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PMID: 8083971 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Journal of virology ·Vol. 68 ·No. 10 ·1994-10-00 ·Pages 6312-22

Hellen CU, Pestova TV, Wimmer E

Abstract

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.

MeSH Terms
Base Sequence Codon/genetics DNA, Ribosomal/genetics DNA, Viral/chemistry,metabolism Genes, Viral HeLa Cells Humans Molecular Sequence Data Mutagenesis, Site-Directed Nucleic Acid Conformation Oligodeoxyribonucleotides Phenotype Plasmids Poliovirus/genetics,metabolism,physiology Protein Biosynthesis RNA, Messenger/metabolism Ribosomes/metabolism Thermodynamics Transcription, Genetic Viral Proteins/biosynthesis Viral Structural Proteins/genetics Virus Replication
Chemicals
Codon DNA, Ribosomal DNA, Viral Oligodeoxyribonucleotides RNA, Messenger Viral Proteins Viral Structural Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Hellen C U
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-8621.
Pestova T V
Wimmer E
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.68.10.6312-6322.1994
Published
1994-10-00
Pages
6312-22
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC237052
Subset
IM
Grants
NIAID NIH HHS · AI-15122 · United States
NCI NIH HHS · CA-28146 · United States
External Links
PubMed source DOI: 10.1128/JVI.68.10.6312-6322.1994 PMC Full Text View journal details
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