부산대학교 도서관

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication. Author summary: Within the pork farming industry, outbreaks of porcine reproductive and respiratory syndrome virus (PRRSV) are extremely costly, and significant effort has gone in to the design and production of vaccines aimed at limiting the impact of PRRSV outbreaks. To better understand the specific mechanisms used by PRRSV to suppress host immune responses during infection, we investigated the structure of a critical multifunctional PRRSV protease, PLP2, which processes the viral polyprotein during replication, and which is capable of suppressing host antiviral immune responses. Using a crystal structure of PLP2 bound to its cellular substrate ubiquitin (Ub), we characterized the residues forming the substrate binding surface. This was used as a platform to guide the design of PLP2 mutants with reduced capacity to bind ubiquitin, and provided a clear link between PLP2 deubiquitinating activity and host innate immune evasion. Using structure-guided mutagenesis and reverse genetics methods for PRRSV, we demonstrate that specific PLP2 residues that are critically involved in substrate recognition are under selective pressure to revert to wild-type during infection. These findings are in contrast to what has been observed with the equine arteritis virus (EAV) PLP2 domain, and uniquely highlight the specific importance of PRRSV PLP2 deubiquitinating activity with respect to viral replication. [ABSTRACT FROM AUTHOR]