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

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that di d not affect the overlapping env reading frame or both a mutated RRE a nd two mutations that eliminated Rev were constructed. The mutations i n the RRE were shown to remove both negative and Rev-inducible positiv e effects of the RRE on gene expression (G. Nasioulas, A. S. Zolotukhi n, 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-acti ng element of simian retrovirus type 1 (SRV-1) into these clones, both RRE(-) and Rev(-)RRE(-) clones were expressed efficiently. The elemen t of SRV-1 has properties similar to those of the recently identified element of Mason-Pfizer monkey virus (M. Pray, 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 prepar ations produced after transfections of these SRV-1 element-containing molecular clones in human cells were infectious after cell-free transm ission, 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 char acteristics and sequence analysis of these viruses after long-term cul ture demonstrated that no RRE(+)Rev(+) revertants developed. These dat a demonstrate that human immunodeficiency virus type 1 Rev and RRE can be replaced by heterologous regulatory systems, resulting in efficien t virus production. The resulting Rev(-)RRE(-) virus can be prepared a nd propagated efficiently in tissue culture and can be used for furthe r studies of the life cycle of the virus. The data also suggest that R ev acts exclusively through the RRE interaction and that it does not h ave any additional essential function in the life cycle of the virus.