Effect of single amino acid mutations in the conserved GDNQ motif of L protein of Rinderpest virus on RNA synthesis in vitro and in vivo.
The paramyxovirus RNA-dependent RNA polymerase consists of two subunits, the transcription co-factor phosphoprotein P and the large protein L, which possesses all the catalytic functions such as RNA synthesis (both transcription replication), methylation, capping and polyadenylation. The L protein has high sequence homology among the negative sense RNA viruses. The domains and residues on the L protein involved in the above-mentioned activities are not well defined, although the role of conserved GDNQ motif of the putative catalytic centre of L protein of few related viruses have been examined. In order to gain insight into the role played by the GDNQ motif of the L protein of Rinderpest virus (RPV), we have examined mutations at each amino acid in this motif of the L protein of Rinderpest virus and tested the biological activity in vivo and in vitro. Site directed mutants were generated and transiently expressed in mammalian cells and were shown to interact with P protein similar to wild type L. The biological activity of mutant L proteins has been tested in an in vitro reconstituted system capable of carrying out cell-free RNA synthesis on synthetic Rinderpest N-RNA template. Further, the role played by individual amino acids has also been defined in vivo using an in vivo minigenome replication/transcription system which indicated the importance of this conserved sequence in viral RNA synthesis.
Chattopadhyay
Anasuya
A
Department of Microbiology and Cell Biology, Indian Institute of Science, 560012, Bangalore, India.
Raha
Tamal
T
Shaila
M S
MS
D020816:Amino Acid Motifs; D020134:Catalytic Domain; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D017124:Conserved Sequence; D015870:Gene Expression; D016679:Genome, Viral; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012302:Rinderpest virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012367:RNA, Viral; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012324:RNA-Dependent RNA Polymerase