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.     

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.     

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.     

The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly.

Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev binds to a cis-acting Rev-responsive element (RRE) located within the env region of HIV-1. It has previously been shown that a 17-amino-acid peptide, corresponding to the basic domain of Rev, specifically inhibited in vitro the splicing of mRNAs containing the RRE. In this reaction, the peptide acts after an ATP-dependent step in the spliceosome assembly resulting in an accumulation of a 45-50S splicing-deficient complex. Characterization of this complex revealed that the basic domain of Rev does not interfere with U1 small nuclear ribonucleoprotein binding but blocks the entry of U4, U5, and U6 small nuclear RNAs into the spliceosome. Binding of U2 small nuclear ribonucleoprotein was partially inhibited. The critical nature of the oligomeric structure of RRE has been investigated both in vitro and in vivo. Reporter genes that contained one, three, or six repeated-monomer high-affinity Rev binding sites (IIB) within an intron yielded a correlation among the oligomeric state of bound Rev; inhibition of splicing; ability to block the assembly of U4, U5, and U6 small nuclear RNAs in the spliceosome in vitro; and level of Rev response in vivo.     

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.     

Inhibition of Human Immunodeficiency Virus Rev and Human T-Cell Leukemia Virus Rex Function, but Not Mason-Pfizer Monkey Virus Constitutive Transport Element Activity, by a Mutant Human Nucleoporin Targeted to Crm1

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.     

Diminished Production of Human Immunodeficiency Virus Type 1 in Astrocytes Results from Inefficient Translation of gag, env, and nef mRNAs despite Efficient Expression of Tat and Rev

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation of gag, env, and nef mRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.     

Regulation of the export of RNA from the nucleus

Transport of macromolecules across the nuclear envelope is an essential activity in eukaryotic cells. RNA molecules within cells are found complexed with proteins and the bound proteins likely contain signals for RNA export. RNAs microinjected into Xenopus oocyte nuclei are readily exported, and their export can be competed by self RNA but not by RNAs of other classes. This indicates that the rate-limiting step in RNA export is the interaction of RNAs with class-specific proteins, at least when substrate RNAs are present at saturating levels. Export of host mRNAs is inhibited following infection by some animal viruses, while the export of viral RNAs occurs. The HIV-1 RNA-binding protein, Rev, mediates the export of intron-containing viral RNAs that would normally be retained in nuclei. This requires a nuclear export signal (NES) within Rev and an element within the RNA to which Rev binds. In yeast, heat shock causes accumulation of poly(A)(+)RNA within nuclei but heat-shock mRNAs are transcribed and exported efficiently. This requires elements within heat shock mRNA that probably interact with a cellular protein to facilitate RNA export. In these cases, the proteins that recognize critical sequences in the RNAs probably direct the RNAs to an RNA export pathway not generally used for mRNA export. This would circumvent the general retention of most poly(A)(+)mRNAs following heat shock in yeast and the need for complete splicing of viral mRNAs that travel through the normal mRNA export pathway.     

The HIV-1 Rev protein: a model system for coupled RNA transport and translation.

The impact of the Rev protein of the human immunodeficiency virus type 1 (HIV-1) on RNA transport, intranuclear RNA distribution, and gene expression was examined for two Rev-dependent expression systems by means of fluorescence in situ hybridization, immunofluorescence, S1 nuclease protection, and functional assays. In the pgTat expression system, which utilizes authentic HIV-1 splice signals, unspliced mRNA remained entrapped in the nucleus in the absence of Rev and was exported to the cytoplasm in its presence, consistent with published findings. In the pSVAR expression system, significant levels of mRNA were found in the nucleus and cytoplasm in both the presence and absence of Rev, but only in the presence of Rev was mRNA translated into protein. The presence of cytoplasmic untranslated mRNA in the absence of Rev was demonstrated by in situ hybridization analysis of individual cells as well as by S1 nuclease analysis of cell populations. The results indicate that Rev has the potential to affect translation as well as transport, suggesting the possibility that cellular mechanisms exist whereby the translational efficiency of an mRNA may be affected by the manner in which it is transported from the nucleus. Fluorescence hybridization also provided high-resolution visualization of the intranuclear distribution of RNAs containing the Rev response element. This demonstrated for both expression systems that mRNA was not highly localized in tracks or around the nucleolus in the presence or absence of Rev, a nucleolar protein, but was more widely distributed throughout the nucleus. In pgTat transfectants, HIV-1 RNA often became localized in 5 to 20 discrete large intranuclear clusters in the presence of Rev, the potential significance of which is discussed.     

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.     

Multiple portions of poly(A)-binding protein stimulate translation in vivo

Translational stimulation of mRNAs during early development is often accompanied by increases in poly(A) tail length. Poly(A)-binding protein (PAB) is an evolutionarily conserved protein that binds to the poly(A) tails of eukaryotic mRNAs. We examined PAB's role in living cells, using both Xenopus laevis oocytes and Saccharomyces cerevisiae, by tethering it to the 3'-untranslated region of reporter mRNAs. Tethered PAB stimulates translation in vivo. Neither a poly(A) tail nor PAB's poly(A)-binding activity is required. Multiple domains of PAB act redundantly in oocytes to stimulate translation: the interaction of RNA recognition motifs (RRMs) 1 and 2 with eukaryotic initiation factor-4G correlates with translational stimulation. Interaction with Paip-1 is insufficient for stimulation. RRMs 3 and 4 also stimulate, but bind neither factor. The regions of tethered PAB required in yeast to stimulate translation and stabilize mRNAs differ, implying that the two functions are distinct. Our results establish that oocytes contain the machinery necessary to support PAB-mediated translation and suggest that PAB may be an important participant in translational regulation during early development.