Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease.

The C-terminal half of the replicase ORF1a polyprotein of the arterivirus equine arteritis virus is processed by a chymotrypsinlike serine protease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864-4871, 1996) located in nonstructural protein 4 (nsp4). Three probable SP cleavage sites had previously been identified in the ORF1a protein. Their proteolysis explained the main processing products generated from the C-terminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755-5764, 1994). By using sequence comparison, ORF1a expression systems, and site-directed mutagenesis, we have now identified two additional SP cleavage sites: Glu-1430 / Gly and Glu-1452 / Ser. This means that the ORF1a protein can be cleaved into eight processing end products: nsp1 to nsp8. By microsequence analysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu / (Gly/Ser) substrates. Importantly, our studies revealed that the C-terminal half of the ORF1a protein (nsp3-8) can be processed by the SP following two alternative pathways, which appear to be mutually exclusive. In the majority of the nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 and nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 site only, whereas the newly identified nsp5/6 and nsp6/7 sites appear to be inaccessible to the protease. In the alternative proteolytic cascade, which is used at a low but significant level in infected cells, it is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp6/7 sites are processed to yield a set of previously unnoticed processing products. Coexpression studies revealed that nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, the first step of the major processing pathway. When the nsp2 cofactor is absent, the nsp4/5 site cannot be processed and nsp3-8 is processed following the alternative, minor pathway.

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription.

Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.

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

The replicase open reading frame lb (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 arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.

Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papainlike cysteine proteases.

Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.

The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases.

The replicase ORF1a polyprotein of equine arteritis virus, a positive-stranded RNA virus, is proteolytically processed into (at least) six nonstructural proteins (Nsp). A papain-like Cys protease in Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this process. In this paper we demonstrate that the Nsp2/3 junction is not cleaved by either of these previously described proteases. Comparative sequence analysis suggested that an additional Cys protease resided in the N-terminal Nsp2 domain. For equine arteritis virus, this domain was shown to induce Nsp2/3 cleavage in a trans-cleavage assay. Processing was abolished when the putative active site residues, Cys-270 and His-332, were replaced. Other Nsp2 domains and three other conserved Cys residues were also shown to be essential. The Nsp2 Cys protease displays sequence similarity with viral papain-like proteases. However, the presumed catalytic Cys-270 is followed by a conserved Gly rather than the characteristic Trp. Replacement of Gly-271 by Trp abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a hallmark of viral chymotrypsin-like Cys proteases. Thus, the arterivirus Nsp2 protease is an unusual Cys protease with amino acid sequence similarities to both papain-like and chymotrypsin-like proteases.

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus.

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.

Proteolytic processing of the N-terminal region of the equine arteritis virus replicase.

A papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase was identified by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C-terminus which leads to the production of an approximately 30K N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys164 and His230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. It was shown that cleavage occurs in cis between Gly260 and Gly261.

The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease.

The presence of a papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase, which had been postulated on the basis of limited sequence similarities with cellular and viral thiol proteases, was confirmed by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C terminus which leads to the production of an approximately 30-kDa N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys-164 and His-230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. By means of N-terminal sequence analysis of a PCP cleavage product, derived from a bacterial expression system, it was shown that cleavage occurs between Gly-260 and Gly-261. No evidence for PCP-directed cleavages at other positions in the EAV replicase was obtained. In cotranslational and posttranslational trans-cleavage assays, neither EAV nsp1 nor its precursor was able to process the PCP cleavage site in trans.

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen.

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.