Stable expression of primary human immunodeficiency virus type 1 structural gene products by use of a noncytopathic sindbis virus vector

Efficient expression of the human immunodeficiency virus type 1 (HIV-1) structural gene products Gag, Pol, and Env involves the regulation by viral Rev and Rev-responsive elements (RRE). Removal of multiple inhibitory sequences (INS) in the coding regions of these structural genes or modification of the codon usage patterns of HIV-1 genes to those used by highly expressed human genes has been found to significantly increase HIV-1 structural protein expression in the absence of Rev and RRE. In this study, we show that efficient and stable expression of the HIV-1 structural gene products Gag and Env could be achieved by transfection with a noncytopathic Sindbis virus expression vector by using HIV-1 sequences from primary isolates without any sequence modification. Stable expression of these Gag and Env proteins was observed for more than 12 months. The fact that the Sindbis virus expression vector replicates its RNA only in the cytoplasm of the transfected cells and the fact that the lack of expression of HIV-1 Gag by the DNA vector containing unmodified HIV-1 gag sequences was associated with a lack of detectable cytoplasmic gag RNA suggest that a major blockage in the expression of HIV-1 structural proteins in the absence of Rev/RRE is caused by inefficient accumulation of mRNA in the cytoplasm. Efficient long-term expression of structural proteins of diverse HIV-1 strains by the noncytopathic Sindbis virus expression system may be a useful tool for functional study of HIV-1 gene products and vaccine research.     

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.     

Regulation of HIV-1 env mRNA translation by Rev protein

We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.     

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.     

RNA interference in human cells is restricted to the cytoplasm

RNA interference (RNAi) is an evolutionarily conserved eukaryotic adaptive response that leads to the specific degradation of target mRNA species in response to cellular exposure to homologous double-stranded RNA molecules. Here, we have analyzed the subcellular location at which RNA degradation occurs in human cells exposed to double-stranded short interfering RNAs. To unequivocally determine whether a given mRNA is subject to degradation in the cytoplasm, the nucleus, or both, we have used the retroviral Rev/RRE system to control whether target mRNAs remain sequestered in the nucleus or are exported to the cytoplasm. In the absence of export, we found that the nuclear level of the RRE-containing target mRNA was not affected by activation of RNAi. In contrast, when nuclear export was induced by expression of Rev, cytoplasmic target mRNAs were effectively and specifically degraded by RNAi. Curiously, when the target mRNA molecule was undergoing active export from the nucleus, induction of RNAi also resulted in a reproducible approximately twofold drop in the level of target mRNA present In the nuclear RNA fraction. As this same mRNA was entirely resistant to RNAi when sequestered in the nucleus, this result suggests that RNAi is able to induce degradation of target mRNAs not only in the cytoplasm but also during the process of nuclear mRNA export. Truly nucleoplasmic mRNAs or pre-mRNAs are, in contrast, resistant to RNAi.     

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.     

Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure.

Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the B1 branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'..CACUAUGGG..3' sequence in the B stem, without affecting the overall structure abolished both in vitro Rev binding and the in vivo Rev response.     

Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.     

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein.

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.     

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.     

Feedback regulation of human immunodeficiency virus type 1 expression by the Rev protein.

Rev is an essential regulatory protein of the human immunodeficiency virus type 1 (HIV-1) that affects the transport and half-life of certain viral mRNAs. Rev exerts its function via a unique element, the Rev-responsive element (RRE), located within the env region of HIV-1. It has been previously demonstrated that Rev affects the relative levels of RRE-containing and RRE-lacking mRNAs. We have studied the effects of Rev on the expression of the three different groups of small, multiply spliced mRNAs that lack the RRE sequence and encode the regulatory proteins Tat, Rev, and Nef. To monitor the tat, rev, and nef mRNAs we generated specific S1 nuclease mapping probes that distinguish among them. Analysis of all the mRNA species producing Tat, Rev, and Nef revealed that their levels are coordinately regulated by Rev. They are increased in the absence of Rev protein and are down regulated in the presence of Rev. The corresponding proteins were measured by immunoprecipitations, and their levels are in agreement with the RNA levels. These results verify the model proposing that Rev is a general regulator indirectly affecting all the multiply spliced mRNAs to a similar extent. Therefore, Rev down regulates its own expression and the expression of Tat and Nef.