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

Abstract

There is growing evidence that porcine reproductive and respiratory syndrome virus (PRRSV) has developed mechanisms to subvert the host innate immune response. PRRSV non-structural protein 2 (Nsp2) was suggested previously as a potential interferon (IFN) antagonist. This study focused on Nsp2 to investigate its inhibitory mechanism of IFN induction. It was demonstrated that Nsp2 strongly inhibited IFN-b production by antagonizing activation of the IFN regulatory factor 3 (IRF-3) pathway induced by the Sendai virus (SeV). Further studies revealed that the cysteine protease domain (PL2) of Nsp2 was necessary for IFN antagonism. Additionally, both full-length Nsp2 and the PL2 protease domain of Nsp2 were found to inhibit SeV-induced phosphorylation and nuclear translocation of IRF-3. These findings suggest that Nsp2 is likely to play an important role in subversion of IRF-3-dependent innate antiviral defences, providing a basis for elucidating the mechanisms underlying PRRSV pathogenesis. INTRODUCTIONThe production of type I interferons (IFN-a/b) plays a pivotal role in the host antiviral immune defence (Bonjardim, 2005). Cellular sensors capable of recognizing various viral determinants initiate IFN signal transduction. Positive-stranded RNA viruses can generate intermediate dsRNA during replication, which is an efficient inducer of type I IFNs. This intermediate dsRNA can trigger a cascade of cellular sensors, resulting in type I IFN production and secretion (Kawai & Akira, 2006a;Randall & Goodbourn, 2008). The induction of type I IFNs is initiated via recognition of a pathogen-associated molecule in dsRNA by cellular pattern recognition receptors, including Tolllike receptor 3 (TLR3), caspase recruitment domain (CARD)-containing proteins, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 . TLR3 signals through the adaptor molecule Toll-interleukin-1-resistance domain-containing adaptor inducing IFN-b (TRIF), whilst MDA-5 and RIG-I signal through the adaptor molecule mitochondrial antiviral signal protein (MAVS; also known as IPS-1, Cardif and VISA) to activate a type I IFN response (Kawai & Akira, 2006b; Kawai et al., 2005; Yamamoto et al., 2003). Despite utilizing different adaptors, both pathways converge to activate two downstream kinases, TANK-binding kinase 1 (TBK-1) and inhibitor of kB kinase e (IKKe), subsequently leading to the activation of latent IFN transcription factors, including IFN regulatory factors (IRF-3 and/or IRF-7), nuclear factor-kB (NF-kB) and activating protein 1 (AP-1) (Fitzgerald et al., 2003;Randall & Goodbourn, 2008; Sharma et al., 2003). Activated IFN transcription factors translocate to the nucleus and activate IFN transcription (Biron, 2001). 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.,...

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Arterivirus nsp1 and N proteins also localize in the cytoplasm and the nucleus of infected cells (84, 85). Porcine reproductive and respiratory syndrome virus (PRRSV) N protein accumulates in the nucleoli of infected cells, where it interacts with the host cell proteins fibrillarin, nucleolin, and poly(A)-binding protein (PABP), the latter of which is transported to the nucleus during infection (86, 87).…”

Genomic characterization and pathogenicity of a strain of type 1 porcine reproductive and respiratory syndrome virus

“…den Boon et al, 1995;van Dinten et al, 1996;Wassenaar et al, 1997). These nsps are not only involved in viral replication and genomic transcription (Bautista et al, 2002;Fang and Snijder, 2010), but also play important roles in the modulation of host innate immune responses (Beura et al, 2010;He et al, 2015;Li et al, 2010a;Shi et al, 2011;Yoo et al, 2010), and in the pathogenicity and virulence of PRRSV (Kwon et al, 2008;Li et al, 2014a;Wang et al, 2008). ORFs 2a, 2b, ORF3 to 7, and ORF5a encode viral structural proteins including GP2a, E, GP3, GP4, GP5, M, N and ORF5a (Johnson et al, 2011;Mardassi et al, 1995;Meng et al, 1994;Meulenberg et al, 1995;Music and Gagnon, 2010;van Nieuwstadt et al, 1991;Wu et al, 2001).…”

HSV-2 Immediate-Early Protein US1 Inhibits IFN-β Production by Suppressing Association of IRF-3 with IFN-β Promoter

“…IRF-3 and IRF-3/5D expression plasmids pIRES-hrGFP/IRF-3-Flag and pIRES-hrGFP/IRF-3/5D-Flag (constitutively active mutant of IRF-3) were provided by Dr. Yi-Ling Lin (Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan) (52). The reporter plasmid PRD (III-I) 4 , and IRF-3 112-427 [residues 112-427]), pcDNA3-MAVS-Flag and the reporter plasmid p125-Luc, internal control plasmid phRL-TK were described previously (30,32).…”

“…For example, viruses can bind directly to IRF-3 (17,18) or interact with CBP/p300 coactivator (19)(20)(21) to impair the type I IFN production. In general, some viruses cause the degradation of activated IRF-3 (22,23), whereas most viruses suppress the production of type I IFNs through inhibition of IRF-3 activation, including phosphorylation, dimerization, and nuclear translocation (24)(25)(26)(27)(28)(29)(30). Only a few viruses have been shown to interfere with the binding of activated IRF-3 to IFN-b promoter, resulting in the suppression of type I IFNs (31,32).…”