Synthesis of the Putative Red Clover Necrotic Mosaic Virus RNA Polymerase by Ribosomal Frameshifting in Vitro

Zhongguo Xiong, K. H. Kim, T. L. Kendall, S. A. Lommel

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5′-p27-p57-p37-3′. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.

Original languageEnglish (US)
Pages (from-to)213-221
Number of pages9
JournalVirology
Volume193
Issue number1
DOIs
StatePublished - Mar 1993

Fingerprint

Tombusviridae
Ribosomal Frameshifting
DNA-Directed RNA Polymerases
RNA
Peptides
Open Reading Frames
Terminator Codon
In Vitro Techniques
Antibodies
Retroviridae
Site-Directed Mutagenesis
Proteins
Genome

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

Cite this

Synthesis of the Putative Red Clover Necrotic Mosaic Virus RNA Polymerase by Ribosomal Frameshifting in Vitro. / Xiong, Zhongguo; Kim, K. H.; Kendall, T. L.; Lommel, S. A.

In: Virology, Vol. 193, No. 1, 03.1993, p. 213-221.

Research output: Contribution to journalArticle

Xiong, Zhongguo ; Kim, K. H. ; Kendall, T. L. ; Lommel, S. A. / Synthesis of the Putative Red Clover Necrotic Mosaic Virus RNA Polymerase by Ribosomal Frameshifting in Vitro. In: Virology. 1993 ; Vol. 193, No. 1. pp. 213-221.
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