Search

SUMOylation is a reversible posttranslational modification involved in the regulation of diverse biological processes. Growing evidence suggests that virus infection can interfere with the SUMOylation system. In the present study, we discovered that apoptosis inhibitor 5 (API5) is a SUMOylated protein. Amino acid substitution further identified that Lys404 of API5 was the critical residue for SUMO3 conjugation. Moreover, we found that Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. In addition, our results further revealed that viral protein VP3 inhibited the SUMOylation of API5 by targeting API5 and promoting UBC9 proteasome-dependent degradation through binding to the ubiquitin E3 ligase TRAF3. Furthermore, we revealed that wild-type but not K404R mutant API5 inhibited IBDV replication by enhancing MDA5-dependent IFN-β production. Taken together, our data demonstrate that API5 is a UBC9-dependent SUMOylated protein and deSUMOylation of API5 by viral protein VP3 aids in viral replication. IMPORTANCE Apoptosis inhibitor 5 (API5) is a nuclear protein initially identified for its antiapoptotic function. However, so far, posttranslational modification of API5 is unclear. In this study, we first identified that API5 K404 can be conjugated by SUMO3, and Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. Mechanically, viral protein VP3 directly interacts with API5 and inhibits SUMOylation of API5. Additionally, the cellular E3 ligase TNF receptor-associated factor 3 (TRAF3) is employed by VP3 to facilitate UBC9 proteasome-dependent degradation, leading to the reduction of API5 SUMOylation. Moreover, our data reveal that SUMOylation of API5 K404 promotes MDA5-dependent beta interferon (IFN-β) induction, and its deSUMOylation contributes to IBDV replication. This work highlights a critical role of conversion between SUMOylation and deSUMOylation of API5 in regulating viral replication.

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, drastically modifies infected cells in an effort to optimize virus replication. Included is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammation and is a central driver of COVID-19 clinical presentations. Inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduces both cytokine production and viral replication. Here, we combined genetic screening with quantitative phosphoproteomics to better understand interactions between the p38/MAPK pathway and SARS-CoV-2. We found that several components of the p38/MAPK pathway impacted SARS-CoV-2 replication and that p38β is a critical host factor for virus replication, and it prevents activation of the type-I interferon pathway. Quantitative phosphoproteomics uncovered several SARS-CoV-2 nucleocapsid phosphorylation sites near the N-terminus that were sensitive to p38 inhibition. Similar to p38β depletion, mutation of these nucleocapsid residues was associated with reduced virus replication and increased activation of type-I interferon signaling. Taken together, this study reveals a unique proviral function for p38β that is not shared with p38α and supports exploring p38β inhibitor development as a strategy towards developing a new class of COVID-19 therapies.ImportanceSARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 mitogen-activated protein kinase (MAPK) pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represseses translation and induces mRNA degradation, while the host elicits anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-meidated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5’UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5’UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5’UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Highlights • Epithelial cell-mimetic (rhACE2-cytomimetic) particles bind SARS-CoV-2 spike RBD. • rhACE2-Cytomimetic particles restrict SARS-CoV-2 spike binding to cell surface. • Particle functionalized rhACE2 is stoichiometrically superior to soluble rhACE2. • Inhaled rhACE2-Cytomimetic particles limit viral spike uptake in airway epithelium. • rhACE2-Cytomimetic particles can be an effective countermeasure for COVID-19., The development of countermeasures that aid in the prevention and propagation of SARS-CoV-2 infections is critical to manage the continuing crisis brought about by COVID-19. Here we present a proof-of-concept study on the use of cell-mimetic microparticles (Cytomimetics) for the interference and sequestration of SARS-CoV-2 virions away from the cellular surfaces required for replication, disease manifestation, and outbreak propagation. Recombinant human ACE2 (rhACE2) functionalized onto the surface of cytomimetic particles binds the receptor binding domain (RBD) of recombinant SARS-CoV-2 spike protein with high affinity and demonstrated a stoichiometric advantage over the use of soluble rhACE2. Inhalation of rhACE2-Cytomimetic particles by mice prior to their exposure to aerosolized spike protein demonstrated the applicability of these cytomimetic particles in preventing viral protein binding to respiratory epithelial cells. Our study demonstrates the potential of an easily deliverable and highly modular technology for the control of viral infections and to complement other prophylactic countermeasures

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

Gammaherpesviruses establish lifelong infections and are associated with B cell lymphomas. Murine gammaherpesvirus 68 (MHV68) infects epithelial and myeloid cells during acute infection, with subsequent passage of the virus to B cells, where physiological B cell differentiation is usurped to ensure the establishment of a chronic latent reservoir. Interferons (IFNs) represent a major antiviral defense system that engages the transcriptional factor STAT1 to attenuate diverse acute and chronic viral infections, including those of gammaherpesviruses. Correspondingly, global deficiency of type I or type II IFN signaling profoundly increases the pathogenesis of acute and chronic gammaherpesvirus infection, compromises host survival, and impedes mechanistic understanding of cell type-specific role of IFN signaling. Here, we demonstrate that myeloid-specific STAT1 expression attenuates acute and persistent MHV68 replication in the lungs and suppresses viral reactivation from peritoneal cells, without any effect on the establishment of viral latent reservoir in splenic B cells. All gammaherpesviruses encode a conserved protein kinase that antagonizes type I IFN signaling in vitro. Here, we show that myeloid-specific STAT1 deficiency rescues the attenuated splenic latent reservoir of the kinase-null MHV68 mutant. However, despite having gained access to splenic B cells, the protein kinase-null MHV68 mutant fails to drive B cell differentiation. Thus, while myeloid-intrinsic STAT1 expression must be counteracted by the gammaherpesvirus protein kinase to facilitate viral passage to splenic B cells, expression of the viral protein kinase continues to be required to promote optimal B cell differentiation and viral reactivation, highlighting the multifunctional nature of this conserved viral protein during chronic infection. IMPORTANCE IFN signaling is a major antiviral system of the host that suppresses replication of diverse viruses, including acute and chronic gammaherpesvirus infection. STAT1 is a critical member and the primary antiviral effector of IFN signaling pathways. Given the significantly compromised antiviral status of global type I or type II IFN deficiency, unabated gammaherpesvirus replication and pathogenesis hinders understanding of cell type-specific antiviral effects. In this study, a mouse model of myeloid-specific STAT1 deficiency unveiled site-specific antiviral effects of STAT1 in the lungs and peritoneal cavity, but not the spleen, of chronically infected hosts. Interestingly, expression of a conserved gammaherpesvirus protein kinase was required to counteract the antiviral effects of myeloid-specific STAT1 expression to facilitate latent infection of splenic B cells, revealing a cell type-specific virus-host antagonism during the establishment of chronic gammaherpesvirus infection.

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

Introduction The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. Areas covered Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestin, GAT and VHS modules, membrane-perturbing annexin, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids and assembled bacterial and viral particles. Expert opinion The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses, and lipidons as lipid "codons" recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.

Nuclear DNA-binding TCF proteins, which act as the main downstream effectors of Wnt signaling, are essential for the regulation of cell fate and innate immunity. However, their role during viral infection in shrimp remains unknown. Herein, we demonstrated that Litopenaeus vannamei TCF (LvTcf) acts independently of Lvβ-catenin to promote interferon-like protein LvVago1 production, thus mounting the response to WSSV infection. Further, we observed that WSV083, a WSSV serine/threonine protein kinase, bound to LvTcf and phosphorylated it. Phosphorylated LvTcf was then recognized and degraded via the ubiquitin-proteasome pathway. Moreover, mass spectrometry analyses indicated that the T39 and T104 residues of LvTcf were target sites phosphorylated by WSV083. Point mutation analyses suggested that additional sites of LvTcf may undergo phosphorylation via WSV083. Taken together, the current work provides valuable insights into host immunity and viral pathogenesis. LvTcf is not only a modulator of shrimp innate immunity but is also an important target for WSSV immune evasion. Thus, the current findings will help improve disease control in shrimps.

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope., Author summary During all herpesvirus infections, the nuclear envelope undergoes deformation in order to enable viral capsids assembled within the nucleus of the infected cell to gain access to the cytoplasm for further maturation and spread to neighbouring cells. These nuclear envelope deformations are orchestrated by the viral nuclear egress complex (NEC), which, in HSV, is composed of two viral proteins, pUL31 and pUL34. How the membrane-deformation activity of the NEC is controlled during infection is incompletely understood. The studies in this communication reveal that the phosphorylation status of pUL31 and pUL34 can determine the nature of nuclear envelope deformations and that the viral protein pUL21 can modulate the phosphorylation status of both NEC components. These findings provide an explanation for why HSV-2 strains lacking pUL21 are defective in nuclear egress. A thorough understanding of how NEC activity is controlled during infection may yield strategies to disrupt this fundamental step in the herpesvirus lifecycle, providing the basis for novel antiviral strategies.

HIV-1 remains incurable due to viral reservoirs, which lead to durably latent HIV infection. Identifying novel host factors and deciphering the molecular mechanisms involved in the establishment and maintenance of latency are critical to discover new targets for the development of novel anti-HIV agents. Here, we show that ubiquitin-like with PHD and RING finger domain 1 (UHRF1) modulates HIV-1 5′-long terminal repeat (LTR)-driven transcription of the viral genome as a novel HIV-1 restriction factor. Correspondingly, UHRF1 depletion reversed the latency of HIV-1 proviruses. Mechanistically, UHRF1 competed with positive transcription factor b (p-TEFb) for the binding to the cysteine-rich motifs of HIV-1 Tat via its TTD, PHD, and RING finger domains. Furthermore, UHRF1 mediated K48-linked ubiquitination and proteasomal degradation of Tat in RING-dependent ways, leading to the disruption of Tat/cyclin T1/CDK9 complex and consequential impediment of transcription elongation. In summary, our findings revealed that UHRF1 is an important mediator of HIV-1 latency by controlling Tat-mediated transcriptional activation, providing novel insights on host-pathogen interaction for modulating HIV-1 latency, beneficial for the development of anti-AIDS therapies. IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. In our work, we identified a critical role of host factor ubiquitin-like with PHD and RING finger domain 1 (UHRF1) in HIV-1 latency via the modulation of the viral protein Tat stability. By disrupting the Tat/cyclin T1/CDK9 complex, UHRF1 promotes the suppression of HIV-1 transcription and maintenance of HIV-1 latency. Our findings provide novel insights in controlling Tat expression via host-pathogen interaction for modulating HIV-1 latency. Based on our results, modulating UHRF1 expression or activity by specific inhibitors is a potential therapeutic strategy for latency reversal in HIV-1 patients.

The current battle against Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2 benefits from the worldwide distribution of different vaccine formulations. All anti-SARS-CoV-2 vaccines in use are conceived to induce anti-Spike neutralizing antibodies. However, this strategy still has unresolved issues, the most relevant of which are: (i) the resistance to neutralizing antibodies of emerging SARS-CoV-2 variants and (ii) the waning of neutralizing antibodies. On the other hand, both pre-clinical evidence and clinical evidence support the idea that the immunity sustained by antigen-specific CD8+ T lymphocytes can complement and also surrogate the antiviral humoral immunity. As a distinctive feature, anti-SARS-CoV-2 CD8+ T-driven immunity maintains its efficacy even in the presence of viral protein mutations. In addition, on the basis of data obtained in survivors of the SARS-CoV epidemic, this immunity is expected to last for several years. In this review, both the mechanisms and role of CD8+ T-cell immunity in viral infections, particularly those induced by SARS-CoV and SARS-CoV-2, are analyzed. Moreover, a CD8+ T-cell-based vaccine platform relying on in vivo engineered extracellular vesicles is described. When applied to SARS-CoV-2, this strategy was proven to induce a strong immunogenicity, holding great promise for its translation into the clinic.

Individuals living with human immunodeficiency virus type 1 (HIV-1) are often plagued by debilitating neurocognitive impairments and affective alterations;the pathophysiology underlying these deficits likely includes dopaminergic system dysfunction. The present review utilized four interrelated aims to critically examine the evidence for dopaminergic alterations following HIV-1 viral protein exposure. First, basal dopamine (DA) values are dependent upon both brain region andexperimental approach (i.e., high-performance liquid chromatography, microdialysis or fast-scan cyclic voltammetry). Second, neurochemical measurements overwhelmingly support decreased DA concentrations following chronic HIV-1 viral protein exposure. Neurocognitive impairments, including alterations in pre-attentive processes and attention, as well as apathetic behaviors, provide an additional line of evidence for dopaminergic deficits in HIV-1. Third, to date, there is no compelling evidence that combination antiretroviral therapy (cART), the primary treatment regimen for HIV-1 seropositive individuals, has any direct pharmacological action on the dopaminergic system. Fourth, the infection of microglia by HIV-1 viral proteins may mechanistically underlie the dopamine deficit observed following chronic HIV-1 viral protein exposure. An inclusive and critical evaluation of the literature, therefore, supports the fundamental conclusion that long-term HIV-1 viral protein exposure leads to a decreased dopaminergic state, which continues to persist despite the advent of cART. Thus, effective treatment of HIV-1-associated apathy/depression and neurocognitive impairments must focus on strategies for rectifying decreases in dopamine function.

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.

The human norovirus (HuNV)-encoded nucleoside-triphosphatase (NTPase) is a multifunctional protein critically involved in viral replication and pathogenesis. Previously, we have shown that the viral NTPase is capable of forming vesicle clusters in cells, interacting with other viral proteins such as P22, and promoting cellular apoptosis. Herein, we demonstrate that NTPase-associated vesicle clusters correspond to lipid droplets (LDs) wrapped by the viral protein and show that NTPase-induced apoptosis is mediated through both caspase-8- and caspase-9-dependent pathways. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of NTPase encompasses two LD-targeting motifs (designated LTM-1 and LTM-2), two apoptosis-inducing motifs, and multiple regulatory regions. Interestingly, the identified LTM-1 and LTM-2, which are located from aa 1 to 50 and from aa 51 to 90, respectively, overlap with the two apoptosis-inducing motifs. Although there was no positive correlation between the extent of LD localization and the degree of cellular apoptosis for NTPase mutants, we noticed that mutant proteins defective in LD-targeting ability could not induce cellular apoptosis. In addition to LD targeting, the amphipathic LTM-1 and LTM-2 motifs could have the potential to direct fusion proteins to the endoplasmic reticulum (ER). Furthermore, we found that the LTM-1 motif is a P22-interacting motif. However, P22 functionally augmented the proapoptotic activity of the LTM-2 fusion protein but not the LTM-1 fusion protein. Overall, our findings propose that NTPase may participate in multiple cellular processes through binding to LDs or to the ER via its N-terminal amphipathic helix motifs. IMPORTANCE Human noroviruses (HuNVs) are the major agent of global gastroenteritis outbreaks. However, due to the lack of an efficient cell culture system for HuNV propagation, functions of the viral-encoded proteins in host cells are still poorly understood. In the current study, we present that the viral NTPase is a lipid droplet (LD)-associated protein, and we identify two LD-targeting motifs, LTM-1 and LTM-2, in its N-terminal domain. In particular, the identified LTM-1 and LTM-2 motifs, which contain a hydrophobic region and an amphipathic helix, are also capable of delivering the fusion protein to the endoplasmic reticulum (ER), promoting cellular apoptosis, and physically or functionally associating with another viral protein P22. Since LDs and the ER have been linked to several biological functions in cells, our study therefore proposes that the norovirus NTPase may utilize LDs or the ER as replication platforms to benefit viral replication and pathogenesis.

The ongoing world-wide Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) pandemic shows the need for new sensing and therapeutic means against the CoV viruses. The SARS-CoV-2 nsp1 protein is important, both for replication and pathogenesis, making it an attractive target for intervention. In recent years nanoparticles have been shown to interact with peptides, ranging in size from single amino acids up to proteins. These nanoparticles can be tailor-made with specific functions and properties including bioavailability. To the best of our knowledge, in this study we show for the first time that a tailored titanium oxide nanoparticle interacts specifically with a unique site of the full-length SARS-CoV-2 nsp1 protein. This can be developed potentially into a tool for selective control of viral protein functions.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface, however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.

In the search for new therapeutic strategies to contrast SARS-CoV-2, we here studied the interaction of polydatin (PD) and resveratrol (RESV)—two natural stilbene polyphenols with manifold, well known biological activities—with Spike, the viral protein essential for virus entry into host cells, and ACE2, the angiotensin-converting enzyme present on the surface of multiple cell types (including respiratory epithelial cells) which is the main host receptor for Spike binding. Molecular Docking simulations evidenced that both compounds can bind Spike, ACE2 and the ACE2:Spike complex with good affinity, although the interaction of PD appears stronger than that of RESV on all the investigated targets. Preliminary biochemical assays revealed a significant inhibitory activity of the ACE2:Spike recognition with a dose-response effect only in the case of PD.

Peroxiredoxin 1 (PRDX1) is a cellular antioxidant enzyme crucial for diverse fundamental biological processes, such as autophagy, inflammation and carcinogenesis. However, molecular mechanisms underpinning its diverse roles are not well understood. Here, we report that PRDX1 positively regulates interferon induction and that pseudorabies virus (PRV) targets PRDX1 to evade interferon (IFN) induction. PRV UL13 encodes a serine/threonine kinase important for PRV infection, although its biological function remains obscure. We identified PRDX1 as a UL13-interacting protein. Virological and biochemical assays demonstrate that PRDX1 promotes IFN induction via interacting with TANK-binding kinase 1 (TBK1) and inhibitor-κb kinase e (IKKe). Conversely, UL13 accelerates PRDX1 degradation via the ubiquitin-proteosome pathway in a kinase-dependent manner. In doing so, PRV inhibits IFN induction during productive infection, which requires PRDX1 expression. This study uncovers an essential role of PRDX1 in innate immune response and reveals a new viral immune evasion strategy to counteract cellular defense. Importance Pseudorabies virus (PRV) interacts with numerous cellular proteins during a productive infection. Here, we demonstrated the interaction of viral protein UL13 with the antioxidant enzyme PRDX1, which functions in multiple signal transduction pathways. We found that PRDX1 participates in the type I interferon pathway by interacting with TBK1 and IKKe, thereby negatively regulating PRV propagation. However, UL13 ubiquitinates PRDX1, which routes PRDX1 into proteasomes for degradation and effectively reduces its expression. These results illuminate the fundamental role that PRDX1 plays in the interferon pathway, and identify a potential target for the control of PRV infection.

The purpose of this work is to provide an in silico molecular rationale of the role eventually played by currently circulating mutations in the receptor binding domain of the SARS-CoV-2 spike protein (S-RBDCoV‑2) in evading the immune surveillance effects elicited by the two Eli Lilly LY-CoV555/bamlanivimab and LY-CoV016/etesevimab monoclonal antibodies. The main findings from this study show that, compared to the wild-type SARS-CoV-2 spike protein, mutations E484A/G/K/Q/R/V, Q493K/L/R, S494A/P/R, L452R and F490S are predicted to be markedly resistant to neutralization by LY-CoV555, while mutations K417E/N/T, D420A/G/N, N460I/K/S/T, T415P, and Y489C/S are predicted to confer LY-CoV016 escaping advantage to the viral protein. A challenge of our global in silico results against relevant experimental data resulted in an overall 90% agreement. Thus, the results presented provide a molecular-based rationale for all relative experimental findings, constitute a fast and reliable tool for identifying and prioritizing all present and newly reported circulating spike SARS-CoV-2 variants with respect to antibody neutralization, and yield substantial structural information for the development of next-generation vaccines and monoclonal antibodies more resilient to viral evolution.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this “viral protein homeostasis” is critical for viral propagation. Furthermore, we clarified that the “convoluted membrane (CM),” which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Marburg virus is a member of filoviridae and spreads severe Marburg hemorrhagic illness in humans and animals. Nowadays, there is no vaccine available that can completely stop the replication of Marburg replication. Therefore, this study is designed to repurpose the effective therapeutic antiviral drug by using a computational approach against exploring the mechanism of Marburg virus Viral protein 35. We have retrieved about 40570 drug-like small compounds from the ZINC database using the “ZINC Pharmer” online tool. Molecular docking of the ligands from the ready-to-dock database has been carried out using MOE. The five drugs have been identified to bind with VP35 possibly. A study was also performed to evaluate the drug-like characteristics of the substances for absorption, distribution, metabolism, and excretion (ADME). The findings clearly showed that ligands are interacting with the MARV VP35 protein. Interestingly, Lipinski’s rule of five was observed by all ligands. These findings provide the foundation for reconstituting and utilizing molecules as a possible therapy for Marburg Virus Disease (MVD).

Cells : open access journal 10(8), 1900 (2021). doi:10.3390/cells10081900, Published by MDPI, Basel

Although nucleos(t)ide analogs and interferons suppress hepatitis B virus (HBV) replication, they must be taken continuously and have a low response rate. Therefore, therapeutics for HBV with novel modes of action are needed. Humanized virus-suppressing factor (hzVSF) is a monoclonal antibody against vimentin that exhibits broad-spectrum antiviral activity. Here, hzVSF significantly inhibited HBV infection. Although hzVSF inhibited HBV RNA production, it did not affect viral transcription from minicircle DNA mimicking covalently closed circular DNA. Additionally, hzVSF did not inhibit viral protein or DNA release from infected cells. Rather, hzVSF inhibited the cell entry of viral preS1 peptides, possibly by altering intracellular vimentin localization, which is important for HBV cell entry. These results suggest that hzVSF has therapeutic potential for HBV infection with a novel mode of action., HBV; hzVSF; Vimentin; Endocytosis.

Motivation Two key steps in the analysis of uncultured viruses recovered from metagenomes are the taxonomic classification of the viral sequences and the identification of putative host(s). Both steps rely mainly on the assignment of viral proteins to orthologs in cultivated viruses. Viral Protein Families (VPFs) can be used for the robust identification of new viral sequences in large metagenomics datasets. Despite the importance of VPF information for viral discovery, VPFs have not yet been explored for determining viral taxonomy and host targets. Results In this work, we classified the set of VPFs from the IMG/VR database and developed VPF-Class. VPF-Class is a tool that automates the taxonomic classification and host prediction of viral contigs based on the assignment of their proteins to a set of classified VPFs. Applying VPF-Class on 731K uncultivated virus contigs from the IMG/VR database, we were able to classify 363K contigs at the genus level and predict the host of over 461K contigs. In the RefSeq database, VPF-class reported an accuracy of nearly 100% to classify dsDNA, ssDNA and retroviruses, at the genus level, considering a membership ratio and a confidence score of 0.2. The accuracy in host prediction was 86.4%, also at the genus level, considering a membership ratio of 0.3 and a confidence score of 0.5. And, in the prophages dataset, the accuracy in host prediction was 86% considering a membership ratio of 0.6 and a confidence score of 0.8. Moreover, from the Global Ocean Virome dataset, over 817K viral contigs out of 1 million were classified. Availability and implementation The implementation of VPF-Class can be downloaded from https://github.com/biocom-uib/vpf-tools. Supplementary information Supplementary data are available at Bioinformatics online.

Background The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. Methods Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. Findings The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922–0.997) (86/88) sensitivity and 100% (95% CI = 0.958–1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. Interpretation Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. Funding This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

Human immunodeficiency virus (HIV)-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs). Most of these transfers resulted in limited spread of these viruses to humans. However, one transmission event involving SIVcpz from chimpanzees gave rise to group M HIV-1, with M being the principal strain of HIV-1 responsible for the AIDS pandemic. Vpu is an HIV-1 accessory protein generated from Env/Vpu encoded bicistronic mRNA and localized in cytosolic and membrane regions of cells capable of being infected by HIV-1 and that regulate HIV-1 infection and transmission by downregulating BST-2, CD4 proteins levels, and immune evasion. This review will focus of critical aspects of Vpu including its zoonosis, the adaptive hurdles to cross-species transmission, and future perspectives and broad implications of Vpu in HIV-1 infection and dissemination.

Background: The SARS-CoV-2 main protease (Mpro) is an attractive target in the COVID-19 drug development process. It catalyzes the polyprotein's translation from viral RNA and specifies a particular cleavage site. Due to the absence of identical cleavage specificity in human cell proteases, targeting Mpro with chemical compounds can obstruct the replication of the virus. Methods: To explore the potential binding mechanisms of 1,2,3-triazole scaffolds in comparison to co-crystallized inhibitors 11a and 11b towards Mpro, we herein utilized molecular dynamics and enhanced sampling simulation studies. Results and conclusion: All the 1,2,3-triazole scaffolds interacted with catalytic residues (Cys145 and His41) and binding pocket residues of Mpro involving Met165, Glu166, Ser144, Gln189, His163, and Met49. Furthermore, the adequate binding free energy and potential mean force of the topmost compound 3h was comparable to the experimental inhibitors 11a and 11b of Mpro. Overall, the current analysis could be beneficial in developing the SARS-CoV-2 Mpro potential inhibitors. © 2022 Elsevier Ltd 5058; Department of Biotechnology, Ministry of Science and Technology, India, DBT; Indian Council of Medical Research, ICMR; Council of Scientific and Industrial Research, India, CSIR; Ministry of Education and Science of the Russian Federation, Minobrnauka: 075-15-2020-777 We gratefully acknowledge to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur for providing the facilities to carry out this work. This research received no specific grant from any funding agency. The work was carried out under the aegis of the Himalayan Centre for High-throughput Computational Biology (HiCHiCoB), a BIC supported by DBT. The CSIR support in the form of projects MLP:0201 and OLP:0043 for bioinformatics studies is highly acknowledged. R. S. expresses gratitude to the Indian Council of Medical Research, New Delhi, India for providing Senior Research Fellowship. G.V.Z. acknowledge the Ministry of Science and Higher Education of the Russian Federation within the framework of the grant agreement as government subsidies from the Federal budget in accordance with paragraph 4 of article 78.1 of the Budget Code of the Russian Federation (Moscow, October 1, 2020, No. 075-15-2020-777). This manuscript represents CSIR-IHBT communication no. 5058. We gratefully acknowledge to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur for providing the facilities to carry out this work. This research received no specific grant from any funding agency. The work was carried out under the aegis of the Himalayan Centre for High-throughput Computational Biology (HiCHiCoB), a BIC supported by DBT. The CSIR support in the form of projects MLP:0201 and OLP:0043 for bioinformatics studies is highly acknowledged. R. S. expresses gratitude to the Indian Council of Medical Research, New Delhi, India for providing Senior Research Fellowship. G.V.Z. acknowledge the Ministry of Science and Higher Education of the Russian Federation within the framework of the grant agreement as government subsidies from the Federal budget in accordance with paragraph 4 of article 78.1 of the Budget Code of the Russian Federation (Moscow, October 1, 2020, No. 075-15-2020-777). This manuscript represents CSIR-IHBT communication no. 5058.

Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the progression of neurological conditions and both the spread and pathogenesis of infectious diseases. It has recently been demonstrated that EVs from Hepatitis C virus (HCV) infected individuals and cells contained replicative-competent viral RNA that was capable of infecting hepatocytes. Being a member of the same viral family, it is likely the Zika virus also hijacks EV pathways to package viral components and secrete vesicles that are infectious and potentially less immunogenic. As EVs have been shown to cross blood-brain and placental barriers, it is possible that Zika virus could usurp normal EV biology to gain access to the brain or developing fetus. Here, we demonstrate that Zika virus infected cells secrete distinct EV sub-populations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with varying sizes and density compared to those released from non-infected cells. We also show that the EV enriched tetraspanin CD63 regulates the release of EVs, and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika infection.ImportanceZika virus is a re-emerging infectious disease that spread rapidly across the Caribbean and South America. Infection of pregnant women during the first trimester has been linked to microcephaly, a neurological condition where babies are born with smaller heads due to abnormal brain development. Babies born with microcephaly can develop convulsions and suffer disabilities as they age. Despite the significance of Zika virus, little is known about how the virus infects the fetus or causes disease. Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells that are present in all biological fluids. EVs carry signaling factors, proteins and microRNAs that mediate intercellular communication. EVs have been shown to be a means by which some viruses can alter cellular environments and cross previously unpassable cellular barriers. Thus gaining a greater understanding of how Zika affects EV cargo may aid in the development of better diagnostics, targeted therapeutics and prophylactic treatments.

By November 2020, over 60 million people in 218 countries have been infected with COVID-19, with the number of deaths rapidly approaching 1.5 million and the second wave of pandemic underway in many countries. The SARS-CoV-2 genome and its organization are similar to other closely related betacoronaviruses, including human SARS-CoV, bat coronavirus, and murine coronavirus. The information on protein binding sites extracted from the viral protein complexes can be also used to elucidate the evolutionary mechanisms and their impact on the viral protein function. In spite of the joint efforts by the scientific community, the people are yet to get a comprehensive structural characterization of all SARS-CoV-2 proteins using experimental methods. The viral proteins of SARS-CoV and SARS-CoV-2 are known to hijack the cellular components responsible for protein transport: endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and Golgi stack are primarily targeted by ORF3a and ORF8 in the current interactome.

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

The interactions between a virus and its host are complex but can be broadly categorized as either viral manipulation of cellular functions or cellular responses to infection. These processes begin at the earliest point of contact between virus and cell and frequently result in changes to cellular gene expression, making genome-wide transcriptomics a useful tool to study them. Several previous studies have used transcriptomics to evaluate the cellular responses to human immunodeficiency virus type 1 (HIV-1) infection; however, none have examined events in primary CD4+ T cells during the first 24 h of infection. Here, we analyzed CD4+ T cells at 4.5, 8, 12, 24, and 48 h following infection. We describe global changes to host gene expression commencing at 4.5 h postinfection and evolving over the ensuing time points. We identify upregulation of genes related to innate immunity, cytokine production, and apoptosis and downregulation of those involved in transcription and translation. We further demonstrate that the viral accessory protein Vpr is necessary for almost all gene expression changes seen at 12 h postinfection and the majority of those seen at 48 h. Identifying this new role for Vpr not only provides fresh perspective on its possible function but also adds further insight into the interplay between HIV-1 and its host at the cellular level. IMPORTANCE HIV-1, while now treatable, remains an important human pathogen causing significant morbidity and mortality globally. The virus predominantly infects CD4+ T cells and, if not treated with medication, ultimately causes their depletion, resulting in AIDS and death. Further refining our understanding of the interaction between HIV-1 and these cells has the potential to inform further therapeutic development. Previous studies have used transcriptomics to assess gene expression changes in CD4+ T cells following HIV-1 infection; here, we provide a detailed examination of changes occurring in the first 24 h of infection. Importantly, we define the viral protein Vpr as essential for the changes observed at this early stage. This finding has significance for understanding the role of Vpr in infection and pathogenesis and also for interpreting previous transcriptomic analyses of HIV-1 infection.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the corona virus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2 infected Vero cells, processed identically. Correlative light and electron microscopy on semi-thick cryo-sections, demonstrated induction of electron-lucent, lipid filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In infected Vero cells and using immuno-electron microscopy, viral proteins were detected in these lipid filled compartments. In addition, several viral proteins were detected in virus particles, Golgi, double membrane spherules and multiple-virus bodies which were not lysosomal. In lung tissue, the non-structural protein 4 and the stable nucleocapsid N-protein, were detected on membranes of lipid filled compartments. The induction of such lipid filled compartments and the localisation of the viral proteins in lung of patients with fatal COVID-19, may explain the extensive inflammatory response. Authors Summary The trafficking of coronaviruses in lung of COVID-19 patients is not well understood and virus particles are difficult to find. Here we have visualized virus particles in SARS-CoV-2 infected cells by focusing on viral protein detection, in combination with ultrastructure. We studied how the virus is altering the cell morphology and determined that in Vero cells, lipid filled compartments contained various viral proteins. In these cells, also membrane enclosed multi-virus bodies were visible that contain a different set of viral proteins. We demonstrated that lipid filled compartments are viral induced compartments, as no known cellular marker such as lipid droplet or lysosomal marker was present. Using this knowledge, we then studied lung tissue from patients with a fatal SARS-Cov-2 infection, processed in a similar manner. Again we detected lipid filled compartments, now with viral proteins nsp4 and the stable nucleocapsid N-protein. The presence of these lipid filled compartments with viral proteins induced by SARS-CoV-2 infections, could be why the immune response of the COVID-19 patients is so strong, resulting in a fatal infection, and should be considered for new therapeutic strategies.

Middle East respiratory syndrome coronavirus (MERS-CoV), a pathogen causing severe respiratory disease in humans that emerged in June 2012, is a novel beta coronavirus similar to severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, immunoprecipitation and proximity ligation assays revealed that the nucleocapsid (N) protein of MERS-CoV interacted with human translation elongation factor 1A (EF1A), an essential component of the translation system with important roles in protein translation, cytokinesis, and filamentous actin (F-actin) bundling. The C-terminal motif (residues 359–363) of the N protein was the crucial domain involved in this interaction. The interaction between the MERS-CoV N protein and EF1A resulted in cytokinesis inhibition due to the formation of inactive F-actin bundles, as observed in an in vitro actin polymerization assay and in MERS-CoV-infected cells. Furthermore, the translation of a CoV-like reporter mRNA carrying the MERS-CoV 5′UTR was significantly potentiated by the N protein, indicating that a similar process may contribute to EF1A-associated viral protein translation. This study highlights the crucial role of EF1A in MERS-CoV infection and provides new insights into the pathogenesis of coronavirus infections.

Viruses are abundant, diverse and ancestral biological entities. Their diversity is high, both in terms of the number of different protein families encountered and in the sequence heterogeneity of each protein family. The recent increase in sequenced viral genomes constitutes a great opportunity to gain new insights into this diversity and consequently urges the development of annotation resources to help functional and comparative analysis. Here, we introduce PHROG (Prokaryotic Virus Remote Homologous Groups), a library of viral protein families generated using a new clustering approach based on remote homology detection by HMM profile-profile comparisons. Considering 17 473 reference (pro)viruses of prokaryotes, 868 340 of the total 938 864 proteins were grouped into 38 880 clusters that proved to be a 2-fold deeper clustering than using a classical strategy based on BLAST-like similarity searches, and yet to remain homogeneous. Manual inspection of similarities to various reference sequence databases led to the annotation of 5108 clusters (containing 50.6 % of the total protein dataset) with 705 different annotation terms, included in 9 functional categories, specifically designed for viruses. Hopefully, PHROG will be a useful tool to better annotate future prokaryotic viral sequences thus helping the scientific community to better understand the evolution and ecology of these entities.

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community – the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of structural heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native mass spectrometry (MS) as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

virusMED (Virus Metal binding sites, Epitopes and Drug binding sites) is a rich internet application based on a database of atomic interactions around hotspots in 7041 experimentally determined viral protein structures., Metal binding sites, antigen epitopes and drug binding sites are the hotspots in viral proteins that control how viruses interact with their hosts. virusMED (virus Metal binding sites, Epitopes and Drug binding sites) is a rich internet application based on a database of atomic interactions around hotspots in 7041 experimentally determined viral protein structures. 25306 hotspots from 805 virus strains from 75 virus families were characterized, including influenza, HIV-1 and SARS-CoV-2 viruses. Just as Google Maps organizes and annotates points of interest, virusMED presents the positions of individual hotspots on each viral protein and creates an atlas upon which newly characterized functional sites can be placed as they are being discovered. virusMED contains an extensive set of annotation tags about the virus species and strains, viral hosts, viral proteins, metal ions, specific antibodies and FDA-approved drugs, which permits rapid screening of hotspots on viral proteins tailored to a particular research problem. The virusMED portal (https://virusmed.biocloud.top) can serve as a window to a valuable resource for many areas of virus research and play a critical role in the rational design of new preventative and therapeutic agents targeting viral infections.

Rotavirus (RV)-encoded non-structural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development.IMPORTANCERotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission in suckling mice. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo, making it an attractable target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.

During a SARS-CoV2 infection, host cells produce large amounts of the viral envelope protein (Ep-CoV2). Ep-CoV2 is partially inserted into the membrane of nascent viral particles and into cellular membranes. To mimic the pathophysiological impact of the cellular protein fraction, Ep-CoV2 was overexpressed in mammalian cells and effects on key signaling parameters were monitored. By tagging with green fluorescent protein (GFP), we found that Ep-CoV2 protein is mostly present in the endoplasmic reticulum with additional trace amounts in the plasma membrane. We observed that wild-type Ep-CoV2 and, to a lesser extent, its mutants (N15A, V25F) corrupted some of the most important homeostatic mechanisms in cells. The same was observed with isolated transmembrane domains of the protein. The Ep-CoV2-evoked elevation of intracellular Ca2+ and pH as well as the induced membrane depolarization produced by the presence of the protein interfere with major signal transduction cascades in host cells. These functions of Ep-CoV2, which likely contribute to the pathogenesis of the viral protein, result from the ion-channel activity of the viral protein. Two independent assays, a functional reconstitution of Ep-CoV2 protein in artificial membranes and a rescue of K+-deficient yeast mutants, confirm that Ep-CoV2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. The data presented here suggest that specific channel function inhibitors of Ep-CoV2 can provide cell protection and virostatic effects.

Human immunodeficiency virus (HIV) is a pathogen that infects blood cells, using CD4 molecule and two cell receptors CCR5 and CXCR4. The other major actor is gp120/gp41 viral protein complex, which interacts with receptors. Here, the presence of synonymous mutations associated with HIV-1 tropism and the related RNA secondary-structure in HIV-1 infected patients was evaluated. The analysis includes gp120-sequences from 340 HIV-1 subtype-B infected patients, all retrieved from Los Alamos database and with phenotypic HIV tropism determination based on recombinant-virus entry-assay. Frequencies of all nucleotide substitutions were calculated. Mfold and RNAfold algorithms were used to predict RNA secondary-structure of HIV-1. Nineteen codons in V2/C2, V3 and C3 domains were found to be closely related to CCR5 and CXCR4. Additionally, in X4-sequences, gp120 gca303gcu and gua222guc synonymous mutations are positively related to the gp120 S11R and T8A/I codons in V3 protein domain. Furthermore, gua222guc increases stability of the viral RNA secondary-structure. Probably, it would not be surprising if a novel escape viral strategy therapy will be related to the gp120 synonymous mutations. Moreover, in relation to the pivotal role played by gp120 in polyvalent vaccine approaches, the impact of gp120 synonymous mutations may play an important role in HIV entry into the cell. Keywords: gp120; tropism; v3; s11r; evolution; vaccine.

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.