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J. Virol., Jul 1997, 4892-4903, Vol 71, No. 7
Copyright © 1997, American Society for Microbiology

Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation

R Schneider, M Campbell, G Nasioulas, BK Felber and GN Pavlakis
Human Retrovirus Section, National Cancer Institute-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Maryland 21702-1201, USA.

The expression of gag, pol, and env of human immunodeficiency virus type 1 (HIV-1) depends on the presence of the viral Rev protein. This dependence is, at least in part, due to the presence of negatively acting sequences (inhibitory or instability elements [INS]) located within unspliced and partially spliced mRNAs. The positive interaction of Rev with the Rev-responsive element in these mRNAs counteracts the negative effects of the inhibitory sequences. Here, we demonstrate that in addition to the previously identified INS1 within p17gag, several other INS elements exist within the gag/pol region of HIV-1. These elements act independently of each other and were eliminated by mutagenesis after the introduction of multiple point mutations not affecting the coding region, leading to constitutive high levels of Gag expression. Expression vectors containing an intact or nearly intact p55gag region allowed the production of immature viral particles in mammalian cells in the absence of any other HIV proteins. The introduction of additional mutations in the protease region allowed efficient production of Gag/protease, which resulted in processing of the Pr55gag precursor and production of mature Gag particles with a lentivirus-like conical-core structure. The elimination of a newly identified INS element within pol and the previously identified CRS located within int was accomplished by the same methodology. Sequence comparisons of the identified inhibitory elements revealed no apparent homologies and demonstrated that these sequences are not splice sites. These results demonstrate that the elimination of INS elements leads to efficient expression of HIV-1 mRNAs in the absence of Rev or any posttranscriptional activating mechanisms.


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