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

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.

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

“…At early timepoints, lower nsp2 frameshift efficiency means more ribosomes continue to translate the remainder of pp1a or pp1ab, which encode components of the replication and transcription complex (RTC), which may be more important for establishing infection than translation of the accessory protein nsp2TF. Later in the replication cycle, higher −2 PRF efficiency likely corresponds to an increased requirement for nsp2TF to prevent degradation of GP5 and M, which are expressed from ~8 hpi and are essential for virion assembly 36,123 . Further, nsp2TF is a more potent innate immune suppressor than nsp2, and down-regulates expression of swine leukocyte antigen class I (swine MHC class I) 32,124 , which may become critical later in infection as viral proteins and double-stranded RNA accumulate due to viral translation and replication.…”

From Recoding to Peptides for MHC Class I Immune Display: Enriching Viral Expression, Virus Vulnerability and Virus Evasion

“…Programmed −1 frameshifting occurs just 5′ of the junction of its long ORF1a with its long ORF1b to synthesize its replicase polyprotein [ 117 , 118 ]. However, within the nsp coding sequence of ORF1a, PRRSV also employs 20% efficient −2 frameshifting, to generate a multifunctional product, nsp2TF ( T rans- F rame), consisting of the N-terminal of nsp2 and a unique C-terminal domain [ 119 , 120 ]. Immediately 3′ of the shift site, there is a stop codon in the -1 frame.…”

“…In vitro transfection experiments showed that their deubiquitinase activity suppresses the innate immune response [ 232 , 233 , 234 ]. Nsp2TF has been directly shown to function as a deubiquitinase that antagonizes ubiquitination of host cell proteins [ 120 , 231 ]. However, in infected cells nsp2 localizes to the virus replication complex for polyprotein processing.…”

“…The C-terminal region of the TF-domain of the −2 frameshift derived Nsp2TF reduces expression of these molecules in alveolar macrophages, PK15-CD163 cells and monocyte-derived dendritic cells [ 237 ]. Nsp2TF promotes arterivirus assembly by interacting with the major viral envelope proteins during their transport along the exocytic pathway and antagonizing host defensive ubiquitination-dependent proteasomal degradation [ 120 ]. While this is just one of the ubiquitin-related strategies viruses have evolved [ 238 ], unlike the many viral encoded deubiquinases that act on relevant cellular substrates, Nsp2TF acts on other viral encoded proteins [ 120 ].…”

“…Nsp2TF promotes arterivirus assembly by interacting with the major viral envelope proteins during their transport along the exocytic pathway and antagonizing host defensive ubiquitination-dependent proteasomal degradation [ 120 ]. While this is just one of the ubiquitin-related strategies viruses have evolved [ 238 ], unlike the many viral encoded deubiquinases that act on relevant cellular substrates, Nsp2TF acts on other viral encoded proteins [ 120 ]. Nsp2TF is targeted to the same compartments of the secretory pathway (ERGIC and Golgi), in which the two major envelope proteins of PRRSV, GP5 and M, accumulate.…”