A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.     

A novel hnRNP specifically interacts with HIV-1 RRE RNA

We have identified and obtained the full-length clone of RREBP49, a human nuclear factor which specifically interacts with the Rev-responsive element (RRE) sequence of human immunodeficiency virus type 1. Sequence analysis revealed that RREBP49 is highly homologous to hnRNP F protein and contains three repeated RNA-binding domains. Binding assays demonstrated that Rev and RREBP49 bind to different subregions on the RRE sequence and that binding is mutually nonexclusive. Blocking of endogenous RREBP49 expression by an antisense construct increases Rev activity in CV-1 cells, indicating that RREBP49 and Rev may play antagonistic roles in HIV-1 replication. RREBP49 may function as a splicing factor or a nuclear retention factor for unspliced mRNAs. However, only a slight decrease of Rev activity was observed when exogenous RREBP49 was introduced into CV-1 cells by pSVL-RREBP49 expression vector. This may be explained by a high endogenous level of RREBP49 which is above optimal. Alternatively, additional cellular factors may be required for RREBP49-mediated inhibition of Rev.     

A specific sequence with a bulged guanosine residue(s) in a stem-bulge-stem structure of Rev-responsive element RNA is required for trans activation by human immunodeficiency virus type 1 Rev.

We demonstrate that both the in vitro RNA binding and in vivo trans activation functions of human immunodeficiency virus type 1 Rev regulatory protein Rev require the presence of a 9-nucleotide 5'-CACUAUGGG-3' RNA motif on its cognate target, the Rev-responsive element RNA. For optimal Rev recognition, this sequence must be presented as a stem-bulge-stem structure and must contain at least two G's, one of which must be unpaired, and include some or all of the CACUAU sequence upstream of the three G's. Distal mutations which result in the base pairing of the G's eliminate the Rev response. The first G is crucial, but changes at the other G's are tolerated if at least one G is unpaired. The secondary structure or the three-dimensional orientation of the B1 and B2 stem-loops of the Rev-responsive element are not relevant as long as the 5'-CACUAUGGG-3' sequence is preserved, with at least one bulged G residue.     

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1.

We have analyzed the action of the Rev and Tev proteins of human immunodeficiency virus type 1 (HIV-1) and of the Rex protein of human T-cell leukemia virus type I (HTLV-I) on a series of Rev-responsive element (RRE) mutants. The minimum continuous RRE region necessary and sufficient for Rev function was determined to be 204 nucleotides. Interestingly, this region was not sufficient for Tev or Rex function. These proteins require additional sequences, which may stabilize the structure of the RRE or may contain additional sequence-specific elements. Internal RRE deletions revealed that the targets for Rev and Rex can be separated, since mutants responding to Rev and not Rex and vice versa were identified. Tev was active on both types of mutants, suggesting that it has a more relaxed specificity than do both Rev and Rex proteins. Although Rev and Rex targets within the RRE appear to be distinct, the trans-dominant mutant RevBL prevents the RRE interaction with Rex. RevBL cannot inhibit the function of Rex on RRE deletions that lack the Rev-responsive portion. These results indicate the presence of distinct sites within the RRE for interaction with these proteins. The binding sites for the different proteins do not function independently and may interfere with one another. Mutations affecting the RRE may change the accessibility and binding characteristics of the different binding sites.     

Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site.     

Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.

Molecular clones of human immunodeficiency virus type 1 that contained either 37 point mutations in the Rev-responsive element (RRE) that did not affect the overlapping env reading frame or both a mutated RRE and two mutations that eliminated Rev were constructed. The mutations in the RRE were shown to remove both negative and Rev-inducible positive effects of the RRE on gene expression (G. Nasioulas, A. S. Zolotukhin, C. Tabernero, L. Solomin, C. P. Cunningham, G. N. Pavlakis, and B. K. Felber, J. Virol. 68:2986-2993, 1994). Upon insertion of a cis-acting element of simian retrovirus type 1 (SRV-1) into these clones, both RRE(-) and Rev(-)RRE(-) clones were expressed efficiently. The element of SRV-1 has properties similar to those of the recently identified element of Mason-Pfizer monkey virus (M. Bray, S. Prasad, J. W. Dubay, E. Hunter, K.-T. Jeang, D. Rekosh, and M.-L. Hammarskjold, Proc. Natl. Acad. Sci. USA 4:1256-1260, 1994). We demonstrated that virus preparations produced after transfections of these SRV-1 element-containing molecular clones in human cells were infectious after cell-free transmission, that they replicated about 5 to 10 times less efficiently than wild-type virus, and that they were propagated continuously for more than 7 months in human peripheral blood mononuclear cells. Growth characteristics and sequence analysis of these viruses after long-term culture demonstrated that no RRE(+)Rev(+) revertants developed. These data demonstrate that human immunodeficiency virus type 1 Rev and RRE can be replaced by heterologous regulatory systems, resulting in efficient virus production. The resulting Rev(-)RRE(-) virus can be prepared and propagated efficiently in tissue culture and can be used for further studies of the life cycle of the virus. The data also suggest that Rev acts exclusively through the RRE interaction and that it does not have any additional essential function in the life cycle of the virus.     

Binding of trans-dominant mutant Rev protein of human immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA

The binding of Rev protein of human immunodeficiency virus type 1 (HIV-1) to the cis-acting Rev-responsive element (RRE) was compared to the binding of a trans-dominant Rev mutant. RevBL, which inhibits Rev function. Rev and RevBL expressed in bacteria were purified and shown to bind in vitro to the RRE with similar affinities. The study of the RRE mutants indicated that Rev and RevBL bind to the same target within the RRE in vitro and in vivo. In vivo experiments demonstrated that RevBL did not increase the steady-state levels of HIV-1 mRNA or protein. These experiments suggested that additional cellular factors interacting with Rev but not with RevBL are necessary for function. The Rex protein of human T-cell leukemia virus type I (HTLV-I) is similar to Rev and acts through a sequence named Rex-responsive element (RXRE) located in the long terminal repeat of HTLV-I. We examined the function of RevBL on a hybrid mRNA molecule containing both the RRE and RXRE. While RevBL prevented Rev function, it did not affect Rex function on the mRNA containing either the RXRE or both the RRE and RXRE. Therefore, binding of RevBL to the RRE had neither positive nor negative effects on the mRNA, since this mRNA could be efficiently utilized in the presence of a functional Rex-RXRE interaction. The results obtained in vivo and in vitro strongly suggest that RevBL inhibits Rev function by binding to the same site as Rev and preventing Rev binding and function.     

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.     

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.     

RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.     

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.     

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.     

Human immunodeficiency virus type 1 Rev is required in vivo for binding of poly(A)-binding protein to Rev-dependent RNAs.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.     

Persistent Infection of Rhesus Macaques by the Rev-Independent Nef(−) Simian Immunodeficiency Virus SIVmac239: Replication Kinetics and Genomic Stability

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.