Nuclear Export Of Viral mRNAs Research Articles

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle.

Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways

The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin α/β-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways.

Implications of the HIV-1 Rev dimer structure at 3.2 Å resolution for multimeric binding to the Rev response element

HIV-1 Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process Rev oligomerizes in association with a highly structured RNA motif, the Rev response element. Crystallographic studies of Rev have been hampered by the protein's tendency to aggregate, but Rev has now been found to form a stable soluble equimolar complex with a specifically engineered monoclonal Fab fragment. We have determined the structure of this complex at 3.2 A resolution. It reveals a molecular dimer of Rev, bound on either side by a Fab, where the ordered portion of each Rev monomer (residues 9-65) contains two coplanar alpha-helices arranged in hairpin fashion. Subunits dimerize through overlapping of the hairpin prongs. Mating of hydrophobic patches on the outer surface of the dimer is likely to promote higher order interactions, suggesting a model for Rev oligomerization onto the viral RNA.

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.

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.

Inhibition of HIV-1 Gene Expression by a Fragment of hnRNP U

Cellular proteins are now appreciated as critically involved in all steps of the human immunodeficiency virus type 1 (HIV-1) life cycle, and disrupting host functions essential for virus replication may provide novel antiviral approaches. Selection from a human complementary DNA (cDNA) library for clones able to induce resistance to infection by recombinant HIV-1 genomes resulted in the identification of a gene fragment that potently restricts HIV-1 activity. The active cDNA encodes an N-terminal fragment of the heterogeneous nuclear ribonuclear protein U (hnRNP U). The gene fragment specifically targets the 3' long terminal repeat (3'LTR) in the viral mRNA and blocks the cytoplasmic accumulation of HIV-1 mRNAs. The results suggest that HIV-1 requires machinery for the nuclear export of viral mRNAs that can be specifically blocked by an interfering gene.

RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA

The human cytomegalovirus protein pUL69 belongs to a family of regulatory factors that is conserved within the Herpesviridae and includes the proteins ICP27 of herpes simplex virus type 1 and EB2 of Epstein–Barr virus. ICP27 and EB2 have been shown to facilitate the nuclear export of viral mRNAs via interacting with the cellular mRNA export factor REF. Furthermore, direct RNA-binding of these proteins was found to be essential for their stimulating effects on mRNA export. Recently, we demonstrated that pUL69 shares common features with ICP27 and EB2 such as (i) nucleocytoplasmic shuttling and (ii) stimulation of nuclear RNA export via binding to the cellular mRNA export machinery. Here, we demonstrate that pUL69 can also interact with RNA both in vivo and in vitro via a complex N-terminal RNA-binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-binding domain of pUL69 overlaps with both the NLS and the binding site of the cellular mRNA export factors UAP56 and URH49. While the deletion of the UAP56/URH49-binding site abolished pUL69-mediated RNA export, an RNA-binding deficient pUL69 mutant which still interacts with UAP56/URH49 retained its RNA export activity. This surprising finding suggests that, in contrast to its homologues, RNA-binding is not a prerequisite for pUL69-mediated nuclear RNA export.

Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo.

In individuals with human immunodeficiency virus type 1 (HIV-1) infection, a small reservoir of resting memory CD4(+) T lymphocytes carrying latent, integrated provirus persists even in patients treated for prolonged periods with highly active antiretroviral therapy (HAART). This reservoir greatly complicates the prospects for eradicating HIV-1 infection with antiretroviral drugs. Therefore, it is critical to understand how HIV-1 latency is established and maintained. In particular, it is important to determine whether transcriptional or posttranscriptional mechanisms are involved. Therefore, HIV-1 DNA and mRNAs were measured in highly purified populations of resting CD4(+) T lymphocytes from the peripheral blood of patients on long-term HAART. In such patients, the predominant form of persistent HIV-1 is latent integrated provirus. Typically, 100 HIV-1 DNA molecules were detected per 10(6) resting CD4(+) T cells. Only very low levels of unspliced HIV-1 RNA ( approximately 50 copies/10(6) resting CD4(+) T cells) were detected using a reverse transcriptase PCR assay capable of detecting a single molecule of RNA standard. Levels of multiply spliced HIV-1 RNA were below the limit of detection (<50 copies/10(6) cells). Only 1% of the HIV-1 DNA-positive lymphocytes in this compartment could be induced to up-regulate HIV-1 mRNAs after cellular activation, indicating that most of the proviral DNA in resting CD4(+) T cells either carries intrinsic defects precluding transcription or is subjected to transcriptional control mechanisms that preclude high-level production of multiply spliced mRNAs. Nevertheless, by inducing T-cell activation, it is possible to isolate replication-competent virus from resting CD4(+) T lymphocytes of all infected individuals, including those on prolonged HAART. Thus, a subset of integrated proviruses (1%) remains competent for high-level mRNA production after cellular activation, and a subset of these can produce infectious virus. Measurements of steady-state levels of multiply spliced and unspliced HIV-1 RNA prior to cellular activation suggest that infected resting CD4(+) T lymphocytes in blood synthesize very little viral RNA and are unlikely to be capable of producing virus. In these cells, latency appears to reflect regulation at the level of mRNA production rather than at the level of splicing or nuclear export of viral mRNAs.

Recruitment of the Crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mRNAs.

Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

Minimal Rev-response element for type 1 human immunodeficiency virus

Rev protein regulates nuclear export of viral mRNAs that contain a 240-base RNA sequence termed the Rev-response element (RRE). We demonstrate that an 88-base truncated RRE encompassing a known Rev binding site can mediate Rev responsiveness in vivo. Two tandem copies of this mutant function as efficiently as the full-length RRE.