Abstract
Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11. Since nsp11 is an endoribonuclease (EndoU), the structure function relationship was examined using a series of nsp11 EndoU mutant plasmids. The mutants that impaired the EndoU activity failed to suppress IFN and led to the normal expression of MAVS. Seven single amino acid substitutions (4 in subdomain A and 3 in subdomain B) plus one insertion (frame-shift in nsp11) were then introduced into PRRSV infectious cDNA clones to generate nsp11 mutant viruses. Unfortunately, all EndoU knock-out nsp11 mutant viruses appeared replication-defective and no progenies were produced. Three mutations in EndoU subdomain A expressed the N and nsp2/3 proteins but their infectivity diminished after 2 passages. Taken together, our data show that PRRSV nsp11 endoribonuclease activity is critical for both viral replication and IFN antagonism. More importantly, the endoribonuclease activity of nsp11 demonstrates the substrate specificity towards MAVS and RIG-I (transcripts and proteins) over p65 and IRF3 in the context of gene transfection and overexpression. This is likely a mechanism of nsp11 suppression of type I IFN production.
Highlights
Type I interferons (IFN-α/β) play a key role for antiviral defense in host cells [1,2,3]
Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) nsp11 contains an IFN promoter suppressive activity but its molecular action is unknown [20, 21, 23,24,25]. To study this function of nsp11, MARC-145 cells were transfected with nsp11, which was Flag-tagged at the N-terminus, and the pIFN-β-luc, pIRF3-luc, or pPRDII-luc reporter plasmids followed by transfection with poly(I:C) for IFN stimulation (Fig 1A). pIRF3-luc and pPRDII-luc contain 4 copies of the interferon regulatory factor 3 (IRF3) binding sequence or 2 copies of the nuclear factor (NF)-κB binding sequence located in the IFN-β promoter, respectively, in front of the luciferase reporter gene
Culture supernatants collected from nsp11-transfected HeLa cells were serially diluted by 2-folds and each diluted supernatant was incubated with MARC-145 cells in 96-well plates, followed by infection with vesicular stomatitis virus expressing GFP (VSV-GFP)
Summary
Type I interferons (IFN-α/β) play a key role for antiviral defense in host cells [1,2,3]. For RNA viruses, the viral genome is first recognized by specific receptors including toll-like receptor 3/ 7 (TLR-3/7) and cytosolic receptors. Activated IRF3 and NF-κB are translocated to the nucleus and form a transcriptionally competent enhanceosome along with cAMP response element-binding (CREB)-binding protein (CBP) and other transcription factors, leading to the expression of type I IFN genes [7]. PRRSV infection causes the suppression of type IFN induction in alveolar macrophages and in lungs of pigs where PRRSV actively replicates [12, 13]. Several viral proteins in PRRSV have been shown to downregulate IFN production, including nsp, nsp, nsp, nsp, and N [14,15,16,17,18,19]. Its IFN antagonism has been previously reported (including by our lab) and its crystal structure has been recently solved, the mechanism of how it inhibits type I IFN production remains unknown [20,21,22,23,24,25]
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