Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains

Abstract

The replicase open reading frame 1b (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a-and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.

The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins

“…1) involves the rapid autoproteolytic release of two or three N-terminal nsps [nsp1 (or nsp1␣/1␤) and nsp2] and the subsequent processing of the remaining polyproteins by the "main protease" residing in nsp4 , together resulting in a set of 13 or 14 individual nsps. It should be noted, however, that many intermediate cleavage products of unknown function have also been detected (Snijder et al, 1994;van Dinten et al, 1996) and that alternative major and minor processing pathways have been defined for the EAV nsp3-8 region .…”

The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro

“…The ORF1a and ORF1b occupying 80% of the genome encode two viral replicase polyproteins-pp1a and pp1ab. The pp1a is processed into ten nonstructural proteins (Nsps) including Nsp1␣, Nsp1␤, Nsp2 to Nsp6, Nsp7␣, Nsp7␤ and Nsp8, while the pp1ab is cleaved into Nsp9, Nsp10, Nsp11 and Nsp12 which are recognized to be involved in viral genome transcription and replication (Bautista et al, 2002;den Boon et al, 1995;Fang and Snijder, 2010;Snijder and Meulenberg, 1998;van Dinten et al, 1996).…”

Expression, purification, and in vitro activity of an arterivirus main proteinase

“…Replicase gene expression ultimately yields (at least) 12 non-structural proteins ( Fig. 1A; Snijder et al, 1994;van Dinten et al, 1996;Wassenaar et al, 1997), which are produced from two primary genome translation products, the large polyproteins pp1a (1728 amino acids) and pp1ab (3175 amino acids), with the latter being expressed following a ribosomal frameshifting event (den Boon et al, 1991). The replicase subunits are released from the polyproteins by three virus-encoded proteinases (Fig.…”