This Article
Right arrow Full Text
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 Murphy, A. M.
Right arrow Articles by Grdzelishvili, V. Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Murphy, A. M.
Right arrow Articles by Grdzelishvili, V. Z.

 Previous Article  |  Next Article 

Journal of Virology, February 2009, p. 1669-1681, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01438-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Sendai Virus L Protein Amino Acid Residues Affecting Viral mRNA Cap Methylation{triangledown}

Andrea M. Murphy and Valery Z. Grdzelishvili*

Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223

Received 10 July 2008/ Accepted 25 November 2008

Viruses of the order Mononegavirales all encode a large (L) polymerase protein responsible for the replication and transcription of the viral genome as well as all posttranscriptional modifications of viral mRNAs. The L protein is conserved among all members of the Mononegavirales and has six conserved regions ("domains"). Using vesicular stomatitis virus (VSV) (family Rhabdoviridae) experimental system, we and others recently identified several conserved amino acid residues within L protein domain VI which are required for viral mRNA cap methylation. To verify that these critical amino acid residues have a similar function in other members of the Mononegavirales, we examined the Sendai virus (SeV) (family Paramyxoviridae) L protein by targeting homologous amino acid residues important for cap methylation in VSV which are highly conserved among all members of the Mononegavirales and are believed to constitute the L protein catalytic and S-adenosylmethionine-binding sites. In addition, an SeV L protein mutant with a deletion of the entire domain VI was generated. First, L mutants were tested for their abilities to synthesize viral mRNAs. While the domain VI deletion completely inactivated L, most of the amino acid substitutions had minor effects on mRNA synthesis. Using a reverse genetics approach, these mutations were introduced into the SeV genome, and recombinant infectious SeV mutants with single alanine substitutions at L positions 1782, 1804, 1805, and 1806 or a double substitution at positions 1804 and 1806 were generated. The mutant SeV virions were purified, detergent activated, and analyzed for their abilities to synthesize viral mRNAs methylated at their cap structures. In addition, further studies were done to examine these SeV mutants for a possible host range phenotype, which was previously shown for VSV cap methylation-defective mutants. In agreement with a predicted role of the SeV L protein invariant lysine 1782 as a catalytic residue, the recombinant virus with a single K1782A substitution was completely defective in cap methylation and showed a host range phenotype. In addition, the E1805A mutation within the putative S-adenosylmethionine-binding site of L resulted in a 60% reduction in cap methylation. In contrast to the homologous VSV mutants, other recombinant SeV mutants with amino acid substitutions at this site were neither defective in cap methylation nor host range restricted. The results of this initial study using an SeV experimental system demonstrate similarities as well as differences between the L protein cap methylation domains in different members of the Mononegavirales.

* Corresponding author. Mailing address: Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223-0001. Phone: (704) 687-7778. Fax: (704) 687-3128. E-mail: vzgrdzel{at}uncc.edu

{triangledown} Published ahead of print on 3 December 2008.

Journal of Virology, February 2009, p. 1669-1681, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01438-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ruedas, J. B., Perrault, J. (2009). Insertion of Enhanced Green Fluorescent Protein in a Hinge Region of Vesicular Stomatitis Virus L Polymerase Protein Creates a Temperature-Sensitive Virus That Displays No Virion-Associated Polymerase Activity In Vitro. J. Virol. 83: 12241-12252 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»