Production of Infectious Recombinant Moloney Murine Leukemia Virus Particles in BHK Cells Using Semliki Forest Virus-derived RNA Expression Vectors

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1996 Oct 15

PMID 8876192

Citations 13

Authors

K J Li

H Garoff

Affiliations

Soon will be listed here.

Abstract

We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney murine leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney murine leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.

Citing Articles

Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env.

Li K, Zhang S, Kronqvist M, Wallin M, Ekstrom M, Derse D J Virol. 2008; 82(14):7135-43.

PMID: 18480461 PMC: 2446982. DOI: 10.1128/JVI.00448-08.

Mobilization of full-length Semliki Forest virus replicon by retrovirus particles.

Piver E, Collin C, Renault N, Bru T, Pages J J Virol. 2006; 80(19):9889-95.

PMID: 16973593 PMC: 1617220. DOI: 10.1128/JVI.00664-06.

Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion.

Wallin M, Ekstrom M, Garoff H EMBO J. 2003; 23(1):54-65.

PMID: 14685283 PMC: 1271652. DOI: 10.1038/sj.emboj.7600012.

Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes.

Muriaux D, Mirro J, Nagashima K, Harvin D, Rein A J Virol. 2002; 76(22):11405-13.

PMID: 12388701 PMC: 136761. DOI: 10.1128/jvi.76.22.11405-11413.2002.

RNA is a structural element in retrovirus particles.

Muriaux D, Mirro J, HARVIN D, Rein A Proc Natl Acad Sci U S A. 2001; 98(9):5246-51.

PMID: 11320254 PMC: 33195. DOI: 10.1073/pnas.091000398.

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