Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.     

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.     

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.     

Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.     

A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.     

Human chromosome 6- and 11-encoded factors support human immunodeficiency virus type 1 Rev function in A9 cells.

The precise mechanism of Rev-mediated expression of human immunodeficiency virus (HIV-1) late genes is not well characterized. We recently proposed a requirement for HIV-1 Rev responsive element (RRE) RNA binding host nuclear proteins in Rev function. In this report, using a transient transfection assay of Rev function, we further demonstrate the role of host cell factors in HIV-1 Rev function. Murine A9 cells, which are inefficient in forming RRE-host protein ribonucleoprotein complexes, are also inefficient in supporting Rev function. We also show that host cell factor(s) encoded by human chromosomes 6 and 11 can support HIV-1 Rev-mediated expression of unspliced viral mRNAs in murine A9 cells.     

Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner

Retroviral gene expression requires nuclear export and translation of incompletely spliced RNA. In the case of human immunodeficiency virus (HIV), this is facilitated by the viral Rev protein binding to its cognate RNA response element (RRE), while other retroviruses contain constitutive transport elements (CTE) binding to cellular factors. These CTE can substitute for the HIV-1 Rev/RRE system, albeit with reduced efficiency. Here, we show that multimeric copies of the CTE restore HIV-1 protein expression to levels comparable to or higher than Rev/RRE in various cell lines from different species. We suggest that multimerization of export factors is important for CTE function, as reported for Rev. CTE function was not affected when the element was displaced from its natural position close to the poly(A) signal, while insertion of an intron into the 3′-untranslated region (3′-UTR) severely reduced CTE activity. In this case, cytoplasmic RNA degradation was observed, which may be mediated by nonsense-mediated RNA decay. In contrast, Rev-dependent gene expression was insensitive to an intron in the 3′-UTR. Finally, we show that the putative CTE-binding protein RNA helicase A is not specifically translocated into the cytoplasm upon overexpression of CTE-containing RNA.     

Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.     

Trafficking through the Rev/RRE pathway is essential for efficient inhibition of human immunodeficiency virus type 1 by an antisense RNA derived from the envelope gene

A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminal-repeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.     

Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.     

Functional Analyses Reveal Extensive RRE Plasticity in Primary HIV-1 Sequences Selected under Selective Pressure

Background

HIV-1 Rev response element (RRE) is a functional region of viral RNA lying immediately downstream to the junction of gp120 and gp41 in the env coding sequence. The RRE is essential for HIV replication and binds with the Rev protein to facilitate the export of viral mRNA from nucleus to cytoplasm. It has been suggested that changes in the predicted secondary structure of primary RRE sequences impact the function of the RREs; however, functional assays have not yet been performed. The aim of this study was to characterize the genetic, structural and functional variation in the RRE primary sequences selected in vivo by Enfuvirtide pressure.

Results

Multiple RRE variants were obtained from viruses isolated from patients who failed an Enfuvirtide-containing regimen. Different alterations were observed in the predicted RRE secondary structures, with the abrogation of the primary Rev binding site in one of the variants. In spite of this, most of the RRE variants were able to bind Rev and promote the cytoplasmic export of the viral mRNAs with equivalent efficiency in a cell-based assay. Only RRE45 and RRE40-45 showed an impaired ability to bind Rev in a gel-shift binding assay. Unexpectedly, this impairment was not reflected in functional capacity when RNA export was evaluated using a reporter assay, or during virus replication in lymphoid cells, suggesting that in vivo the RRE would be highly malleable.

Conclusions

The Rev-RRE functionality is unaffected in RRE variants selected in patients failing an ENF-containing regimen. Our data show that the current understanding of the Rev-RRE complex structure does not suffice and fails to rationally predict the function of naturally occurring RRE mutants. Therefore, this data should be taken into account in the development of antiviral agents that target the RRE-Rev complex.