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. 1994 Dec;68(12):7944–7952. doi: 10.1128/jvi.68.12.7944-7952.1994

Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.

A S Zolotukhin 1, A Valentin 1, G N Pavlakis 1, B K Felber 1
PMCID: PMC237257  PMID: 7966585

Abstract

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that did not affect the overlapping env reading frame or both a mutated RRE and two mutations that eliminated Rev were constructed. The mutations in the RRE were shown to remove both negative and Rev-inducible positive effects of the RRE on gene expression (G. Nasioulas, A. S. Zolotukhin, C. Tabernero, L. Solomin, C. P. Cunningham, G. N. Pavlakis, and B. K. Felber, J. Virol. 68:2986-2993, 1994). Upon insertion of a cis-acting element of simian retrovirus type 1 (SRV-1) into these clones, both RRE(-) and Rev(-)RRE(-) clones were expressed efficiently. The element of SRV-1 has properties similar to those of the recently identified element of Mason-Pfizer monkey virus (M. Bray, S. Prasad, J. W. Dubay, E. Hunter, K.-T. Jeang, D. Rekosh, and M.-L. Hammarskjold, Proc. Natl. Acad. Sci. USA 4:1256-1260, 1994). We demonstrated that virus preparations produced after transfections of these SRV-1 element-containing molecular clones in human cells were infectious after cell-free transmission, that they replicated about 5 to 10 times less efficiently than wild-type virus, and that they were propagated continuously for more than 7 months in human peripheral blood mononuclear cells. Growth characteristics and sequence analysis of these viruses after long-term culture demonstrated that no RRE(+)Rev(+) revertants developed. These data demonstrate that human immunodeficiency virus type 1 Rev and RRE can be replaced by heterologous regulatory systems, resulting in efficient virus production. The resulting Rev(-)RRE(-) virus can be prepared and propagated efficiently in tissue culture and can be used for further studies of the life cycle of the virus. The data also suggest that Rev acts exclusively through the RRE interaction and that it does not have any additional essential function in the life cycle of the virus.

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Selected References

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