Without sufficient herd immunity through either vaccination or natural infection, the coronavirus disease 2019 pandemic is unlikely to be controlled. Waning immunity with the currently approved ...vaccines suggests the need to evaluate vaccines causing the induction of long-term responses. Here, we report the immunogenicity and efficacy of our adjuvanted single-dose Rabies-vectored SARS-CoV-2 S1 vaccine, CORAVAX, in hamsters. CORAVAX induces high SARS-CoV-2 S1-specific and virus-neutralizing antibodies (VNAs) that prevent weight loss, viral loads, disease, lung inflammation, and the cytokine storm in hamsters. We also observed high Rabies VNA titers. In summary, CORAVAX is a promising dual-antigen vaccine candidate for clinical evaluation against SARS-CoV-2 and Rabies virus.
Abstract The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis ...energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open ‘valve’ conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a ‘valve’ for the ATP-pocket, presenting a promising target for antiviral development.
•Mass spectrometry (MS)-based proteomics is a powerful tool to investigate flavivirus-host interactions.•MS coupled with genetic screens can identify host proteins that physically interact with ...flaviviruses and impact viral replication.•MS can elucidate mechanisms of dysregulation and post-translational modifications of host proteins in flavivirus-infected cells.
Flaviviruses are a group of important emerging and re-emerging human pathogens that cause worldwide epidemics with thousands of deaths annually. Flaviviruses are small, enveloped, positive-sense, single-stranded RNA viruses that are obligate intracellular pathogens, relying heavily on host cell machinery for productive replication. Proteomic approaches have become an increasingly powerful tool to investigate the mechanisms by which viruses interact with host proteins and manipulate cellular processes to promote infection. Here, we review recent advances in employing quantitative proteomics techniques to improve our understanding of the complex interplay between flaviviruses and host cells. We describe new findings on our understanding of how flaviviruses impact protein–protein interactions, protein–RNA interactions, protein abundance, and post-translational modifications to modulate viral infection.
Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 ...patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.
Flaviviruses infect hundreds of millions of people annually, and no antiviral therapy is available. We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that, when edited, ...resulted in reduced flavivirus infection. Here, we validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum functions including translocation, protein degradation, and N-linked glycosylation. In particular, a subset of endoplasmic reticulum-associated signal peptidase complex (SPCS) proteins was necessary for proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, Dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), but had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I major histocompatibility complex (MHC) antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with unknown host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection by the expanding number of flaviviruses of medical concern.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to ...other antigenically distinct common seasonal human coronaviruses (hCoVs) before the coronavirus disease 2019 (COVID-19) pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection.
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•Some humans possessed cross-reactive SARS-CoV-2 antibodies prior to the pandemic•Pre-pandemic SARS-CoV-2 reactive antibodies are not associated with protection•Antibodies to a related betacoronavirus are boosted upon SARS-CoV-2 infection
Analysis of human serum samples before and after the onset of the COVID-19 pandemic show that antibodies against common seasonal human coronaviruses are cross-reactive against SARS-CoV-2 but do not confer cross-protection against infection or hospitalization.
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, infecting over 100 million people and claiming over 2 million lives globally. This drastically ...highlights the need for understanding the host-virus protein-protein interactions and immune evasion strategies governing SARS-CoV-2 pathogenesis. Our overall aim was to identify antiviral immune targeting during SARS-CoV-2 infection and protein overexpression. To understand if SARS-CoV-2 can block immune signaling, we first examined nuclear translocation of the transcription factors, IRF3 and NF-κB, as nuclear translocation is one step in interferon induction, during SARS-CoV-2 infection of the human airway epithelial cells. We found that in infected cells, there was limited IRF3 and NF-κB nuclear translocation, suggesting SARS-CoV-2 can limit immune activation. Additionally, through promoter luciferase signaling assays, the viral proteins NSP1, the host translation shutoff factor, and NSP13, the RNA helicase, can block signaling to the IFN-β and NF-κB promoters. To further investigate this reduction in promoter signaling, we examined nuclear translocation of NF-κB in the context of NSP1 and NSP13 plasmid overexpression. Surprisingly, we found that while NSP13 reduced NF-κB nuclear translocation, NSP1 was unable to despite its ability to limit promoter signaling. Instead, we found, utilizing Click-it chemistry, that NSP1 reduces host protein synthesis. Last, we found NSP13 targets TBK1 activation by limiting its phosphorylation in a dose-dependent manner. Collectively, our data illustrate that SARS-CoV-2 can block multiple innate immune signaling pathways through distinct mechanisms.
Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders ...and replicates efficiently in reproductive tissues
. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7
mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.
Post-acute sequelae of COVID-19 (PASC, “Long COVID”) pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several ...hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.
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•Long COVID is associated with reduced circulating serotonin levels•Serotonin depletion is driven by viral RNA-induced type I interferons (IFNs)•IFNs reduce serotonin through diminished tryptophan uptake and hypercoagulability•Peripheral serotonin deficiency impairs cognition via reduced vagal signaling
Post-viral syndromes are associated with serotonin reduction, which may contribute to the neurological and cognitive symptoms seen in individuals with Long COVID.
The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid ...protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 0.35-0.74,
= 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of
and
demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.
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