A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny

Author summary Arteriviruses are a rapidly expanding family of positive-stranded RNA viruses, which includes economically important veterinary pathogens like equine arteritis virus (EAV) and two species of porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2). In our previous studies, we uncovered an unprecedented arterivirus gene expression mechanism: a highly efficient -2 programmed ribosomal frameshift (PRF) that is controlled by an interaction of viral protein nsp1ss with specific RNA sequences and host poly(C) binding proteins. It is used by PRRSVs, and most other arteriviruses, to efficiently produce a previously unknown nonstructural protein variant, nsp2TF. In this study, we demonstrate that PRRSV nsp2TF interacts with the two major arteriviral envelope proteins, GP5 and M, whose heterodimerization in the secretory pathway is a key step in envelope protein trafficking and virus assembly. Our findings suggest that nsp2TF promotes arterivirus assembly by antagonizing the ubiquitination-dependent proteasomal degradation of GP5 and M proteins. This mechanism is based on the DUB activity of the PLP2 protease domain located within the N-terminal region of nsp2TF. To our knowledge, this is the first study to demonstrate that viruses can express a DUB that functions specifically to counteract the ubiquitination and degradation of key viral structural proteins. Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.