JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sleat, D E
Right arrow Articles by Banerjee, A K
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sleat, D E
Right arrow Articles by Banerjee, A K
J Virol. 1993 March; 67(3): 1334-1339

Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase.

D E Sleat and A K Banerjee

Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.

ABSTRACT

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.

J Virol. 1993 March; 67(3): 1334-1339




This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 1993 by the American Society for Microbiology. All rights reserved.