Mutational definition of the human immunodeficiency virus type 1 Rev activation domain.

Replication of human immunodeficiency virus type 1 requires the functional expression of the virally encoded Rev protein. The binding of this nuclear trans activator to its viral target sequence, the Rev-response element, induces the cytoplasmic expression of unspliced viral mRNAs. Mutation of the activation domain of Rev generates inactive proteins with normal RNA binding capabilities that inhibit wild-type Rev function in a trans-dominant manner. Here, we report that the activation domain comprises a minimum of nine amino acids, four of which are critically spaced leucines. The preservation of this essential sequence in other primate and nonprimate lentivirus Rev proteins indicates that this leucine-rich motif has been highly conserved during evolution. This conclusion, taken together with the observed permissiveness of a variety of eukaryotic cell types for Rev function, suggests that the target for the activation domain of Rev is likely to be a highly conserved cellular protein(s) intrinsic to nuclear mRNA transport or splicing.     

Some human immunodeficiency virus type 1 Vpu proteins are able to antagonize macaque BST-2 in vitro and in vivo: Vpu-negative simian-human immunodeficiency viruses are attenuated in vivo

Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by targeting BST-2/tetherin, a cellular protein inhibiting virus release. The widely used HIV-1(NL4-3) Vpu functionally inactivates human BST-2 but not murine or monkey BST-2, leading to the notion that Vpu antagonism is species specific. Here we investigated the properties of the CXCR4-tropic simian-human immunodeficiency virus DH12 (SHIV(DH12)) and the CCR5-tropic SHIV(AD8), each of which carries vpu genes derived from different primary HIV-1 isolates. We found that virion release from infected rhesus peripheral blood mononuclear cells was enhanced to various degrees by the Vpu present in both SHIVs. Transfer of the SHIV(DH12) Vpu transmembrane domain to the HIV-1(NL4-3) Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIV(DH12) and SHIV(AD8) vpu genes impaired virus replication in 6 of 8 inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4(+) T cells, and delayed disease progression. The expanded host range of the SHIV(DH12) Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1(DH12) Vpu protein. These results demonstrate that the species-specific inhibition of BST-2 by HIV-1(NL4-3) Vpu is not characteristic of all HIV-1 Vpu proteins; some HIV-1 isolates encode a Vpu with a broader host range.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

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.     

Nuclear preservation and cytoplasmic degradation of human immunodeficiency virus type 1 Rev protein.

Rev, a major regulatory protein of human immunodeficiency virus type 1, has been demonstrated to shuttle between the nucleus and cytoplasm of infected cells. The fate of the Rev protein in living cells was evaluated by pulse-chase experiments using a transient Rev expression system. Sixteen hours after chasing with unlabelled amino acids, 45% of the labelled Rev was still present, which clearly indicates a long half-life of Rev in living cells. A Rev mutant which is deficient in the ability to migrate from the nucleus to the cytoplasm was degraded more slowly than the wild-type Rev protein. As well, another Rev mutant protein, which lacks a functional nucleolar targeting signal (NOS) and is unable to enter the cell nucleus, was rapidly degraded and undetectable 16 h after chasing. Nuclear-nucleolar targeting properties provided by a divergent NOS from a related retrovirus, which was used to substitute for the NOS of Rev, increased the intracellular half-life of this Rev mutant. Moreover, coexpression of an intracellular anti-Rev single-chain antibody (SFv), which has been shown to interfere with the nuclear translocation of Rev, accelerated the degradation of the wild-type Rev protein. Differential degradation of Rev in the nucleus and cytoplasm may play a critical role in determining and maintaining different stages of human immunodeficiency virus type 1 infection, in conjunction with the shuttling properties of the Rev protein.     

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.     

Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins

The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev-RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C(2)H(2)-type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (K(d)'s) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the alpha-helical portion of the zinc fingers and target the bulge region of RREIIB-TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements.     

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.     

Circularization of human immunodeficiency virus type 1 DNA in vitro.

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Two immunodominant domains of gp41 bind antibodies which enhance human immunodeficiency virus type 1 infection in vitro.

Four of eight human monoclonal antibodies (huMAbs) to gp41 were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp41 transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp41 which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection.     

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.     

cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.