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PMID: 7666541 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

Journal of virology ·Vol. 69 ·No. 10 ·1995-10-00 ·Pages 6400-7

Berlioz C, Torrent C, Darlix JL

Abstract

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

Related Genes
Ha-ras v-ras
MeSH Terms
3T3 Cells Animals Base Sequence Cysteine Endopeptidases/metabolism Genes, Viral Genes, ras Genetic Vectors Harvey murine sarcoma virus/genetics Mice Protein Biosynthesis RNA, Messenger/analysis,biosynthesis,genetics,metabolism RNA, Viral/biosynthesis,genetics,metabolism Rabbits Rats Recombinant Proteins/analysis,biosynthesis Reticulocytes/metabolism Retroviridae Ribosomes/metabolism Transfection Viral Proteins Viral Structural Proteins/genetics beta-Galactosidase/analysis,biosynthesis
Chemicals
RNA, Messenger RNA, Viral Recombinant Proteins Viral Proteins Viral Structural Proteins beta-Galactosidase Cysteine Endopeptidases picornain 2A, Picornavirus
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Berlioz C
LaboRétro, Unité de Virologie Humaine-U412, Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure de Lyon, France.
Torrent C
Darlix J L
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.69.10.6400-6407.1995
Published
1995-10-00
Pages
6400-7
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC189539
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.69.10.6400-6407.1995 PMC Full Text View journal details
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