Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase polyproteins pp1a and pp1ab are proteolytically processed by four proteases encoded in ORF1a. In this study, a large set of PRRSV replicase cleavage products were identified and pp1a cleavage sites were verified by using a combination of bioinformatics, proteomics, immunoprecipitation, and site-directed mutagenesis. For genotype 1 PRRSV (isolate SD01-08), proteomic analysis identified H180/S181, G385/A386, and G1446/A1447 as the cleavage sites separating nsp1α/1β, nsp1β/nsp2, and nsp2/nsp3, respectively. Transient expression of nsp2-8, nsp3-8, nsp4-8, nsp5-8 (using the recombinant vaccinia virus/T7 RNA polymerase system) and immunoprecipitation identified the cleavage end products nsp2, nsp3, nsp4, nsp7α and nsp7β, and various processing intermediates. Our studies also revealed the existence of alternative proteolytic processing pathways for the processing of the nsp3-8 region, depending on the presence or absence of nsp2 as a co-factor. The identity of most cleavage products was further corroborated by site-directed mutagenesis of individual cleavage sites in constructs expressing nsp3-8 or nsp4-8. This study constitutes the first in-depth experimental analysis of PRRSV replicase processing and the data are discussed against the background of the processing scheme previously derived for the arterivirus prototype, the distantly related equine arteritis virus (EAV). Despite several differences between the two viruses, of which the functional significance remains to be studied, our study demonstrates the general conservation of the replicase pp1a processing scheme between EAV and PRRSV, and likely also the other members of the arterivirus family.

Biogenesis and architecture of arterivirus replication organelles

“…This event, which results in the synthesis of pp1ab, is promoted by a specific heptanucleotide shift site and an RNA pseudoknot structure (Brierley et al, 1989;den Boon et al, 1991). The polyproteins pp1a and pp1ab contain the viral nsps, which are released by multiple internal proteases (Li et al, 2014;Ziebuhr et al, 2000). Together they are often referred to as the replicase, as they either are part of or support the functions of the so-called replication and transcription complex (RTC), the aggregate enzymes that replicate the viral genome and produce the sg mRNAs used to express the structural proteins.…”

“…When free nsp2 is present, the nsp4/nsp5 junction can be cleaved, which yields the nsp5-7 and nsp5-8 cleavage products that, apart from the slow processing of the nsp7/nsp8 junction, do not seem to undergo further processing. In the absence of nsp2, the nsp4/5 junction remains uncleaved, but instead the nsp5/6, nsp6/7 and possibly the nsp7␣/7␤ sites are processed (Li et al, , 2014van Aken et al, 2006;Wassenaar et al, 1997). In infected cells, the nsp4/5 junction is mostly cleaved, resulting in the majority of nsp5 being present in nsp5-7 and nsp5-8 cleavage products; however, cleavage of sites in nsp5-7, even though occurring in a minority of cases, is critical for virus replication (van Aken et al, 2006).…”

“…To investigate this, using the same approach as above, we created a stable inducible HuH-7 cell line expressing nsp2-7. Since most nsp5 molecules in infected cells are present in the form of nsp5-7 or nsp5-8 processing intermediates (Li et al, 2014;Wassenaar et al, 1997) (Section 2.1), expression of a self-cleaving nsp2-7 was chosen to mimic the course of events in infected cells most closely (Supplementary Fig. 1).…”

Structural Analysis of Porcine Reproductive and Respiratory Syndrome Virus Non-structural Protein 7α (NSP7α) and Identification of Its Interaction with NSP9

“…Of these, ORF1a and ORF1b occupy 75% of the genome and encode polyproteins pp1a and pp1ab. Thereafter, the two polyproteins could be cleaved into non-structural proteins (NSP1-12) by proteases NSP1α, NSP1β, NSP2, and NSP4 (van Aken et al, 2006; Fang and Snijder, 2010; Li et al, 2015). …”

Epitope mapping and characterization of a novel Nsp10-specific monoclonal antibody that differentiates genotype 2 PRRSV from genotype 1 PRRSV

“…The PRRSV RNA genome is about 15 kb in length, containing at least 11 open reading frames (ORFs) [7]. The ORF1a and ORF1b encode replication-related non-structural proteins (Nsps), whereas ORFs 2–7 are translated from a nested set of subgenomic RNA (sgRNA) encoding the structural proteins [8, 9].…”