financial support was provided by Robert H. Lurie Comprehensice Cancer Center. Karla J. F. Satchell reports financial support was provided by National Institute of Allergy and Infectious Diseases. Judd F. Hultquist reports financial support was provided by Successful Clinical Response to Pneumonia Therapy Center. Karla J. F. Satchell reports financial support was provided by Institute for Quantitative Biosciences Institute Coronavirus Research Group Antiviral Drug Discovery Center. Karla J. F. Satchell reports a relationship with Situ Biosciences that includes: equity or stocks. Judd F. Hultquist reports a relationship with Gilead Sciences Inc that includes: funding grants. Judd F. Hultquist reports a relationship with Merck that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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16. Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells.
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Stranford DM, Simons LM, Berman KE, Cheng L, DiBiase BN, Hung ME, Lucks JB, Hultquist JF, and Leonard JN
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- Humans, CD4-Positive T-Lymphocytes metabolism, T-Lymphocytes immunology, T-Lymphocytes metabolism, HEK293 Cells, Genetic Engineering methods, CRISPR-Cas Systems genetics, Drug Delivery Systems methods, Extracellular Vesicles metabolism, Biological Products
- Abstract
The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)-which naturally encapsulate and transfer proteins and nucleic acids between cells-for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9-single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4
+ T cells. The strategy is amenable to the targeted delivery of biologics to other cell types., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2024
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17. Genomic characterization of SARS-CoV-2 in Guinea, West Africa.
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Sow MS, Togo J, Simons LM, Diallo ST, Magassouba ML, Keita MB, Somboro AM, Coulibaly Y, Ozer EA, Hultquist JF, Murphy RL, Maiga AI, Maiga M, and Lorenzo-Redondo R
- Subjects
- Humans, Guinea epidemiology, Pandemics, Retrospective Studies, Africa, Western epidemiology, Genomics, SARS-CoV-2 genetics, COVID-19 epidemiology
- Abstract
SARS-CoV-2 has claimed several million lives since its emergence in late 2019. The ongoing evolution of the virus has resulted in the periodic emergence of new viral variants with distinct fitness advantages, including enhanced transmission and immune escape. While several SARS-CoV-2 variants of concern trace their origins back to the African continent-including Beta, Eta, and Omicron-most countries in Africa remain under-sampled in global genomic surveillance efforts. In an effort to begin filling these knowledge gaps, we conducted retrospective viral genomic surveillance in Guinea from October 2020 to August 2021. We found that SARS-CoV-2 clades 20A, 20B, and 20C dominated throughout 2020 until the coincident emergence of the Alpha and Eta variants of concern in January 2021. The Alpha variant remained dominant throughout early 2021 until the arrival of the Delta variant in July. Surprisingly, despite the small sample size of our study, we also found the persistence of the early SARS-CoV-2 clade 19B as late as April 2021. Together, these data help fill in our understanding of the SARS-CoV-2 population dynamics in West Africa early in the COVID-19 pandemic., Competing Interests: The authors have declared that no competing interests., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)
- Published
- 2024
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18. Host ZCCHC3 blocks HIV-1 infection and production through a dual mechanism.
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Yi B, Tanaka YL, Cornish D, Kosako H, Butlertanaka EP, Sengupta P, Lippincott-Schwartz J, Hultquist JF, Saito A, and Yoshimura SH
- Abstract
Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. Several host restriction factors that inhibit human immunodeficiency virus type 1 (HIV-1) have been identified, but most of them are antagonized by viral proteins. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel HIV-1 restriction factor that suppresses the production of HIV-1 and other retroviruses, but does not appear to be directly antagonized by viral proteins. It acts by binding to Gag nucleocapsid (GagNC) via zinc-finger motifs, which inhibits viral genome recruitment and results in genome-deficient virion production. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. This distinct, dual-acting antiviral mechanism makes upregulation of ZCCHC3 a novel potential therapeutic strategy., Competing Interests: J.F.H. has received research support, paid to Northwestern University, from Gilead Sciences and is a paid consultant for Merck., (© 2024 The Author(s).)
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- 2024
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19. Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.
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Ling-Hu T, Simons LM, Dean TJ, Rios-Guzman E, Caputo MT, Alisoltani A, Qi C, Malczynski M, Blanke T, Jennings LJ, Ison MG, Achenbach CJ, Larkin PM, Kaul KL, Lorenzo-Redondo R, Ozer EA, and Hultquist JF
- Subjects
- Humans, SARS-CoV-2 genetics, Molecular Epidemiology, Retrospective Studies, COVID-19 Testing, COVID-19 epidemiology
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk., Competing Interests: Declaration of interests M.G.I. has received research support, paid to Northwestern University, from AiCuris, GlaxoSmithKline Janssen, and Shire. M.G.I. is a paid consultant for Adagio, AlloVir, Celltrion, Cidara, Genentech, Roche, Janssen, Shionogi, Takeda, and Viracor Eurofins. M.G.I. is a paid member of the data and safety monitoring boards (DSMBs) of CSL Berhring, Janssen, Merck, SAB Biotherapeutics, Sequiris, and Takeda. J.F.H. has received research support, paid to Northwestern University, from Gilead Sciences and is a paid consultant for Merck., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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20. Social/Sexual Networks of People Newly Diagnosed with HIV in Ibadan, Nigeria.
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Phillips Ii G, Rodriguez-Ortiz AE, Adewumi OM, Banner K, Adetunji A, Awolude OA, Olayinka OA, Simons LM, Hultquist JF, Ozer EA, Kapogiannis B, Kuhns LM, Garofalo R, Taiwo B, Birkett M, and Lorenzo-Redondo R
- Subjects
- Male, Humans, Homosexuality, Male, Nigeria epidemiology, Sexual Behavior, HIV Infections diagnosis, HIV Infections epidemiology, Sexual and Gender Minorities, HIV Seropositivity
- Abstract
Young men who have sex with men (YMSM) in Nigeria are ten times more likely to be living with HIV-1 than other young men. Due to stigma and criminalization of same-sex sexual behavior, YMSM sexual networks are likely to overlap with those of the general population, leading to a generalized HIV-1 epidemic. Due to limited research on social/sexual network dynamics related to HIV-1 in Nigeria, our study focused on YMSM and sought to assess the feasibility and acceptability of collecting social and sexual network data in Network Canvas from individuals newly diagnosed with HIV-1 in Ibadan, Nigeria. The Network Canvas software was piloted at three sites in Ibadan, Nigeria to collect social/sexual network data from 151 individuals newly diagnosed with HIV-1. Our study sample included 37.7% YMSM; participants reported a mean of 2.6 social alters and 2.6 sexual alters. From the 151 egos and 634 alters, 85 potential unique individuals (194 total) were identified; 65 egos/alters were collapsed into 25 unique individuals. Our success collecting network data from individuals newly diagnosed with HIV-1 in Ibadan demonstrates clear feasibility and acceptability of the approach and the use of Network Canvas to capture and manage these data., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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21. A distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.
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Markov NS, Ren Z, Senkow KJ, Grant RA, Gao CA, Malsin ES, Sichizya L, Kihshen H, Helmin KA, Jovisic M, Arnold JM, Pérez-Leonor XG, Abdala-Valencia H, Swaminathan S, Nwaezeapu J, Kang M, Rasmussen L, Ozer EA, Lorenzo-Redondo R, Hultquist JF, Simons LM, Rios-Guzman E, Misharin AV, Wunderink RG, Budinger GRS, Singer BD, and Morales-Nebreda L
- Abstract
Pathogen clearance and resolution of inflammation in patients with pneumonia require an effective local T cell response. Nevertheless, local T cell activation may drive lung injury, particularly during prolonged episodes of respiratory failure characteristic of severe SARS-CoV-2 pneumonia. While T cell responses in the peripheral blood are well described, the evolution of T cell phenotypes and molecular signatures in the distal lung of patients with severe pneumonia caused by SARS-CoV-2 or other pathogens is understudied. Accordingly, we serially obtained 432 bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia and respiratory failure, including 74 unvaccinated patients with COVID-19, and performed flow cytometry, transcriptional, and T cell receptor profiling on sorted CD8
+ and CD4+ T cell subsets. In patients with COVID-19 but not pneumonia secondary to other pathogens, we found that early and persistent enrichment in CD8+ and CD4+ T cell subsets correlated with survival to hospital discharge. Activation of interferon signaling pathways early after intubation for COVID-19 was associated with favorable outcomes, while activation of NF-κB-driven programs late in disease was associated with poor outcomes. Patients with SARS-CoV-2 pneumonia whose alveolar T cells preferentially targeted the Spike and Nucleocapsid proteins tended to experience more favorable outcomes than patients whose T cells predominantly targeted the ORF1ab polyprotein complex. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize patients who recover, yet these responses progress to NF-κB activation against non-structural proteins in patients who go on to experience poor clinical outcomes., Competing Interests: Competing Interest Statement: BDS holds United States Patent No. US 10,905,706 B2, “Compositions and Methods to Accelerate Resolution of Acute Lung Inflammation”, and serves on the Scientific Advisory Board of Zoe Biosciences, outside of the submitted work. The other authors have no competing interests to declare.- Published
- 2023
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22. SARS-CoV-2 genomics and impact on clinical care for COVID-19.
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Lorenzo-Redondo R, de Sant'Anna Carvalho AM, Hultquist JF, and Ozer EA
- Subjects
- Humans, SARS-CoV-2 genetics, Antiviral Agents, Pandemics, Genomics, COVID-19, Hepatitis C, Chronic
- Abstract
The emergence and worldwide spread of SARS-CoV-2 during the COVID-19 pandemic necessitated the adaptation and rapid deployment of viral WGS and analysis techniques that had been previously applied on a more limited basis to other viral pathogens, such as HIV and influenza viruses. The need for WGS was driven in part by the low mutation rate of SARS-CoV-2, which necessitated measuring variation along the entire genome sequence to effectively differentiate lineages and characterize viral evolution. Several WGS approaches designed to maximize throughput and accuracy were quickly adopted by surveillance labs around the world. These broad-based SARS-CoV-2 genomic sequencing efforts revealed ongoing evolution of the virus, highlighted by the successive emergence of new viral variants throughout the course of the pandemic. These genomic insights were instrumental in characterizing the effects of viral mutations on transmissibility, immune escape and viral tropism, which in turn helped guide public health policy, the use of monoclonal antibody therapeutics and vaccine development strategies. As the use of direct-acting antivirals for the treatment of COVID-19 became more widespread, the potential for emergence of antiviral resistance has driven ongoing efforts to delineate resistance mutations and to monitor global sequence databases for their emergence. Given the critical role of viral genomics in the international effort to combat the COVID-19 pandemic, coordinated efforts should be made to expand global genomic surveillance capacity and infrastructure towards the anticipation and prevention of future pandemics., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)
- Published
- 2023
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23. Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.
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Samer S, Thomas Y, Araínga M, Carter C, Shirreff LM, Arif MS, Avita JM, Frank I, McRaven MD, Thuruthiyil CT, Heybeli VB, Anderson MR, Owen B, Gaisin A, Bose D, Simons LM, Hultquist JF, Arthos J, Cicala C, Sereti I, Santangelo PJ, Lorenzo-Redondo R, Hope TJ, Villinger FJ, and Martinelli E
- Subjects
- Animals, Immunity, Transforming Growth Factor beta, HIV-1, Simian Acquired Immunodeficiency Syndrome, Simian Immunodeficiency Virus
- Published
- 2023
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24. Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.
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Behrens RT, Rajashekar JK, Bruce JW, Evans EL 3rd, Hansen AM, Salazar-Quiroz N, Simons LM, Ahlquist P, Hultquist JF, Kumar P, and Sherer NM
- Subjects
- Humans, Mice, Animals, Rodentia, Cell Line, Cyclin T genetics, Cyclin T metabolism, Gene Expression, T-Lymphocytes, HIV-1 physiology, HIV Seropositivity, HIV Infections
- Abstract
Importance: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
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25. Resurgence of SARS-CoV-2 Delta after Omicron variant superinfection in an immunocompromised pediatric patient.
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Alisoltani A, Simons LM, Agnes MFR, Heald-Sargent TA, Muller WJ, Kociolek LK, Hultquist JF, Lorenzo-Redondo R, and Ozer EA
- Subjects
- Humans, Child, SARS-CoV-2 genetics, Immunocompromised Host, COVID-19, Superinfection, Coinfection
- Abstract
Background: Persistent SARS-CoV-2 infection in immunocompromised hosts is thought to contribute to viral evolution by facilitating long-term natural selection and viral recombination in cases of viral co-infection or superinfection. However, there are limited data on the longitudinal intra-host population dynamics of SARS-CoV-2 co-infection/superinfection, especially in pediatric populations. Here, we report a case of Delta-Omicron superinfection in a hospitalized, immunocompromised pediatric patient., Methods: We conducted Illumina whole genome sequencing (WGS) for longitudinal specimens to investigate intra-host dynamics of SARS-CoV-2 strains. Topoisomerase PCR cloning of Spike open-reading frame and Sanger sequencing of samples was performed for four specimens to validate the findings. Analysis of publicly available SARS-CoV-2 sequence data was performed to investigate the co-circulation and persistence of SARS-CoV-2 variants., Results: Results of WGS indicate the patient was initially infected with the SARS-CoV-2 Delta variant before developing a SARS-CoV-2 Omicron variant superinfection, which became predominant. Shortly thereafter, viral loads decreased below the level of detection before resurgence of the original Delta variant with no residual trace of Omicron. After 54 days of persistent infection, the patient tested negative for SARS-CoV-2 but ultimately succumbed to a COVID-19-related death. Despite protracted treatment with remdesivir, no antiviral resistance mutations emerged. These results indicate a unique case of persistent SARS-CoV-2 infection with the Delta variant interposed by a transient superinfection with the Omicron variant. Analysis of publicly available sequence data suggests the persistence and ongoing evolution of Delta subvariants despite the global predominance of Omicron, potentially indicative of continued transmission in an unknown population or niche., Conclusion: A better understanding of SARS-CoV-2 intra-host population dynamics, persistence, and evolution during co-infections and/or superinfections will be required to continue optimizing patient care and to better predict the emergence of new variants of concern., (© 2023. The Author(s).)
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- 2023
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26. Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.
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Haas KM, McGregor MJ, Bouhaddou M, Polacco BJ, Kim EY, Nguyen TT, Newton BW, Urbanowski M, Kim H, Williams MAP, Rezelj VV, Hardy A, Fossati A, Stevenson EJ, Sukerman E, Kim T, Penugonda S, Moreno E, Braberg H, Zhou Y, Metreveli G, Harjai B, Tummino TA, Melnyk JE, Soucheray M, Batra J, Pache L, Martin-Sancho L, Carlson-Stevermer J, Jureka AS, Basler CF, Shokat KM, Shoichet BK, Shriver LP, Johnson JR, Shaw ML, Chanda SK, Roden DM, Carter TC, Kottyan LC, Chisholm RL, Pacheco JA, Smith ME, Schrodi SJ, Albrecht RA, Vignuzzi M, Zuliani-Alvarez L, Swaney DL, Eckhardt M, Wolinsky SM, White KM, Hultquist JF, Kaake RM, García-Sastre A, and Krogan NJ
- Subjects
- Humans, Influenza A Virus, H3N2 Subtype metabolism, Proteomics, Virus Replication genetics, SARS-CoV-2, Antiviral Agents metabolism, Host-Pathogen Interactions genetics, Influenza A virus genetics, Influenza, Human genetics, Influenza A Virus, H5N1 Subtype genetics, COVID-19
- Abstract
Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT., (© 2023. Springer Nature Limited.)
- Published
- 2023
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27. Quantity of SARS-CoV-2 RNA copies exhaled per minute during natural breathing over the course of COVID-19 infection.
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Lane G, Zhou G, Hultquist JF, Simons LM, Redondo RL, Ozer EA, McCarthy DM, Ison MG, Achenbach CJ, Wang X, Wai CM, Wyatt E, Aalsburg A, Yang Q, Noto T, Alisoltani A, Ysselstein D, Awatramani R, Murphy R, Theron G, and Zelano C
- Abstract
SARS-CoV-2 is spread through exhaled breath of infected individuals. A fundamental question in understanding transmission of SARS-CoV-2 is how much virus an individual is exhaling into the environment while they breathe, over the course of their infection. Research on viral load dynamics during COVID-19 infection has focused on internal swab specimens, which provide a measure of viral loads inside the respiratory tract, but not on breath. Therefore, the dynamics of viral shedding on exhaled breath over the course of infection are poorly understood. Here, we collected exhaled breath specimens from COVID-19 patients and used RTq-PCR to show that numbers of exhaled SARS-CoV-2 RNA copies during COVID-19 infection do not decrease significantly until day 8 from symptom-onset. COVID-19-positive participants exhaled an average of 80 SARS-CoV-2 viral RNA copies per minute during the first 8 days of infection, with significant variability both between and within individuals, including spikes over 800 copies a minute in some patients. After day 8, there was a steep drop to levels nearing the limit of detection, persisting for up to 20 days. We further found that levels of exhaled viral RNA increased with self-rated symptom-severity, though individual variation was high. Levels of exhaled viral RNA did not differ across age, sex, time of day, vaccination status or viral variant. Our data provide a fine-grained, direct measure of the number of SARS-CoV-2 viral copies exhaled per minute during natural breathing-including 312 breath specimens collected multiple times daily over the course of infection-in order to fill an important gap in our understanding of the time course of exhaled viral loads in COVID-19.
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- 2023
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28. Author Correction: Africa-specific human genetic variation near CHD1L associates with HIV-1 load.
- Author
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McLaren PJ, Porreca I, Iaconis G, Mok HP, Mukhopadhyay S, Karakoc E, Cristinelli S, Pomilla C, Bartha I, Thorball CW, Tough RH, Angelino P, Kiar CS, Carstensen T, Fatumo S, Porter T, Jarvis I, Skarnes WC, Bassett A, DeGorter MK, Sathya Moorthy MP, Tuff JF, Kim EY, Walter M, Simons LM, Bashirova A, Buchbinder S, Carrington M, Cossarizza A, De Luca A, Goedert JJ, Goldstein DB, Haas DW, Herbeck JT, Johnson EO, Kaleebu P, Kilembe W, Kirk GD, Kootstra NA, Kral AH, Lambotte O, Luo M, Mallal S, Martinez-Picado J, Meyer L, Miro JM, Moodley P, Motala AA, Mullins JI, Nam K, Obel N, Pirie F, Plummer FA, Poli G, Price MA, Rauch A, Theodorou I, Trkola A, Walker BD, Winkler CA, Zagury JF, Montgomery SB, Ciuffi A, Hultquist JF, Wolinsky SM, Dougan G, Lever AML, Gurdasani D, Groom H, Sandhu MS, and Fellay J
- Published
- 2023
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29. Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors.
- Author
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Fink EA, Bardine C, Gahbauer S, Singh I, Detomasi TC, White K, Gu S, Wan X, Chen J, Ary B, Glenn I, O'Connell J, O'Donnell H, Fajtová P, Lyu J, Vigneron S, Young NJ, Kondratov IS, Alisoltani A, Simons LM, Lorenzo-Redondo R, Ozer EA, Hultquist JF, O'Donoghue AJ, Moroz YS, Taunton J, Renslo AR, Irwin JJ, García-Sastre A, Shoichet BK, and Craik CS
- Subjects
- Humans, SARS-CoV-2 metabolism, Pandemics, Protease Inhibitors pharmacology, Protease Inhibitors chemistry, Molecular Docking Simulation, Viral Nonstructural Proteins chemistry, Antiviral Agents pharmacology, Antiviral Agents chemistry, COVID-19
- Abstract
Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (M
Pro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 μM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself., (© 2023 The Protein Society.)- Published
- 2023
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30. Africa-specific human genetic variation near CHD1L associates with HIV-1 load.
- Author
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McLaren PJ, Porreca I, Iaconis G, Mok HP, Mukhopadhyay S, Karakoc E, Cristinelli S, Pomilla C, Bartha I, Thorball CW, Tough RH, Angelino P, Kiar CS, Carstensen T, Fatumo S, Porter T, Jarvis I, Skarnes WC, Bassett A, DeGorter MK, Sathya Moorthy MP, Tuff JF, Kim EY, Walter M, Simons LM, Bashirova A, Buchbinder S, Carrington M, Cossarizza A, De Luca A, Goedert JJ, Goldstein DB, Haas DW, Herbeck JT, Johnson EO, Kaleebu P, Kilembe W, Kirk GD, Kootstra NA, Kral AH, Lambotte O, Luo M, Mallal S, Martinez-Picado J, Meyer L, Miro JM, Moodley P, Motala AA, Mullins JI, Nam K, Obel N, Pirie F, Plummer FA, Poli G, Price MA, Rauch A, Theodorou I, Trkola A, Walker BD, Winkler CA, Zagury JF, Montgomery SB, Ciuffi A, Hultquist JF, Wolinsky SM, Dougan G, Lever AML, Gurdasani D, Groom H, Sandhu MS, and Fellay J
- Subjects
- Humans, Cell Line, Africa, Chromosomes, Human, Pair 1 genetics, Alleles, RNA, Long Noncoding genetics, Virus Replication, DNA Helicases genetics, DNA Helicases metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Genetic Variation, HIV Infections genetics, HIV-1 growth & development, HIV-1 physiology, Viral Load genetics
- Abstract
HIV-1 remains a global health crisis
1 , highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2 , we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3 . We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10 -transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4 . Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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31. Analysis of the Contribution of 6-mer Seed Toxicity to HIV-1-Induced Cytopathicity.
- Author
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Vaidyanathan A, Taylor HE, Hope TJ, D'Aquila RT, Bartom ET, Hultquist JF, and Peter ME
- Subjects
- Humans, Virus Latency genetics, RNA-Induced Silencing Complex metabolism, HIV Infections, HIV-1 physiology, MicroRNAs genetics, MicroRNAs metabolism
- Abstract
HIV-1 (HIV) infects CD4
+ T cells, the gradual depletion of which can lead to AIDS in the absence of antiretroviral therapy (ART). Some cells, however, survive HIV infection and persist as part of the latently infected reservoir that causes recurrent viremia after ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could lead to a way to clear the latent reservoir. Death induced by survival gene elimination (DISE), an RNA interference (RNAi)-based mechanism, kills cells through short RNAs (sRNAs) with toxic 6-mer seeds (positions 2 to 7 of sRNA). These toxic seeds target the 3' untranslated region (UTR) of mRNAs, decreasing the expression of hundreds of genes critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded nontoxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. HIV has been shown to inhibit the biogenesis of host miRNAs in multiple ways. We now report that HIV infection of cells deficient in miRNA expression or function results in enhanced RISC loading of an HIV-encoded miRNA HIV-miR-TAR-3p, which can kill cells by DISE through a noncanonical (positions 3 to 8) 6-mer seed. In addition, cellular RISC-bound sRNAs shift to lower seed viability. This also occurs after latent HIV provirus reactivation in J-Lat cells, suggesting independence of permissiveness of cells to viral infection. More precise targeting of the balance between protective and cytotoxic sRNAs could provide new avenues to explore novel cell death mechanisms that could be used to kill latent HIV. IMPORTANCE Several mechanisms by which initial HIV infection is cytotoxic to infected cells have been reported and involve various forms of cell death. Characterizing the mechanisms underlying the long-term survival of certain T cells that become persistent provirus reservoirs is critical to developing a cure. We recently discovered death induced by survival gene elimination (DISE), an RNAi-based mechanism of cell death whereby toxic short RNAs (sRNAs) containing 6-mer seed sequences (exerting 6-mer seed toxicity) targeting essential survival genes are loaded into RNA-induced silencing complex (RISC) complexes, resulting in inescapable cell death. We now report that HIV infection in cells with low miRNA expression causes a shift of mostly cellular RISC-bound sRNAs to more toxic seeds. This could prime cells to DISE and is further enhanced by the viral microRNA (miRNA) HIV-miR-TAR-3p, which carries a toxic noncanonical 6-mer seed. Our data provide multiple new avenues to explore novel cell death mechanisms that could be used to kill latent HIV., Competing Interests: The authors declare no conflict of interest.- Published
- 2023
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32. Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions.
- Author
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Vandervaart JP, Inniss NL, Ling-Hu T, Minasov G, Wiersum G, Rosas-Lemus M, Shuvalova L, Achenbach CJ, Hultquist JF, Satchell KJF, and Bachta KER
- Subjects
- Humans, SARS-CoV-2, Antibodies, Viral, Epitopes, Nucleocapsid, Peptides, Intrinsically Disordered Proteins, COVID-19
- Abstract
The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design., Competing Interests: The authors declare a conflict of interest. K.J.F.S. has significant financial interest in Situ Biosciences LLC, a contract research organization that conducts coronavirus research unrelated to this work. K.J.F.S. also received payments from Buoy Health during the time of this investigation for consultation on COVID-19 topics unrelated to this work. All other authors declare no conflicts of interest.
- Published
- 2023
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33. Enhancing HIV-1 latency reversal through regulating the elongating RNA Pol II pause-release by a small-molecule disruptor of PAF1C.
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Soliman SHA, Cisneros WJ, Iwanaszko M, Aoi Y, Ganesan S, Walter M, Zeidner JM, Mishra RK, Kim EY, Wolinsky SM, Hultquist JF, and Shilatifard A
- Subjects
- Humans, Molecular Docking Simulation, Cell Line, Transcription, Genetic, Transcription Factors genetics, RNA Polymerase II metabolism, HIV-1 genetics, HIV-1 metabolism
- Abstract
The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus-1 (HIV-1) latency. Using a molecular docking-based compound screen in silico and global sequencing-based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock-down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.
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- 2023
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34. Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4 + T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.
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Fernandez-de Céspedes MV, Hoffman HK, Carter H, Simons LM, Naing L, Ablan SD, Scheiblin DA, Hultquist JF, van Engelenburg SB, and Freed EO
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- Humans, HeLa Cells, HIV Envelope Protein gp41 metabolism, Virus Assembly, env Gene Products, Human Immunodeficiency Virus metabolism, CD4-Positive T-Lymphocytes virology, HIV-1 physiology, Virus Replication, Adaptor Proteins, Signal Transducing metabolism, Membrane Proteins metabolism
- Abstract
The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4
+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795 W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.- Published
- 2022
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35. ViralVar: A Web Tool for Multilevel Visualization of SARS-CoV-2 Genomes.
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Alisoltani A, Jaroszewski L, Godzik A, Iranzadeh A, Simons LM, Dean TJ, Lorenzo-Redondo R, Hultquist JF, and Ozer EA
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- Humans, Child, Aged, Genome, Viral, Mutation, Phylogeny, SARS-CoV-2 genetics, COVID-19 genetics
- Abstract
The unprecedented growth of publicly available SARS-CoV-2 genome sequence data has increased the demand for effective and accessible SARS-CoV-2 data analysis and visualization tools. The majority of the currently available tools either require computational expertise to deploy them or limit user input to preselected subsets of SARS-CoV-2 genomes. To address these limitations, we developed ViralVar, a publicly available, point-and-click webtool that gives users the freedom to investigate and visualize user-selected subsets of SARS-CoV-2 genomes obtained from the GISAID public database. ViralVar has two primary features that enable: (1) the visualization of the spatiotemporal dynamics of SARS-CoV-2 lineages and (2) a structural/functional analysis of genomic mutations. As proof-of-principle, ViralVar was used to explore the evolution of the SARS-CoV-2 pandemic in the USA in pediatric, adult, and elderly populations (n > 1.7 million genomes). Whereas the spatiotemporal dynamics of the variants did not differ between these age groups, several USA-specific sublineages arose relative to the rest of the world. Our development and utilization of ViralVar to provide insights on the evolution of SARS-CoV-2 in the USA demonstrates the importance of developing accessible tools to facilitate and accelerate the large-scale surveillance of circulating pathogens.
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- 2022
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36. De novo emergence of SARS-CoV-2 spike mutations in immunosuppressed patients.
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Simons LM, Ozer EA, Gambut S, Dean TJ, Zhang L, Bhimalli P, Schneider JR, Mamede JI, Ison MG, Karmali R, Gordon LI, Lorenzo-Redondo R, and Hultquist JF
- Subjects
- Humans, Mutation, Antiviral Agents, Immunocompromised Host, Antibodies, Neutralizing, Antibodies, Viral, SARS-CoV-2 genetics, COVID-19
- Abstract
Background: The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts., Methods: Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro., Results: Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry., Conclusions: These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings., (© 2022 The Authors. Transplant Infectious Disease published by Wiley Periodicals LLC.)
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- 2022
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37. Incidence and Clinical Characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 Infection After Vaccination in Children.
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Reyna ME, Edward PR, Ozer EA, Simons LM, Hultquist JF, Lorenzo-Redondo R, Patel AB, and Kociolek LK
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- Child, Humans, Cohort Studies, COVID-19 epidemiology, COVID-19 prevention & control, Incidence, SARS-CoV-2, Vaccination, COVID-19 Vaccines, Vaccine Efficacy
- Abstract
The objective of this single-center cohort study was to characterize the frequency, clinical characteristics, and molecular epidemiology of pediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection after vaccination. Between May 15, 2021, and January 1, 2022, 171 children experienced SARS-CoV-2 infection postvaccination, 146 (86%) following the Omicron variant predominance. Outcomes were generally mild and comparable before and after Omicron predominance., (Copyright © 2022 Elsevier Inc. All rights reserved.)
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- 2022
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38. Challenges and Opportunities for Global Genomic Surveillance Strategies in the COVID-19 Era.
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Ling-Hu T, Rios-Guzman E, Lorenzo-Redondo R, Ozer EA, and Hultquist JF
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- Humans, SARS-CoV-2 genetics, Genomics, Pandemics prevention & control, Public Health, COVID-19 epidemiology, COVID-19 prevention & control
- Abstract
Global SARS-CoV-2 genomic surveillance efforts have provided critical data on the ongoing evolution of the virus to inform best practices in clinical care and public health throughout the pandemic. Impactful genomic surveillance strategies generally follow a multi-disciplinary pipeline involving clinical sample collection, viral genotyping, metadata linkage, data reporting, and public health responses. Unfortunately, current limitations in each of these steps have compromised the overall effectiveness of these strategies. Biases from convenience-based sampling methods can obfuscate the true distribution of circulating variants. The lack of standardization in genotyping strategies and bioinformatic expertise can create bottlenecks in data processing and complicate interpretation. Limitations and inconsistencies in clinical and demographic data collection and sharing can slow the compilation and limit the utility of comprehensive datasets. This likewise can complicate data reporting, restricting the availability of timely data. Finally, gaps and delays in the implementation of genomic surveillance data in the public health sphere can prevent officials from formulating effective mitigation strategies to prevent outbreaks. In this review, we outline current SARS-CoV-2 global genomic surveillance methods and assess roadblocks at each step of the pipeline to identify potential solutions. Evaluating the current obstacles that impede effective surveillance can improve both global coordination efforts and pandemic preparedness for future outbreaks.
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- 2022
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39. Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.
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Samer S, Thomas Y, Araínga M, Carter C, Shirreff LM, Arif MS, Avita JM, Frank I, McRaven MD, Thuruthiyil CT, Heybeli VB, Anderson MR, Owen B, Gaisin A, Bose D, Simons LM, Hultquist JF, Arthos J, Cicala C, Sereti I, Santangelo PJ, Lorenzo-Redondo R, Hope TJ, Villinger FJ, and Martinelli E
- Subjects
- Animals, Copper Radioisotopes pharmacology, Copper Radioisotopes therapeutic use, Anti-Retroviral Agents therapeutic use, Positron Emission Tomography Computed Tomography, Macaca mulatta, Virus Replication, Transforming Growth Factor beta, Immunity, HIV-1, Simian Acquired Immunodeficiency Syndrome drug therapy, Simian Immunodeficiency Virus, HIV Infections
- Abstract
TGF-β plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-β-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-β and a TGF-β type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab')2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-β reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1-infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected macaques. This increase correlated with an increase in SIV RNA in the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-β signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.
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- 2022
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40. CCL2: a Chemokine Potentially Promoting Early Seeding of the Latent HIV Reservoir.
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Packard TA, Schwarzer R, Herzig E, Rao D, Luo X, Egedal JH, Hsiao F, Widera M, Hultquist JF, Grimmett ZW, Messer RJ, Krogan NJ, Deeks SG, Roan NR, Dittmer U, Hasenkrug KJ, and Greene WC
- Subjects
- Animals, Mice, Virus Latency physiology, Chemokine CCL2, Receptors, CCR2, Virus Replication, CD4-Positive T-Lymphocytes, Antiviral Agents therapeutic use, Chemokines, Inflammation, HIV Infections, HIV-1 physiology
- Abstract
HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5
+ cells and (ii) CCR2/5+ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5+ CD4+ T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.- Published
- 2022
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41. Severity of Illness Caused by Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern in Children: A Single-Center Retrospective Cohort Study.
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Edward PR, Lorenzo-Redondo R, Reyna ME, Simons LM, Hultquist JF, Patel AB, Ozer EA, Muller WJ, Heald-Sargent T, McHugh M, Dean T, Dalal RM, John J, Manz SC, and Kociolek LK
- Subjects
- Child, Humans, Adolescent, COVID-19 Vaccines, Retrospective Studies, Patient Acuity, SARS-CoV-2, COVID-19
- Abstract
Background: Recent COVID-19 surges are attributed to emergence of more transmissible SARS-CoV-2 variants of concern (VOCs). The relative severity of VOCs in children is unknown., Methods: We performed a single-center retrospective cohort study of children ≤18 years old diagnosed with COVID-19 from October 2020-February 2022 and whose SARS-CoV-2 isolate underwent Illumina sequencing. We measured the frequency of five markers of COVID-19 severity. Logistic regression models were fitted to estimate the odds of each severity marker with each VOC., Results: Among 714 children, 471 (66.0%) were infected with a VOC: 96 (13.4%) alpha, 38 (5.3%) gamma, 119 (16.7%) delta, and 215 (30.1%) omicron. High-risk medical conditions and increasing age were independently associated with COVID-19 severity. After adjusting for age, race, ethnicity, high-risk medical conditions, and COVID-19 community incidence, neither alpha, delta, nor omicron was associated with severe COVID-19. Gamma was independently associated with hospitalization (OR 6.7, 95% CI 2.0-22.1); pharmacologic treatment (OR 5.7, 95% CI 1.2-26.8); respiratory support (OR 11.9, 95% CI 2.7-62.4); and severe disease per the WHO Clinical Progression Scale (OR 11.7, 95% CI 2.1-90.5). Upon subgroup analyses, omicron was independently associated with ICU admission and severe disease per the WHO Clinical Progression Scale in children without SARS-CoV-2 immunization or prior COVID-19 infection., Conclusions: Compared to non-VOC COVID-19, the gamma VOC was independently associated with increased COVID-19 severity, as was omicron in children without SARS-CoV-2 immunization or prior COVID-19 infection. SARS-CoV-2 vaccination and prior COVID-19 prevented severe outcomes during the omicron surge., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2022
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42. Clinical and biological consequences of respiratory syncytial virus genetic diversity.
- Author
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Rios Guzman E and Hultquist JF
- Abstract
Respiratory syncytial virus (RSV) is one of the most common etiological agents of global acute respiratory tract infections with a disproportionate burden among infants, individuals over the age of 65, and immunocompromised populations. The two major subtypes of RSV (A and B) co-circulate with a predominance of either group during different epidemic seasons, with frequently emerging genotypes due to RSV's high genetic variability. Global surveillance systems have improved our understanding of seasonality, disease burden, and genomic evolution of RSV through genotyping by sequencing of attachment (G) glycoprotein. However, the integration of these systems into international infrastructures is in its infancy, resulting in a relatively low number (~2200) of publicly available RSV genomes. These limitations in surveillance hinder our ability to contextualize RSV evolution past current canonical attachment glycoprotein (G)-oriented understanding, thus resulting in gaps in understanding of how genetic diversity can play a role in clinical outcome, therapeutic efficacy, and the host immune response. Furthermore, utilizing emerging RSV genotype information from surveillance and testing the impact of viral evolution using molecular techniques allows us to establish causation between the clinical and biological consequences of arising genotypes, which subsequently aids in informed vaccine design and future vaccination strategy. In this review, we aim to discuss the findings from current molecular surveillance efforts and the gaps in knowledge surrounding the consequence of RSV genetic diversity on disease severity, therapeutic efficacy, and RSV-host interactions., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2022.)
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- 2022
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43. Viral whole-genome sequencing to assess impact of universal masking on SARS-CoV-2 transmission among pediatric healthcare workers.
- Author
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Kociolek LK, Patel AB, Hultquist JF, Ozer EA, Simons LM, McHugh M, Muller WJ, and Lorenzo-Redondo R
- Subjects
- Humans, Child, Infectious Disease Transmission, Patient-to-Professional prevention & control, Phylogeny, Health Personnel, SARS-CoV-2 genetics, COVID-19 diagnosis, COVID-19 epidemiology, COVID-19 prevention & control
- Abstract
Objective: To identify the impact of universal masking on COVID-19 incidence and putative SARS-CoV-2 transmissions events among children's hospital healthcare workers (HCWs)., Design: Quasi-experimental study., Setting: Single academic free-standing children's hospital., Methods: We performed whole-genome sequencing of SARS-CoV-2- PCR-positive samples collected from HCWs 3 weeks before and 6 weeks after implementing a universal masking policy. Phylogenetic analyses were performed to identify clusters of clonally related SARS-CoV-2 indicative of putative transmission events. We measured COVID-19 incidence, SARS-CoV-2 test positivity rates, and frequency of putative transmission events before and after the masking policy was implemented., Results: HCW COVID-19 incidence and test positivity declined from 14.3 to 4.3 cases per week, and from 18.4% to 9.0%, respectively. Putative transmission events were only identified prior to universal masking., Conclusions: A universal masking policy was associated with reductions in HCW COVID-19 infections and occupational acquisition of SARS-CoV-2.
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- 2022
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44. Application of CRISPR-Cas9 Gene Editing for HIV Host Factor Discovery and Validation.
- Author
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Cisneros WJ, Cornish D, and Hultquist JF
- Abstract
Human Immunodeficiency Virus (HIV) interacts with a wide array of host factors at each stage of its lifecycle to facilitate replication and circumvent the immune response. Identification and characterization of these host factors is critical for elucidating the mechanism of viral replication and for developing next-generation HIV-1 therapeutic and curative strategies. Recent advances in CRISPR-Cas9-based genome engineering approaches have provided researchers with an assortment of new, valuable tools for host factor discovery and interrogation. Genome-wide screening in a variety of in vitro cell models has helped define the critical host factors that play a role in various cellular and biological contexts. Targeted manipulation of specific host factors by CRISPR-Cas9-mediated gene knock-out, overexpression, and/or directed repair have furthermore allowed for target validation in primary cell models and mechanistic inquiry through hypothesis-based testing. In this review, we summarize several CRISPR-based screening strategies for the identification of HIV-1 host factors and highlight how CRISPR-Cas9 approaches have been used to elucidate the molecular mechanisms of viral replication and host response. Finally, we examine promising new technologies in the CRISPR field and how these may be applied to address critical questions in HIV-1 biology going forward.
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- 2022
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45. Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA.
- Author
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Yoh SM, Mamede JI, Lau D, Ahn N, Sánchez-Aparicio MT, Temple J, Tuckwell A, Fuchs NV, Cianci GC, Riva L, Curry H, Yin X, Gambut S, Simons LM, Hultquist JF, König R, Xiong Y, García-Sastre A, Böcking T, Hope TJ, and Chanda SK
- Subjects
- Capsid metabolism, DNA metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Humans, Immunity, Innate, Nucleotidyltransferases metabolism, Pathogen-Associated Molecular Pattern Molecules metabolism, HIV-1 genetics
- Abstract
We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP., Competing Interests: Declaration of interests The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, Paratus, CureLab Oncology, CureLab Veterinary, Synairgen, and Pfizer, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Sequirus, Janssen, and Astrazeneca outside of the reported work. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. The S.K.C. laboratory has received research support from Eli Lilly & Co., Boehringer Ingelheim, Merck & Co., Cidara Therapeutics, outside of the reported work. S.K.C. has consulting agreements for the following companies involving cash and/or stock: Pagoda Genomics, Cidara Therapeutics, and Samsara Biocapital, outside of the reported work. S.K.C. is inventor on patents and patent applications on the use of antivirals, adjuvants, and immunotherapies for the treatment and prevention of virus infections and cancer, owned by Sanford Burnham Prebys Medical Discovery Institute and Scripps Research, outside of the reported work., (Copyright © 2022 Elsevier Inc. All rights reserved.)
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- 2022
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46. Covid-19: is omicron less lethal than delta?
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Lorenzo-Redondo R, Ozer EA, and Hultquist JF
- Subjects
- Humans, COVID-19
- Abstract
Competing Interests: Competing interests: The BMJ has judged that there are no disqualifying financial ties to commercial companies. The authors declare the following other interests: none. Further details of The BMJ policy on financial interests is here: https://www.bmj.com/sites/default/files/attachments/resources/2016/03/16-current-bmj-education-coi-form.pdf.
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- 2022
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47. Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.
- Author
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Agarwal DK, Hunt AC, Shekhawat GS, Carter L, Chan S, Wu K, Cao L, Baker D, Lorenzo-Redondo R, Ozer EA, Simons LM, Hultquist JF, Jewett MC, and Dravid VP
- Subjects
- COVID-19 Testing, Clinical Laboratory Techniques, Humans, Sensitivity and Specificity, COVID-19 diagnosis, SARS-CoV-2 genetics
- Abstract
New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.
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- 2022
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48. Overlapping Delta and Omicron Outbreaks During the COVID-19 Pandemic: Dynamic Panel Data Estimates.
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Lundberg AL, Lorenzo-Redondo R, Hultquist JF, Hawkins CA, Ozer EA, Welch SB, Prasad PVV, Achenbach CJ, White JI, Oehmke JF, Murphy RL, Havey RJ, and Post LA
- Subjects
- Disease Outbreaks, Humans, Pandemics, Public Health Surveillance methods, COVID-19 epidemiology, SARS-CoV-2
- Abstract
Background: The Omicron variant of SARS-CoV-2 is more transmissible than prior variants of concern (VOCs). It has caused the largest outbreaks in the pandemic, with increases in mortality and hospitalizations. Early data on the spread of Omicron were captured in countries with relatively low case counts, so it was unclear how the arrival of Omicron would impact the trajectory of the pandemic in countries already experiencing high levels of community transmission of Delta., Objective: The objective of this study is to quantify and explain the impact of Omicron on pandemic trajectories and how they differ between countries that were or were not in a Delta outbreak at the time Omicron occurred., Methods: We used SARS-CoV-2 surveillance and genetic sequence data to classify countries into 2 groups: those that were in a Delta outbreak (defined by at least 10 novel daily transmissions per 100,000 population) when Omicron was first sequenced in the country and those that were not. We used trend analysis, survival curves, and dynamic panel regression models to compare outbreaks in the 2 groups over the period from November 1, 2021, to February 11, 2022. We summarized the outbreaks in terms of their peak rate of SARS-CoV-2 infections and the duration of time the outbreaks took to reach the peak rate., Results: Countries that were already in an outbreak with predominantly Delta lineages when Omicron arrived took longer to reach their peak rate and saw greater than a twofold increase (2.04) in the average apex of the Omicron outbreak compared to countries that were not yet in an outbreak., Conclusions: These results suggest that high community transmission of Delta at the time of the first detection of Omicron was not protective, but rather preluded larger outbreaks in those countries. Outbreak status may reflect a generally susceptible population, due to overlapping factors, including climate, policy, and individual behavior. In the absence of strong mitigation measures, arrival of a new, more transmissible variant in these countries is therefore more likely to lead to larger outbreaks. Alternately, countries with enhanced surveillance programs and incentives may be more likely to both exist in an outbreak status and detect more cases during an outbreak, resulting in a spurious relationship. Either way, these data argue against herd immunity mitigating future outbreaks with variants that have undergone significant antigenic shifts., (©Alexander L Lundberg, Ramon Lorenzo-Redondo, Judd F Hultquist, Claudia A Hawkins, Egon A Ozer, Sarah B Welch, P V Vara Prasad, Chad J Achenbach, Janine I White, James F Oehmke, Robert L Murphy, Robert J Havey, Lori A Post. Originally published in JMIR Public Health and Surveillance (https://publichealth.jmir.org), 03.06.2022.)
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- 2022
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49. Longitudinal Analysis of SARS-CoV-2 Vaccine Breakthrough Infections Reveals Limited Infectious Virus Shedding and Restricted Tissue Distribution.
- Author
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Ke R, Martinez PP, Smith RL, Gibson LL, Achenbach CJ, McFall S, Qi C, Jacob J, Dembele E, Bundy C, Simons LM, Ozer EA, Hultquist JF, Lorenzo-Redondo R, Opdycke AK, Hawkins C, Murphy RL, Mirza A, Conte M, Gallagher N, Luo CH, Jarrett J, Conte A, Zhou R, Farjo M, Rendon G, Fields CJ, Wang L, Fredrickson R, Baughman ME, Chiu KK, Choi H, Scardina KR, Owens AN, Broach J, Barton B, Lazar P, Robinson ML, Mostafa HH, Manabe YC, Pekosz A, McManus DD, and Brooke CB
- Abstract
Background: The global effort to vaccinate people against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during an ongoing pandemic has raised questions about how vaccine breakthrough infections compare with infections in immunologically naive individuals and the potential for vaccinated individuals to transmit the virus., Methods: We examined viral dynamics and infectious virus shedding through daily longitudinal sampling in 23 adults infected with SARS-CoV-2 at varying stages of vaccination, including 6 fully vaccinated individuals., Results: The durations of both infectious virus shedding and symptoms were significantly reduced in vaccinated individuals compared with unvaccinated individuals. We also observed that breakthrough infections are associated with strong tissue compartmentalization and are only detectable in saliva in some cases., Conclusions: Vaccination shortens the duration of time of high transmission potential, minimizes symptom duration, and may restrict tissue dissemination., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)
- Published
- 2022
- Full Text
- View/download PDF
50. Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling.
- Author
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Johnson JR, Crosby DC, Hultquist JF, Kurland AP, Adhikary P, Li D, Marlett J, Swann J, Hüttenhain R, Verschueren E, Johnson TL, Newton BW, Shales M, Simon VA, Beltrao P, Frankel AD, Marson A, Cox JS, Fregoso OI, Young JAT, and Krogan NJ
- Subjects
- Humans, Protein Processing, Post-Translational, Proteomics, Ubiquitination, HIV Infections, HIV Seropositivity, HIV-1 genetics
- Abstract
Viruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We use mass spectrometry-based proteomics to quantify changes in protein abundance and two PTM types-phosphorylation and ubiquitination-in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis reveals a requirement for Aurora kinase activity in HIV-1 infection and identified putative substrates of a phosphatase that is degraded during infection. Finally, we demonstrate that the HIV-1 Vpr protein inhibits histone H1 ubiquitination, leading to defects in DNA repair., Competing Interests: Declaration of interests The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. N.J.K. has consulting agreements with the Icahn School of Medicine at Mount Sinai, Maze Therapeutics, and Interline Therapeutics, is a shareholder in Tenaya Therapeutics, Maze Therapeutics, and Interline Therapeutics, and is a financially compensated scientific advisory board member for GEn1E Lifesciences, Inc. A.M. is a compensated cofounder, a member of the board of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. is a cofounder, a member of the board of directors, and a member of the scientific advisory board of Survey Genomics. A.M. is a compensated member of the scientific advisory board of NewLimit. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, and Merck. A.M. has received fees from 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
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