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.     

Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1)

The Rev protein of human immunodeficiency virus type 1 (HIV-1) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in the cytoplasm. The cis-acting RNA target sequence for the Rev protein, termed the Rev response element (RRE), is present in the env gene and is predicted to form a highly ordered RNA secondary structure. Recent data indicate that Rev directly binds to RRE and, further, that this binding can be mapped to a 90-nucleotide subfragment at the 5' end of RRE. We now report that RRE also binds specifically and predominantly to a nuclear factor of approximately 56 kD. Mapping of the binding site reveals that the same subfragment that binds Rev also binds this nuclear factor. We designate this protein as NFRRE for nuclear factor, RRE binding. Rev and NFRRE appear to bind simultaneously to RRE. NFRRE is widely distributed in various mammalian cells. We speculate that this factor plays an important role in Rev-mediated transactivation and is likely to be involved in the processing or transport of cellular mRNA.     

Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron.

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regulation of HIV env expression is critically dependent on whether the env region is defined as an intron. This strengthens the hypothesis that Rev interacts with components of the splicing machinery to release splicing factors and enable export of the mRNA before splicing occurs.     

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.     

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.     

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.     

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.     

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.     

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.     

A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.