Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame (ORF) 3b, ORF 6, and Nucleocapsid Proteins Function as Interferon Antagonists

Abstract

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...

Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation

“…The 3b, ORF6 and nucleocapsid proteins of SARS-CoV function as interferon antagonists by inhibiting the expression of beta interferon (Kopecky-Bromberg et al, 2007). Of these, the ORF6 protein inhibits nuclear translocation of STAT1, a transcription factor that is required for the activation of interferon-responsive promoters.…”

The cysteine protease domain of porcine reproductive and respiratory syndrome virus non-structural protein 2 antagonizes interferon regulatory factor 3 activation

“…It has been demonstrated that many viral IFN antagonists interfere with the IRF-3 and/or NF-kB signalling pathways (Basler et al, 2003;Boxer et al, 2009;Kochs et al, 2007;Kopecky-Bromberg et al, 2007). We next determined the impact of Nsp2 expression on the IRF-3-or NF-kBdependent, SeV-induced activation of the IFN-b promoter.…”

“…Others can induce proteasomedependent degradation of IRF-3. For example, Ebola virus VP35 protein and severe acute respiratory syndrome coronavirus (SARS-CoV) ORF3b, ORF6, N and papain-like protease (PLP) proteins inhibit IRF-3 activation (Basler et al, 2003; Clementz et al, 2010;Devaraj et al, 2007;Frieman et al, 2009;Kopecky-Bromberg et al, 2007). Classical swine fever virus N pro and rotavirus NSP1 induce IRF-3 degradation (Bauhofer et al, 2007;Graff et al, 2007).…”

“…Secreted IFN binds to the IFN receptor and activates a JAK/STAT signalling pathway, upregulating the expression of a subset of genes that impede virus replication (Randall & Goodbourn, 2008). However, many viruses have evolved mechanisms to circumvent the IFN response (Blakqori et al, 2007;Jennings et al, 2005;Kopecky-Bromberg et al, 2007;Narayanan et al, 2008).…”

“…Secreted IFN binds to the IFN receptor and activates a JAK/STAT signalling pathway, upregulating the expression of a subset of genes that impede virus replication (Randall & Goodbourn, 2008). However, many viruses have evolved mechanisms to circumvent the IFN response (Blakqori et al, 2007;Jennings et al, 2005;Kopecky-Bromberg et al, 2007;Narayanan et al, 2008).Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of porcine reproductive and respiratory syndrome, is a small enveloped, singlestranded, positive-sense RNA virus belonging to the family Arteriviridae within the order Nidovirales (Meulenberg, 2000). The genome is about 15.4 kb and contains at least nine ORFs.…”