Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function.

Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species.

The known primate lentiviruses can be divided into two subgroups consisting of the human immunodeficiency virus type 1 (HIV-1) isolates and the related HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) isolates. HIV-1 has been shown to encode a post-transcriptional trans-activator of viral structural gene expression, termed Rev, that is essential for viral replication in culture. Here, we demonstrate that HIV-2 and SIVmac also encode functional Rev proteins. As in the case of HIV-1, these Rev trans-activators are shown to induce the cytoplasmic expression of the unspliced viral transcripts that encode the viral structural proteins. Unexpectedly, the Rev proteins of HIV-2 and SIVmac proved incapable of activating the cytoplasmic expression of unspliced HIV-1 transcripts, whereas HIV-1 Rev was fully functional in the HIV-2/SIV system. This nonreciprocal complementation may imply a direct role for Rev in mediating the recognition of its viral RNA target sequence.

Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function.

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat.

Retrovirus mRNAs are normally polyadenylated within the proviral 3' long terminal repeat (LTR). The site of retrovirus transcript polyadenylation is flanked 3' by an LTR-specific sequence termed the U5 region, but the role of U5 in the determination of polyadenylation efficiency has not been addressed. We have used site-directed mutagenesis of a human immunodeficiency virus LTR to map U5 sequences which are required for efficient polyadenylation within the LTR. These LTR U5 region sequences display homology to a motif termed the G-T cluster, which is known to facilitate the efficient polyadenylation of mRNAs encoded by several cellular and viral genes. These results suggest that the LTR U5 region functions in vivo to permit efficient polyadenylation within the proviral 3' LTR.