SARS-CoV Pathogenesis Is Regulated by a STAT1 Dependent but a Type I, II and III Interferon Receptor Independent Mechanism

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1−/− mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1−/− mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation

SARS Coronavirus 3b Accessory Protein Modulates Transcriptional Activity of RUNX1b

BACKGROUND: The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells. CONCLUSIONS/SIGNIFICANCE: These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection

Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction

Exacerbated Innate Host Response to SARS-CoV in Aged Non-Human Primates

The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-κB as central player, whereas expression of type I interferon (IFN)-β is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins

Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus

The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity

BACKGROUND: The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. METHODS: We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. RESULTS: Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro’s DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. CONCLUSION: These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis