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.
Snijder
E J
EJ
Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Wassenaar
A L
AL
Spaan
W J
WJ
D000595:Amino Acid Sequence; D007240:Equartevirus / Q000201:enzymology*; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012995:Solubility; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D010455:Peptides; D014764:Viral Proteins; D015678:Viral Structural Proteins; D012324:RNA-Dependent RNA Polymerase