Abstract
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.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the order Nidovirales, family Arteriviridae
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
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
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the order Nidovirales, family Arteriviridae. Besides PRRSV, the arterivirus family includes equine arteritis virus (EAV), mouse lactate dehydrogenase elevating virus (LDV), simian hemorrhagic fever virus (SHFV) and a variety of more recently identified members, many of which are of simian origin [2,3]. PRRSV is a small, enveloped virus, with a ~15 kb genome that contains 11 known open reading frames (ORFs). The 3’-proximal quarter of the viral genome encodes four envelope glycoproteins (GP2a, GP3, GP4 and GP5), three non-glycosylated trans-membrane proteins (E, ORF5a, and M) and the nucleocapsid protein (N). Studies of EAV and LDV have shown that GP5 and the non-glycosylated M protein, which spans the membrane three times, form a disulfide-linked heterodimer [5,6,7]. Heterodimerization triggers a series of important events, including transport of the GP5-M complex from the ER
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