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Journal of Virology, February 2009, p. 1930-1940, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02162-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis{triangledown}

Jianrong Li,* Amal Rahmeh, Vesna Brusic, and Sean P. J. Whelan*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 13 October 2008/ Accepted 28 November 2008

The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3'-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3' termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5' cap formation and 3' polyadenylation.

* Corresponding author. Mailing address for Sean P. J. Whelan: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1923. Fax: (617) 738-7664. E-mail: swhelan{at}hms.harvard.edu. Present address for Jianrong Li: Department of Food Science and Technology, The Ohio State University, 233 Parker Food Science & Tech Bldg., 2015 Fyffe Road, Columbus, OH 43210. Phone: (614) 688-5728. Fax: (614) 292-0218. E-mail: li.926{at}osu.edu

{triangledown} Published ahead of print on 10 December 2008.

Journal of Virology, February 2009, p. 1930-1940, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02162-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rahmeh, A. A., Li, J., Kranzusch, P. J., Whelan, S. P. J. (2009). Ribose 2'-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein. J. Virol. 83: 11043-11050 [Abstract] [Full Text]  


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