Search

Abstract Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann–Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.

Abstract During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNAi fMet) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (Pi) release, changes conformation facilitating fMet-tRNAi fMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNAi fMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.

The COVID-19 pandemic has led to contentious discourse regarding unproven COVID-19 therapies (UCTs),(e.g. ivermectin). Despite recommendations against it, ivermectin remains, in some areas, highly demanded. The goal of this study is to understand patient and provider perspectives about UCTs (e.g., ivermectin) and how responses to requests for UCTs impact healthcare distrust. This mixed methods observational study was conducted in a rural healthcare system in the Southern United States. Adults (n = 26) with a history of COVID-19 or clinicians (n = 8) from the same system were interviewed using questionnaires assessing healthcare distrust and qualitatively interviewed exploring perceptions about UCTs. Patient themes were: 1) Importance of anecdotal stories for decision-making; 2) Use of haphazard approaches to ‘research’; 3) Strong distrust of government and healthcare organizations; 4) Inherent trust in local healthcare; 5) Decision-making as weighing pros/cons; and 6) Feeling a right to try medications. High survey medians indicated high distrust with differences of 8.5 points for those who requested/used ivermectin versus those who did not (p = 0.027). Clinician themes were: 1) Frustration when patients trust social media over clinicians; 2) Acceptance of community beliefs about UCTs; 3) Distrust originating outside of the healthcare system; 4) Feeling torn about prescribing UCTs to build trust; and 5) Variable educational strategies. When clinicians are perceived as aligned with government, this may void patients’ trust of clinicians. Clinicians should leverage trust in local healthcare and distance themselves from distrusted information sources. Ethical questions arise regarding appropriateness of acquiescing to patient requests for ivermectin for building trust.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Summary: The major heat shock protein Hsp70 forms a complex with a scaffold protein Bag3 that links it to components of signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, to search for pathways regulated by the complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. RNAseq followed by the pathway analysis indicated that several signaling pathways including UPR were activated by JG-98. Surprisingly, only the eIF2α-associated branch of the UPR was activated, while other UPR branches were not induced, suggesting that the response was unrelated to the ER proteotoxicity and ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was driven by a distinct eIF2α kinase HRI. Hsp70-Bag3 directly interacted with HRI and regulated eIF2α phosphorylation upon cytoplasmic proteotoxicity. Therefore, cytosolic proteotoxicity can activate certain UPR genes via Hsp70-Bag3-HRI-eIF2α axis.

Finding synergistic drug combinations is an important area of cancer research. Here, we sought to rationally design synergistic drug combinations with an inhibitor of BTK kinase, ibrutinib, which is used for the treatment of several types of leukemia. We (a) used a pooled shRNA screen to identify genes that protect cells from the drug, (b) identified protective pathways via bioinformatics analysis of these gene sets, and (c) identified drugs that inhibit these pathways. Based on this analysis, we established that inhibitors of proteasome and mTORC1 could synergize with ibrutinib both in vitro and in vivo. We suggest that FDA-approved inhibitors of these pathways could be effectively combined with ibrutinib for the treatment of chronic lymphocytic leukemia (CLL).

ABSTRACTThe harmful effects of ZIKA virus (ZIKV) infection are reflected by severe neurological manifestations such as microcephaly in neonates and other complications associated with Guillain-Barré syndrome in adults. The transmission dynamics of ZIKV in or between neurons, or within the developing brains of the foetuses are not fully understood. Using primary cultures of murine cortical neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Cryo-electron microscopy showed heterogeneous population of neuronal exosomes with a size range of 30–200 nm. Increased production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing antibodies treatment studies suggest the presence of viral RNA/proteins inside exosomes. Exosomes derived from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene expression of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly reduced ZIKV loads in both cortical neurons and in exosomes derived from these neuronal cells. Taken together, our results suggest that ZIKV modulates SMPD3 activity in cortical neurons for its infection and transmission through exosomes perhaps leading to severe neuronal death that may result in neurological manifestations such as microcephaly in the developing embryonic brains.

Medicinal properties of plants are usually identified based on knowledge of traditional medicine or using low-throughput screens for specific pharmacological activities. The former is very biased since it requires prior knowledge of plants’ properties, while the latter depends on a specific screening system and will miss medicinal activities not covered by the screen. We sought to enrich our understanding of the biological activities of Sarcopoterium spinosum L. root extract based on transcriptome changes to uncover a plurality of possible pharmacological effects without the need for prior knowledge or functional screening. We integrated Gene Set Enrichment Analysis of the RNAseq data to identify pathways affected by the treatment of cells with the extract and perturbational signatures in the CMAP database to enhance the validity of the results. Activities of signaling pathways were measured using immunoblotting with phospho-specific antibodies. Mitochondrial membrane potential was assessed using JC-1 staining. SARS-CoV-2-induced cell killing was assessed in Vero E6 and A549 cells using an MTT assay. Here, we identified transcriptome changes following exposure of cultured cells to the medicinal plant Sarcopoterium spinosum L. root extract. By integrating algorithms of GSEA and CMAP, we confirmed known anti-cancer activities of the extract and predicted novel biological effects on oxidative phosphorylation and interferon pathways. Experimental validation of these pathways uncovered strong activation of autophagy, including mitophagy, and excellent protection from SARS-CoV-2 infection. Our study shows that gene expression analysis alone is insufficient for predicting biological effects since some of the changes reflect compensatory effects, and additional biochemical tests provide necessary corrections. This study defines the advantages and limitations of transcriptome analysis in predicting the biological and medicinal effects of the Sarcopoterium spinosum L. extract. Such analysis could be used as a general approach for predicting the medicinal properties of plants.

In recent years, mathematical models have developed into an important tool for cancer research, combining quantitative analysis and natural processes. We have focused on Chronic Lymphocytic Leukemia (CLL), since it is one of the most common adult leukemias, which remains incurable. As the first step toward the mathematical prediction of in vivo drug efficacy, we first found that logistic growth best described the proliferation of fluorescently labeled murine A20 leukemic cells injected in immunocompetent Balb/c mice. Then, we tested the cytotoxic efficacy of Ibrutinib (Ibr) and Cytarabine (Cyt) in A20-bearing mice. The results afforded calculation of the killing rate of the A20 cells as a function of therapy. The experimental data were compared with the simulation model to validate the latter’s applicability. On the basis of these results, we developed a new ordinary differential equations (ODEs) model and provided its sensitivity and stability analysis. There was excellent accordance between numerical simulations of the model and results from in vivo experiments. We found that simulations of our model could predict that the combination of Cyt and Ibr would lead to approximately 95% killing of A20 cells. In its current format, the model can be used as a tool for mathematical prediction of in vivo drug efficacy, and could form the basis of software for prediction of personalized chemotherapy.

Ticks secrete various anti-coagulatory, anti-vasoconstrictory, anti-inflammatory, and anti-platelet aggregation factors in their saliva at the bite site during feeding to evade host immunological surveillance and responses. For the first time, we report successful isolation of exosomes (small membrane-bound extracellular signaling vesicles) from saliva and salivary glands of partially fed or unfed ixodid ticks. Our data showed a novel role of these in vivo exosomes in the inhibition of wound healing via downregulation of C-X-C motif chemokine ligand 12 (CXCL12) and upregulation of interleukin-8 (IL-8). Cryo-electron microscopy (cryo-EM) analysis revealed that tick saliva and salivary glands are composed of heterogeneous populations of in vivo exosomes with sizes ranging from 30 to 200 nm. Enriched amounts of tick CD63 ortholog protein and heat shock protein 70 (HSP70) were evident in these exosomes. Treatment of human skin keratinocytes (HaCaT cells) with exosomes derived from tick saliva/salivary glands or ISE6 cells dramatically delayed cell migration, wound healing, and repair process. Wound healing is a highly dynamic process with several individualized processes including secretion of cytokines. Cytokine array profiling followed by immunoblotting and quantitative-PCR analysis revealed that HaCaT cells treated with exosomes derived from tick saliva/salivary glands or ISE6 cells showed enhanced IL-8 levels and reduced CXCL12 loads. Inhibition of IL-8 or CXCL12 further delayed exosome-mediated cell migration, wound healing, and repair process, suggesting a skin barrier protection role for these chemokines at the tick bite site. In contrast, exogenous treatment of CXCL12 protein completely restored this delay and enhanced the repair process. Taken together, our study provides novel insights on how tick salivary exosomes secreted in saliva can delay wound healing at the bite site to facilitate successful blood feeding.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

Protein-shelled viruses have been thought as “tin cans” that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids “breathing”, waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a “living” protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host.

BACKGROUND:Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. METHODOLOGY/PRINCIPAL FINDINGS:To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. CONCLUSIONS/SIGNIFICANCE:Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases.

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

A unique biological safety level (BSL)-3 cryo-electron microscopy facility with a 200 keV high-end cryo-electron microscope has been commissioned at the University of Texas Medical Branch (UTMB) to study the structure of viruses and bacteria classified as select agents. We developed a microscope decontamination protocol based on chlorine dioxide gas with a continuous flow system. In this paper we report on testing digital camera sensors (both CCD and CMOS direct detector) in a BSL-3 environment, and microscope performance after chlorine dioxide (ClO2) decontamination cycles.

ABSTRACT Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development.