Analysis of murine hepatitis virus strain A59 temperature-sensitive mutant TS-LA6 suggests that nsp10 plays a critical role in polyprotein processing.
نویسندگان
چکیده
Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 ( approximately 150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.
منابع مشابه
Murine hepatitis virus replicase protein nsp10 is a critical regulator of viral RNA synthesis.
Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually approximately 15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/re...
متن کاملThe murine coronavirus nucleocapsid gene is a determinant of virulence.
The murine coronavirus, mouse hepatitis virus (MHV) strain A59, causes acute encephalitis and chronic demyelinating disease as well as hepatitis in mice. The JHM strain (also called MHV-4 or JHM.SD) causes fatal encephalitis and only minimal hepatitis. Previous analysis of chimeric recombinant MHVs in which the spike gene, encoding the protein that mediates viral entry and cell-to-cell fusion, ...
متن کاملGenetic analysis of murine hepatitis virus non-structural protein 16.
MHV-Wüts18 is an RNA-negative, temperature-sensitive mutant of mouse coronavirus, strain murine hepatitis virus (MHV)-A59. We have previously identified the putative causal mutation of MHV-Wüts18 as a C to U transition at codon 2446 in ORF1b, which results in a substitution of proline 12 with serine in non-structural protein 16. Here, we have used a vaccinia virus-based reverse genetic system t...
متن کاملNew antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp15.
The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-A resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have ...
متن کاملMutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59.
The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV recep...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Journal of virology
دوره 81 13 شماره
صفحات -
تاریخ انتشار 2007