Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3

As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5\u27- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3

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

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed

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