The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-␤), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-␤, IRF-3. N protein dramatically inhibited expression from an NF-B-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) infected thousands of people throughout the world, killing hundreds. The molecular mechanisms governing virus-induced pathology have not been fully elucidated. The first immune challenge a virus must surmount in order to cause disease symptoms in people is the innate immune response. A major component of innate immunity is the interferon response. Infection of cells with virus causes the activation of several cellular transcription factors, such as IRF-3 and NF-B, which activate the expression of the interferon genes. Once interferon is synthesized and released from cells, it binds to interferon receptors, initiating a signaling cascade of the JAK/ STAT pathway that results in activated transcription factors translocating to the nucleus. These transcription factors bind to and activate genes containing an interferon-stimulated response element (ISRE) in their promoters. Activation of these genes enables the cell to combat the virus infection and can prevent viral replication (8). Many viruses have developed mechanisms to subvert the interferon response. Infection of cells with SARS-CoV does not result in the production of interferon, and pretreatment of cells with interferon prevents growth of 33). These results indicate that SARS-CoV has evolved to overcome the interferon response.SARS-CoV contains a 29.7-kb single-stranded RNA genome wrapped in a helical nucleocapsid composed of multiple copies of N protein, which in turn is surrounded by an envelope containing a 180-to 190-kDa S glycoprotein, a 23-kDa M glycoprotein, an ϳ30-kDa 3a glycoprotein, and a small E protein. The viral gene order is similar to that in other known coronavi...

The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-␣/␤) system. This virus-host interplay is dependent on the production of IFN-␣/␤ and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-␤ promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.The genome of influenza A virus (FLUAV) consists of eight RNA segments that encode nine structural proteins and two nonstructural proteins, called NS1 (41) and PB1-F2 (11). NS1 is a virulence factor of FLUAV by virtue of conferring resistance to the antiviral effects of the host interferon (IFN) system (21,40,63). Previous studies with recombinant FLUAV carrying deletions in the NS1 gene (delNS1) showed a strong attenuation in IFN-competent systems, whereas the NS1-deleted virus replicated to levels similar to those of wild-type virus in cell culture and in mice with a defect in the IFN system (16,23,39).The expression of type I IFNs (IFN-␣/␤) is induced in response to viral infection. Viral single-stranded and doublestranded RNAs (dsRNAs) with phosphorylated 5Ј ends are among the viral products that induce IFN-␣/␤ (32, 42, 55). These viral RNA molecules activate a variety of cellular signaling pathways, resulting in the activation of transcription factors, such as the IFN regulatory factors (IRFs) and the stress-induced transcription factors NF-B and c-Jun/ATF2 (34, 60, 64). Upon activation, these latent transcription factors move from the cytoplasm into the nucleus and initiate the expression of type I IFNs. IFN-␣/␤ subtypes bind to a common type I IFN receptor, thus activating the JAK-STAT signaling ...

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The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-␣/␤) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitrotranscribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or doublestranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-␤ promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKK and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.