Human Immunodeficiency Virus Type 1 Rev Protein Research Articles

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

Among eukaryotic transcription trans-activators, the human immunodeficiency virus type 1 (HIV-1) Tat protein is exceptional in that its target site TAR is an RNA rather than a DNA sequence. Here, we confirm that fusion of Tat to the RNA-binding domain of the HIV-1 Rev protein permits the efficient activation of an HIV-1 long terminal repeat (LTR) promoter in which critical TAR sequences have been replaced by RNA sequences derived from the HIV-1 Rev response element (RRE). An RRE target sequence as small as 13 nucleotides is shown to form an effective in vivo target for Rev binding. More important, a fusion protein consisting of Rev attached to the VP16 transcription activation domain was also observed to efficiently activate the HIV-1 LTR from this nascent RNA target. These data demonstrate that trans-activation of transcription by acidic activation domains does not require a stable interaction with the promoter DNA and suggest that VP16, like Tat, can act on steps subsequent to the formation of the HIV-1 LTR preinitiation complex. The finding that the activation domains of VP16 and Tat are functionally interchangeable raises the possibility that these apparently disparate viral trans-activators may nevertheless act via similar mechanisms.

In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.

Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex protein activation domain by functional exchange.

The human retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are characterized by complex regulation of gene expression. Each virus encodes a posttranscriptional regulator, the 19-kDa HIV-1 Rev protein and the 27-kDa HTLV-I Rex protein, which is required for viral replication. Expression of these trans activators results in the cytoplasmic accumulation of unspliced or singly spliced viral mRNA which encode the gag, pol, and env gene products. The finding that the HTLV-I Rex protein is able to functionally substitute for the Rev protein of HIV-1 indicates that HIV-1 Rev and HTLV-I Rex may interact with the same component of a cellular pathway involved in either mRNA splicing or transport. In this study, we have generated functional Rev/Rex hybrid proteins by domain exchange. We have defined, using in vivo and in vitro analyses, the activation domains of Rev and Rex which are the putative targets of a common host cell factor(s) required for Rev and Rex function.

The Rex regulatory protein of human T-cell lymphotropic virus type I binds specifically to its target site within the viral RNA.

The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified. Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE). Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE. The binding results obtained in vitro are therefore in agreement with the nonreciprocal function of Rev and Rex in vivo. Rex binds specifically to both RRE and RXRE and activates expression in both HIV-1 and HTLV-I, while Rev binds to RRE and activates only HIV-1. Binding of Rex to RRE deletion mutants previously shown to lack either the Rev-responsive or the Rex-responsive portion suggested preferential binding of Rex to a distinct target within the RRE. These results demonstrated that Rex, like Rev, acts by binding to a specific RNA target.

The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators

Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.

Sequence-specific RNA binding by the HIV-1 Rev protein

The human immunodeficiency virus type 1 (HIV-1) Rev protein acts post-transcriptionally to increase the amounts of the viral gag-pol and env messenger RNAs in the cytoplasm of infected cells. The mechanism of Rev action is uncertain. Possibilities include an accelerating effect on the rate of export of its mRNA targets from the nucleus and/or modulation of the splicing of pre-mRNAs. Both the gag-pol and env mRNAs contain a sequence that is required for responsiveness to Rev--the Rev responsive element, RRE. Here we show that Rev is a sequence-specific binding protein, whose binding site is the RRE. This information should help to clarify the mechanism by which Rev acts.

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.