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PMID: 2072446 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.

Journal of virology ·Vol. 65 ·No. 8 ·1991-08-00 ·Pages 4107-13

Liljeström P, Lusa S, Huylebroeck D, Garoff H

Abstract

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.

MeSH Terms
Animals Cell Line Cloning, Molecular Gene Expression Regulation, Viral Membrane Proteins/genetics Mutagenesis RNA, Viral/biosynthesis Semliki forest virus/genetics,physiology Transcription, Genetic Transfection Viral Envelope Proteins/biosynthesis,genetics,physiology Virion/genetics,physiology
Chemicals
Membrane Proteins RNA, Viral Viral Envelope Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Liljeström P
Department of Molecular Biology, Karolinska Institute, Huddinge, Sweden.
Lusa S
Huylebroeck D
Garoff H
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.65.8.4107-4113.1991
Published
1991-08-00
Pages
4107-13
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC248843
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.65.8.4107-4113.1991 PMC Full Text View journal details
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