The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Mapping of susceptibility loci for Ebola virus pathogenesis in mice.

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies.

Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P- and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.

Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology.

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen, herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-α/ß receptors (Ifnar1-/-Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions without affecting viral loads. We used RNAseq to define IFN-λ- and IFN-ß-induced transcriptional responses in primary mouse keratinocytes. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.IMPORTANCEType III interferons (IFN-λ) have been shown to have antiviral and immunomodulatory effects at epithelial barriers such as the respiratory and gastrointestinal tracts, but their effects on the skin have not been extensively investigated. We used mice lacking IFN-λ signaling to investigate the skin-specific effects of IFN-λ against the herpes simplex virus (HSV), which targets epithelial tissues to cause cold sores and genital herpes. We found that IFN-λ limited the severity of HSV skin lesions without affecting viral load and that this protective effect required IFN-λ signaling in both keratinocytes and neutrophils. We found that IFN-λ signaling in keratinocytes suppressed neutrophil recruitment to the skin and that depleting neutrophils protected against severe HSV skin lesions in the absence of IFN-λ. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.

Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

A nano-luciferase expressing human coronavirus OC43 for countermeasure development.

The genetic diversity of the coronavirus (CoV) family poses a significant challenge for drug discovery and development. Traditional antiviral drugs often target specific viral proteins from specific viruses which limits their use, especially against novel emerging viruses. Antivirals with broad-spectrum activity overcome this limitation by targeting highly conserved regions or catalytic domains within viral proteins that are essential for replication. For rapid identification of small molecules with broad antiviral activity, assays with viruses representing family-wide genetic diversity are needed. Viruses engineered to express a reporter gene (i.e. luminescence, fluorescence, etc.) can increase the efficiency, sensitivity or precision of drug screening over classical measures of replication like observation of cytopathic effect or measurement of infectious titers. We have previously developed reporter virus systems for multiple other endemic, pandemic, epidemic and enzootic CoV. Human CoV OC43 (HCoV-OC43) is a human endemic CoV that causes respiratory infection with age-related exacerbations of pathogenesis. Here, we describe the development of a novel recombinant HCoV-OC43 reporter virus that expresses nano-luciferase (HCoV-OC43 nLuc), and its potential application for screening of antivirals against CoV.

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Vaccine-mediated protection against Merbecovirus and Sarbecovirus challenge in mice.

The emergence of three highly pathogenic human coronaviruses-severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, Middle Eastern respiratory syndrome (MERS)-CoV in 2012, and SARS-CoV-2 in 2019-underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines protect against severe COVID-19, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor-binding domains (RBDs), which elicited live-virus neutralizing antibody responses. The trivalent RBD scNP elicited serum neutralizing antibodies against bat zoonotic Wuhan Institute of Virology-1 (WIV-1)-CoV, SARS-CoV, SARS-CoV-2 BA.1, SARS-CoV-2 XBB.1.5, and MERS-CoV live viruses. The monovalent SARS-CoV-2 RBD scNP vaccine only protected against Sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both Merbecovirus and Sarbecovirus challenge in highly pathogenic and lethal mouse models. This study demonstrates proof of concept for a single pan-sarbecovirus/pan-merbecovirus vaccine that protects against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology.

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-αß receptor (Ifnar1-/- Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions, independent of a direct effect on viral load. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity, and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection, and suggest potential applications for IFN-λ in treating viral skin infections.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential.

Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.

Vaccine-mediated protection against merbecovirus and sarbecovirus challenge in mice.

The emergence of three distinct highly pathogenic human coronaviruses - SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019 - underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines are highly protective against severe COVID-19 disease, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor binding domains (RBDs), which elicited live-virus neutralizing antibody responses and broad protection. Specifically, a monovalent SARS-CoV-2 RBD scNP vaccine only protected against sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both merbecovirus and sarbecovirus challenge in highly pathogenic and lethal mouse models. Moreover, the trivalent RBD scNP elicited serum neutralizing antibodies against SARS-CoV, MERS-CoV and SARS-CoV-2 BA.1 live viruses. Our findings show that a trivalent RBD nanoparticle vaccine displaying merbecovirus and sarbecovirus immunogens elicits immunity that broadly protects mice against disease. This study demonstrates proof-of-concept for a single pan-betacoronavirus vaccine to protect against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

Human ACE2 expression, a major tropism determinant for SARS-CoV-2, is regulated by upstream and intragenic elements.

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19.

Multiplexing mRNAs builds a "broad-immunity wall" against the flu.

Delivery of mRNAs encoding influenza HA antigens covering all known subtypes and lineages elicits cross-reactive and protective immunity.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Induction of the unfolded protein response (UPR) suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication.

Replication of most RNA viruses is closely associated with the endoplasmic reticulum (ER) within permissive cells. As such, viruses often induce tremendous amounts of stress on cells during viral replication. To cope with the stress, cells initiate the unfolded protein response (UPR) to promote cellular survival. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen, was previously shown to induce cellular stress at late stages post-infection resulting in the formation of stress granules (SGs). Here in this study, we demonstrate that PRRSV also induces additional cellular response pathways, including the UPR. Confocal microscopy analysis demonstrated significant morphological changes in the ER of PRRSV-infected cells, indicative of pronounced ER stress. Further investigation revealed induction of all three branches of the UPR, including eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), serine-threonine protein kinase/endoribonuclease (IRE1), and cyclic AMP-dependent transcription factor (ATF6). Activation of these sensors resulted in significant transcriptional upregulation of downstream UPR effectors. Additionally, UPR activation was shown to be detrimental to PRRSV replication, as treatment of cells with chemical ER stress inducers potently suppressed viral replication and RNA synthesis. Further investigation into the molecular mechanisms of UPR suppression of PRRSV replication revealed that PERK exacerbates the PRRSV-induced cytokine response. Collectively, these results demonstrate that PRRSV infection induces UPR activation through all three branches, and that UPR signaling may play a role in PRRSV pathogenesis. The results of this study further our understanding of the underlying molecular mechanisms of PRRSV replication and host-pathogen interactions.

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced stress granules are associated with viral replication complexes and suppression of host translation.

Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions.

Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing Membrane-Bound Interleukin-15 as an Immunomodulatory Adjuvant Enhances NK and γδ T Cell Responses and Confers Heterologous Protection.

Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-γ)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN-γ-producing CD4- CD8+ T cells and γδ T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of γδ T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and γδ T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis.

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.