Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing.
Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus Equine arteritis virus (order Nidovirales), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-beta-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
Danny
D
van Aken
Eric J
EJ
Snijder
Alexander E
AE
Gorbalenya
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D018360:Crystallography, X-Ray; D007240:Equartevirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D010948:Viral Plaque Assay
D020815:Polyproteins; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases