SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Suppression of TRIM19 by arterivirus nonstructural protein 1 promotes viral replication.

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

Reprogramming viral immune evasion for a rational design of next-generation vaccines for RNA viruses.

Type I interferons (IFNs-α/ß) are antiviral cytokines that constitute the innate immunity of hosts to fight against viral infections. Recent studies, however, have revealed the pleiotropic functions of IFNs, in addition to their antiviral activities, for the priming of activation and maturation of adaptive immunity. In turn, many viruses have developed various strategies to counteract the IFN response and to evade the host immune system for their benefits. The inefficient innate immunity and delayed adaptive response fail to clear of invading viruses and negatively affect the efficacy of vaccines. A better understanding of evasion strategies will provide opportunities to revert the viral IFN antagonism. Furthermore, IFN antagonism-deficient viruses can be generated by reverse genetics technology. Such viruses can potentially serve as next-generation vaccines that can induce effective and broad-spectrum responses for both innate and adaptive immunities for various pathogens. This review describes the recent advances in developing IFN antagonism-deficient viruses, their immune evasion and attenuated phenotypes in natural host animal species, and future potential as veterinary vaccines.

Mechanisms of Non-segmented Negative Sense RNA Viral Antagonism of Host RIG-I-Like Receptors.

The pattern recognition receptors RIG-I-like receptors (RLRs) are critical molecules for cytosolic viral recognition and for subsequent activation of type I interferon production. The interferon signaling pathway plays a key role in viral detection and generating antiviral responses. Among the many pathogens, the non-segmented negative sense RNA viruses target the RLR pathway using a variety of mechanisms. Here, I review the current state of knowledge on the molecular mechanisms that allow non-segmented negative sense RNA virus recognition and antagonism of RLRs.

Phage display technique identifies the interaction of severe acute respiratory syndrome coronavirus open reading frame 6 protein with nuclear pore complex interacting protein NPIPB3 in modulating Type I interferon antagonism.

BACKGROUND/PURPOSE: Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins including ORF6 inhibit Type I interferon (IFN) signaling. METHODS: This study identified SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries, and examined the association of ORF6-host factor interaction with Type I IFN antagonism. After the fifth round of biopanning with Escherichia coli-synthesized ORF6-His tagged protein, the relative binding affinity of phage clones to ORF6 was determined using direct enzyme-linked immunosorbent assay. RESULTS: The highest affinity clone to ORF6 displayed the C-terminal domain of NPIPB3 (nuclear pore complex interacting protein family, member B3; also named as phosphatidylinositol-3-kinase-related kinase SMG-1 isoform 1 homolog). The coimmunoprecipitation assay demonstrated the direct binding of ORF6 to the C-terminal domain of NPIPB3 in vitro. Confocal imaging revealed a close colocalization of SARS-CoV ORF6 protein with NPIPB3 in human promonocytes. The dual luciferase reporter assay showed that the C-terminal domain of NPIPB3 attenuated the antagonistic activity of SARS-CoV ORF6 on IFN-ß-induced ISRE (IFN stimulated response element)-responsive firefly luciferase activity. In addition, confocal imaging and Western blotting assays revealed that the increases in STAT-1 nuclear translocation and phosphorylation occurred in the transfected cells expressing both genes of ORF6 and NPIPB3, but not in the ORF6-expressing cells in response to IFN-ß. CONCLUSION: The overexpression of NPIPB3 restored the IFN-ß responses in SARS-CoV ORF6 expressing cells, indicating that the interaction of SARS CoV ORF6 and NPIPB3 reduced Type I IFN antagonism by SARS-CoV ORF6.

Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses.

Previous in vitro studies have demonstrated that Ebola and Marburg virus (EBOV and MARV) VP35 antagonize the host cell immune response. Moreover, specific mutations in the IFN inhibitory domain (IID) of EBOV and MARV VP35 that abrogate their interaction with virus-derived dsRNA, lack the ability to inhibit the host immune response. To investigate the role of MARV VP35 in the context of infectious virus, we used our reverse genetics system to generate two recombinant MARVs carrying specific mutations in the IID region of VP35. Our data show that wild-type and mutant viruses grow to similar titers in interferon deficient cells, but exhibit attenuated growth in interferon-competent cells. Furthermore, in contrast to wild-type virus, both MARV mutants were unable to inhibit expression of various antiviral genes. The MARV VP35 mutants exhibit similar phenotypes to those previously described for EBOV, suggesting the existence of a shared immune-modulatory strategy between filoviruses.

Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses.

Type I interferons (IFNs-α/ß) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV-nsp1 and LDV-nsp1 were auto-cleaved to produce the nsp1α and nsp1ß subunits, EAV-nsp1 remained uncleaved. SHFV-nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1ß, and nsp1γ), but only two subunits were generated as SHFV-nsp1αß and SHFV-nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1ß motif of nsp1ß was functional to generate SHFV-nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV-nsp1ß, LDV-nsp1ß, EAV-nsp1, and SHFV-nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV-nsp1α, CBP degradation was evident in cells expressing LDV-nsp1α and SHFV-nsp1γ, but no such degradation was observed for EAV-nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other.