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PMID: 9971783 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication.

Journal of virology ·Vol. 73 ·No. 3 ·1999-03-00 ·Pages 2027-37

van Dinten LC, Rensen S, Gorbalenya AE, Snijder EJ

Abstract

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.

MeSH Terms
Amino Acid Sequence Equartevirus/enzymology,physiology Mutagenesis, Site-Directed Open Reading Frames RNA, Viral/biosynthesis RNA-Dependent RNA Polymerase/genetics,metabolism Serine Endopeptidases/physiology Structure-Activity Relationship Virus Replication
Chemicals
RNA, Viral RNA-Dependent RNA Polymerase Serine Endopeptidases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
van Dinten L C
Department of Virology, Leiden University Medical Center, Leiden, The Netherlands.
Rensen S
Gorbalenya A E
Snijder E J
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.73.3.2027-2037.1999
Published
1999-03-00
Pages
2027-37
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC104445
Subset
IM
Grants
NCI NIH HHS · N01-CO-5600 · United States
External Links
PubMed source DOI: 10.1128/JVI.73.3.2027-2037.1999 PMC Full Text View journal details
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