Human Immunodeficiency Virus Rev Protein Research Articles

Roles of microRNAs and long-noncoding RNAs in human immunodeficiency virus replication.

MicroRNAs (miRNAs) and long-noncoding RNAs (lncRNAs) are involved in many biological processes, including viral replication. In this review, the role of miRNAs and lncRNAs in human immunodeficiency virus (HIV) replication will be discussed. The review focuses on miRNAs that target cellular proteins involved in HIV replication-proteins that mediate steps in the viral life cycle, as well as proteins of the innate immune system that inhibit HIV replication. Given the large number of miRNAs encoded in the human genome, as well as the large number of cellular proteins involved in HIV replication, the number of miRNAs identified to date that affect viral replication are certainly only the 'tip of the iceberg'. The review also discusses two lncRNAs that are involved in HIV gene regulation-7SK RNA and NEAT1 RNA. 7SK RNA is involved in HIV Tat protein stimulation of RNA polymerase II elongation of the integrated provirus, while NEAT1 RNA is involved in HIV Rev protein export of incompletely spliced viral transcripts.

Diverse mutants of HIV RRE IIB recognize wild-type Rev ARM or Rev ARM R35G-N40V.

The binding of human immunodeficiency virus Rev protein via its arginine-rich motif (ARM) to an internal loop in the Rev-response element region IIB (RRE IIB) is necessary for viral replication. Many variant RNAs and ARMs that bind Rev and RRE IIB have been found. Despite the essential role of Rev asparagine 40 in recognition, the Rev ARM double-mutant R35G-N40V functions well in a Rev-RRE IIB reporter assay, indicating R35G-N40V uses a distinct recognition strategy. To examine how RRE IIB may evolve specificity to wild-type Rev ARM and R35G-N40V, 10 RRE IIB libraries, each completely randomized in overlapping regions, were screened with wild-type Rev ARM and R35G-N40V using a reporter system based on bacteriophage λ N antitermination. Consistent with previous studies, a core element of RRE IIB did not vary, and substitutions occurred at conserved residues only in the presence of other substitutions. Notably, the groove-widening, non-canonical base-pair G48:G71 was mutable to U48:G71 without strong loss of binding to wild-type Rev ARM, suggesting U48:G71 performs the same role by adopting the nearly isosteric, reverse wobble base pair. Originating from RRE IIB, as few as one or two substitutions are sufficient to confer specificity to wild-type Rev or Rev R35G-N40. The diversity of RRE IIB mutants that maintain binding to wild-type Rev ARM and R35G-N40V supports neutral theories of evolution and illustrates paths by which viral RNA-protein interactions can evolve new specificities. Rev-RRE offers an excellent model with which to study the fine structure of how specificity evolves.

The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication.

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.

Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging

The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.

Identification of a novel Rev-interacting cellular protein

BackgroundHuman cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies.ResultsYeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev.ConclusionThese results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors.

Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion.

The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

Analysis of nuclear targeting activities of transport signals in the human immunodeficiency virus Rev protein

The human immunodeficiency Rev protein shuttles between the nucleus and cytoplasm, while accumulating to high levels in the nucleus. Rev has a nuclear localization signal (NLS; AA 35–50) with an arginine-rich motif (ARM) that interacts with importin β and a leucine-rich nuclear export signal (NES; AA 75–84) recognized by CRM1/exportin 1. Here we explore nuclear targeting activities of the transport signals of Rev. GFP tagging and quantitative fluorescence microscopy were used to study the localization behavior of Rev NLS/ARM mutants under conditions inhibiting the export of Rev. Rev mutant M5 was actively transported to the nucleus, despite its known failure to bind importin β. Microinjection of transport substrates with Rev–NES peptides revealed that the Rev–NES has both nuclear import and export activities. Replacement of amino acid residues “PLER” (77–80) of the NES with alanines abolished bidirectional transport activity of the Rev–NES. These results indicate that both transport signals of Rev have nuclear import capabilities and that the Rev NLS has more than one nuclear targeting activity. This suggests that Rev is able to use various routes for nuclear entry rather than depending on a single pathway.

Role of Protein Kinase CK2 Phosphorylation in the Molecular Chaperone Activity of Nucleolar Protein B23

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.

Heterogeneous Nuclear Ribonucleoprotein A1 Interferes with the Binding of the Human T Cell Leukemia Virus Type 1 Rex Regulatory Protein to Its Response Element

The human T cell leukemia virus, type 1 (HTLV-1), Rex protein mediates the nuclear export of unspliced and incompletely spliced viral mRNAs. This post-transcriptional activity is dependent in part on the binding of this protein to cis-regulatory sequences termed the Rex-response element (XRE). We have proposed previously that the decreased functionality exhibited by Rex in human lymphoblastoid Jurkat T cells may be linked to alterations in the Rex/XRE interactions. The analysis of the ribonucleoprotein complexes formed between Jurkat nuclear proteins and XRE-RNA led to the identification of a 36-kDa protein as heterogeneous nuclear ribonucleoprotein (hnRNP) A1. In vitro binding assays revealed that hnRNP A1 proteins were found to interfere with the binding of Rex to XRE, whereas nuclear extracts depleted of these proteins were unable to disrupt Rex-XRE complexes. Furthermore, A1 proteins from Jurkat cells were acting in a concentration-dependent manner, suggesting that the amount of these RNA-binding proteins is a critical parameter in controlling Rex activity. We indeed observed a lower level of hnRNP A1 in in vitro HTLV-1-transformed virus-producing T cells than that detected in Jurkat cells. Likewise, overexpression of hnRNP A1 proteins in 293T cells and in Jurkat cells led to a decrease in the expression of a reporter gene dependent on Rex/XRE interactions. Such a decrease was not observed when the expression of the same reporter gene by cells overexpressing hnRNP A1 was dependent on the interactions of human immunodeficiency virus Rev protein with the Rev-response element. These findings indicate that hnRNP A1 by competing with Rex for the formation of REX-XRE complexes is specifically involved in the modulation of the post-transcriptional activity of Rex.

Basic Residues within the Kruppel Zinc Finger DNA Binding Domains Are the Critical Nuclear Localization Determinants of EKLF/KLF-1

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.

Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening

A high throughput scintillation proximity assay with biotinylated human immunodeficiency virus (HIV) Rev protein and tritiated Rev response element RNA was used to screen over 500 000 small molecules. Several chemical classes of inhibitors and two chemical classes of enhancers of binding were identified, with the molecular weight range being 400–600. The most common structural motif of inhibitor was an acidic moiety at the end of a linear aromatic system. Most of these modulators had EC 50 values in the 1–10 μM potency range, with several below 1 μM. Several classes displayed structure–activity relationships suggesting specific molecular interactions between small molecule and macromolecule. Several molecules were confirmed as inhibitors in a gel shift assay and by surface plasmon resonance analysis. Furthermore, one inhibitor was shown to bind the Rev protein with a binding constant equal to its IC 50 value, consistent with the mechanism of inhibition being binding Rev. Thus, small molecules can modulate this macromolecular protein–RNA interaction in vitro. However, no compound demonstrated HIV antiviral activity in a relevant cell-based assay.

Adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway.

Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.

Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene.

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1(SF2) p55(Gag) were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gag protease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

Nucleo-cytoplasmic shuttling of the HDM2 oncoprotein regulates the levels of P53 protein VIA a pathway USED by the human immunodeficiency virus REV protein

Nucleo-cytoplasmic shuttling of the HDM2 oncoprotein regulates the levels of P53 protein VIA a pathway USED by the human immunodeficiency virus REV protein

Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein.

The hdm2 gene is overexpressed in a variety of human tumors. Its gene product localizes predominantly to the nucleus, where it acts as an inhibitor of the p53 tumor suppressor gene product. It is shown here that the hdm2 oncoprotein constantly shuttles between the nucleus and the cytoplasm. Shuttling of hdm2 does not depend on its interaction with p53. Nuclear export of hdm2 is mediated by a signal sequence similar to the nuclear export signal of the rev protein from human immunodeficiency virus and other lentiviruses. Mutation of this signal sequence abolishes detectable nucleo-cytoplasmic shuttling. When fused to a carrier protein, the hdm2 signal sequence can mediate nuclear export after intranuclear microinjection into HeLa cells. The export of hdm2 can be blocked by a competitive inhibitor of rev export, arguing that the export pathways for hdm2 and rev are either overlapping or identical. Inhibition of its export modifies the ability of hdm2 to block p53-mediated transcriptional activation, and hdm2's export function is required to accelerate the degradation of p53. Thus the rev nuclear export pathway may be used to regulate an oncogene product's activity and modulate cellular growth.

Structural variety of arginine-rich RNA-binding peptides.

Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an alpha-helical conformation. Here we show that related peptides from splicing factors, viral coat proteins, and bacteriophage antiterminators (the N proteins) also have propensities to form alpha-helices and that the N peptides require helical conformations to bind to their cognate RNAs. In contrast, introducing proline mutations into the arginine-rich domain of the human immunodeficiency virus Tat protein abolishes its potential to form an alpha-helix but does not affect RNA-binding affinity in vitro or in vivo. Based on results from several peptide-RNA model systems, we suggest that helical peptides may be used to recognize RNA structures having particularly wide major grooves, such as those found near loops or large bulges, and that nonhelical or extended peptides may be used to recognize less accessible grooves.

Costabilization of peptide and RNA structure in an HIV Rev peptide-RRE complex.

An arginine-rich peptide corresponding to amino acids 34-50 of the human immunodeficiency virus Rev protein has been shown to bind specifically to its RNA-binding site (RRE) when the peptide is in an alpha-helical conformation. Mutation of any one of six amino acids (Thr34, Arg35, Arg38, Arg39, Asn40, or Arg44) was shown to strongly decrease specific RNA-binding affinity in vitro, suggesting that these residues may contact specific bases or distinct structural features of the RNA. We now show that the four arginine side chains, and not just their charge, are important for specific binding in vivo, and present evidence that three additional arginines (Arg46, Arg48, and Arg50) may make electrostatic contacts to the RRE. RNA-binding specificity of the Rev peptide is temperature-dependent in vitro, correlating with alpha-helix unfolding. Circular dichroism experiments indicate that the peptide helical structure is stabilized when bound specifically to the RRE and that the RNA undergoes a conformational change upon binding. Because the structures of the peptide and RNA in this model system appear to be mutually stabilized upon binding, it is suggested that the entire complex may be viewed as a single folding unit.

Cationization of a monoclonal antibody to the human immunodeficiency virus REV protein enhances cellular uptake but does not impair antigen binding of the antibody

Replication of the human immunodeficiency virus (HIV) within cells may be blocked by neutralization of viral-specific proteins that are absolutely required for growth of the virus. One such viral-specific protein is REV, and a monoclonal antibody (mAb) against the REV protein is a potential therapeutic for acquired immune deficiency syndrome (AIDS). However, in order to effect ‘intracellular immunization’, mAbs must be enabled to target the intracellular compartment. One strategy for transcellular drug delivery of mAb-based therapeutics is cationization, and the present studies describe the cationization of a murine mAb specific to the REV protein of HIV-1. The isoelectric point (pI) of the mAb was raised from 6.6 to more than 9.5. There was virtually no difference in binding to wild-type REV protein between the native or cationized anti-REV mAb, based on studies with a solid-phase immunoradiometric assay. The uptake of the [ 125I] native anti-REV mAb by human peripheral blood lymphocytes (PBLs) was negligible; however, there was a marked increase in both total cell binding and endocytosis by the human PBLs of the [ 125I] cationized anti-REV mAb. In conclusion, these studies show that an anti-REV mAb may be cationized to markedly increase endocytosis of the antibody and that this cationization reaction does not significantly alter the affinity of the antibody for its target protein. Cationized anti-REV mAbs may allow for intracellular immunization of the virus and are potential therapeutics for the treatment of HIV.

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.

Small molecules that selectively block RNA binding of HIV-1 rev protein inhibit rev function and viral production

Replication of RNA viruses, such as the human immunodeficiency virus (HIV), is dependent upon multiple specific interactions between viral RNAs and viral and cellular proteins. A small molecule that interferes specifically with one or more of these RNA-protein interactions could be an efficacious antiviral agent. Here we show that certain aminoglycoside antibiotics, in particular neomycin B, can block binding of the HIV Rev protein to its viral RNA recognition element. Inhibition appears to be highly selective, resulting from competitive binding of the drug to a small viral RNA region within the Rev-binding site. We further demonstrate that neomycin B can specifically antagonize Rev function in vitro and in vivo and can inhibit production of HIV. Our results establish the feasibility for developing antiviral drugs that act by selectively blocking RNA-protein interactions.