The vOTU domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference

Abstract

Arterivirus genus member Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically devastating disease, recently exacerbated by the emergence of highly pathogenic strains (HP-PRRSV). Within the nonstructural protein 2 of PRRSV is a deubiquitinating enzyme domain belonging to the viral ovarian tumor (vOTU) protease superfamily. vOTUs, which can greatly vary in their preference for their host ubiquitin (Ub) and Ub-like substrates such as interferon stimulated gene 15 (ISG15), have been implicated as a potential virulence factor. Since various strains of PRRSV have large variations in virulence, the specificity of vOTUs from two PRRSV strains of varying virulence were determined. While both vOTUs showed de-ubiquitinating activity and markedly low deISGylating activity, HP-PRRSV demonstrated a strong preference for lysine 63-linked poly-Ubiquitin, tied to innate immune response regulation. This represents the first report of biochemical activity unique to HP-PRRSV that has implications for a potential increase in immunosuppression and virulence.

Structure and Function of Viral Deubiquitinating Enzymes

“…The greatest difference between these PLP2s was that JXwn06 was more active toward Lys63-linked diUb than modified live virus. In vitro, PLP2 cleaved neither h u m a n I S G 1 5 -7 -a m i n o -4 -m e t h y l c o u m a r i n (ISG15-AMC) nor the pro-forms of human ISG15 or porcine ISG15, which contradicts the earlier observation of ISG15 conjugate cleavage upon ectopic expression of PLP2 [198,207,209]. With respect to the different isoforms of PPRSV nsp2 that include PLP2, no studies have been carried out to assess the specific DUB activities of these variants and their functional contribution during infection.…”

“…RIG-I was also deubiquitinated upon overexpression of PRRSV PLP2, suggesting that the DUB activity of PLP2 is responsible for evading RIG-Iinduced innate immune responses [196]. PLP2 of JXwn06, a highly pathogenic PRRSV strain, and that of a modified live virus vaccine strain, were shown to cleave all diUb chains linked through the different Lys in Ub, but not linear diUb in vitro [209]. The greatest difference between these PLP2s was that JXwn06 was more active toward Lys63-linked diUb than modified live virus.…”

Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro

“…PRRSV primarily infects PAMs and is characterized by the high rate of mutation and recombination (Deaton et al, 2014). Moreover, it causes the delayed appearance and low titer of neutralizing antibodies and initiates a comprehensive campaign against the innate immune response (Beura et al, 2010;Fang et al, 2012;Song et al, 2010;Sun et al, 2010).…”

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

“…However, a core function of Bat3 is to bind hydrophobic regions of proteins in order to maintain solubility while a triage (protein quality control) decision is made regarding ER targeting or ubiquitin-dependent destruction (Lee and Ye, 2013). While it is postulated that Bat3 would function to directly target nsp2 for post-translational membrane insertion, the PLP2 protease possesses deubiquitinating functionality (Deaton et al, 2014;Frias-Staheli et al, 2007) that, if targeted for destruction, may rescue nsp2 from ULB4 ubiquitin-dependent degradation and further potentiate ER membrane targeting.…”

“…Of the replicase proteins, nsp2 is unique due to its large size ($ 1196 amino acids (aa)), genetic heterogeneity, its participation in diverse roles supporting the viral replication cycle, and its packaging within the PRRSV virion (Fang et al, 2012a(Fang et al, , 2012bKappes et al, 2013;Li et al, 2014;Sun et al, 2012;van Kasteren et al, 2013). The amino (N)-terminal PLP2 protease domain of nsp2 is recognized for its dual functionality between viral pp1a/b processing (Fang and Snijder, 2010;Han et al, 2009) and its immunomodulatory deubiquitinating activity (Deaton et al, 2014;Frias-Staheli et al, 2007;van Kasteren et al, 2012). However, outside of the context of the PLP2 protease domain, very little is known about the potential functional roles for the large central and C-terminal domains of nsp2.…”