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Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation of cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.
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Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.
Assuntos
Infecções por Caliciviridae , Norovirus , Camundongos , Humanos , Animais , Macrófagos , Apoptose , Imunidade Inata , Norovirus/fisiologia , Replicação ViralRESUMO
Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.
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Vacinas Virais , Infecção por Zika virus , Zika virus , Animais , Camundongos , Anticorpos Neutralizantes , Anticorpos Antivirais , AdenoviridaeRESUMO
Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC50]: 0.009 µg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC50 of 0.090 µg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs.
Assuntos
Infecções por HIV , HIV-1 , Vacinas , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/química , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , Bovinos , Feminino , Anticorpos Anti-HIV , Infecções por HIV/prevenção & controle , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009800.].
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Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.
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Interferon beta/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/farmacologia , SARS-CoV-2/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Células Vero , Proteínas Virais/genéticaRESUMO
The COVID-19 pandemic has impacted and enforced significant restrictions within our societies, including the attendance of public and professional athletes in gyms. Liquid chalk is a commonly used accessory in gyms and is comprised of magnesium carbonate and alcohol that quickly evaporates on the hands to leave a layer of dry chalk. We investigated whether liquid chalk is an antiseptic against highly pathogenic human viruses, including SARS-CoV-2, influenza virus, and noroviruses. Chalk was applied before or after virus, inoculum and recovery of infectious virus was determined to mimic the use in the gym. We observed that addition of chalk before or after virus contact led to a significant reduction in recovery of infectious SARS-CoV-2 and influenza virus but had little impact on norovirus. These observations suggest that the use and application of liquid chalk can be an effective and suitable antiseptic for major sporting events, such as the Olympic Games. IMPORTANCE To restrict the potential transmission and infectivity of SARS-CoV-2, the use of liquid chalk has been a requirement in an active gym setting. However, its effectiveness has not been scientifically proven. Here, we show that the application of liquid chalk before or after virus inoculum significantly impacts recovery of infectious SARS-CoV-2 and influenza viruses but not noroviruses. Thus, our study has shown that the implementation and application of liquid chalk in communal social gym settings is effective in reducing the infectivity of respiratory viruses, and this supports the use of liquid chalk in major sporting events to restrict the impact of COVID-19 on our communities.
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Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous "organelles" that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.
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The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study, we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in the suppression of host translation, and yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signaling cytokines such as beta interferon, interleukin-6, and tumor necrosis factor alpha. Our subsequent analyses revealed that the phosphorylation of eIF2α was mediated via protein kinase R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2α, and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection and that MNV can restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears that MNV utilizes G3BP1 to enhance replication but equally to prevent SG formation, suggesting an anti-MNV property of SGs. Overall, this study highlights MNV manipulation of SGs, PKR, and translational control to regulate cytokine translation and to promote viral replication.IMPORTANCE Viruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate, and cause disease. In retaliation, cells possess various defense mechanisms to detect, destroy, and clear infecting viruses, as well as signal to neighboring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and proinflammatory cytokines. However, virus replication and protein translation still ensue. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SGs), by recruiting the key host protein G3BP1 to the MNV replication complex, a recruitment that is crucial to establishing and maintaining virus replication. Thus, MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.
Assuntos
Infecções por Caliciviridae/imunologia , Grânulos Citoplasmáticos/virologia , DNA Helicases/imunologia , Fator de Iniciação 2 em Eucariotos/imunologia , Evasão da Resposta Imune , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , RNA Helicases/imunologia , Proteínas com Motivo de Reconhecimento de RNA/imunologia , eIF-2 Quinase/imunologia , Animais , Grânulos Citoplasmáticos/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Camundongos , Fosforilação , Biossíntese de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.
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Regulação Viral da Expressão Gênica , Antígenos HLA-A/biossíntese , Antígenos HLA-B/biossíntese , Antígenos HLA-C/biossíntese , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Genes MHC Classe I , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Humanos , Influenza Humana/imunologia , Influenza Humana/virologia , Transporte Proteico , Receptores de Antígenos de Linfócitos T/imunologia , Células THP-1RESUMO
Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here, we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus-induced organelles characteristic of infection.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Animais , Humanos , Metabolismo dos LipídeosRESUMO
Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.
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Retículo Endoplasmático/virologia , Rim/enzimologia , Lipídeos de Membrana/metabolismo , Fosfolipases A2/metabolismo , Replicação Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Retículo Endoplasmático/enzimologia , Rim/virologia , Células Vero , Febre do Nilo Ocidental/enzimologiaRESUMO
Flaviviruses are enveloped, positive-sensed single-stranded RNA viruses that remodel host membranes, incorporating both viral and host factors facilitating viral replication. In this study, we identified a key role for the membrane-bending host protein Reticulon 3.1 (RTN3.1A) during the replication cycle of three flaviviruses: West Nile virus (WNV), Dengue virus (DENV), and Zika virus (ZIKV). We observed that, during infection, RTN3.1A is redistributed and recruited to the viral replication complex, a recruitment facilitated via the WNV NS4A protein, however, not DENV or ZIKV NS4A. Critically, small interfering RNA (siRNA)-mediated knockdown of RTN3.1A expression attenuated WNV, DENV, and ZIKV replication and severely affected the stability and abundance of the NS4A protein, coinciding with a significant alternation and reduction of viral membrane structures in the endoplasmic reticulum. These observations identified a crucial role of RTN3.1A for the viral remodelling of host membranes during efficient flavivirus replication and the stabilization of viral proteins within the endoplasmic reticulum.
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Proteínas de Transporte/metabolismo , Vírus da Dengue/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vírus do Nilo Ocidental/fisiologia , Zika virus/fisiologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
It is well established that +ssRNA viruses manipulate cellular lipid homoeostasis and distribution to facilitate efficient replication. Here, we show that the cellular lipid ceramide is redistributed to the West Nile virus strain Kunjin virus (WNVKUN) replication complex (RC) but not to the dengue virus serotype 2 strain New Guinea C (DENVNGC) RC. We show that prolonged chemical inhibition of serine palmitoyltransferase with myriocin had a significant deleterious effect on WNVKUN replication but enhanced DENVNGC replication. Additionally, inhibition of ceramide synthase with Fumonisin B1 had a detrimental effect on WNVKUN replication and release of infectious virus particles but contrastingly an enhancing effect on DENVNGC replication and virus production. These observations suggest that ceramide production via the de novo and salvage pathway is a requirement for WNVKUN replication but inhibitory for DENVNGC replication. Thus, although these two viruses are from the same genus, they have a differential ceramide requirement for replication.