Transcriptional and Posttranscriptional Regulation of HIV-1 Gene Expression

Immediately after infection, HIV-1 produces only short completely spliced mRNAs encoding Tat and Rev.

“Immediately after infection, HIV-1 produces only short completely spliced mRNAs encoding the viral regulatory proteins Tat and Rev.”

Tat stimulates transcription elongation by recruiting the cellular elongation factor P-TEFb to nascent RNA polymerases.

“Tat stimulates transcription elongation by directing the cellular transcriptional elongation factor P-TEFb to nascent RNA polymerases.”

There is a strict correlation between TAR RNA binding to Tat in vitro and the ability of TAR sequences to support transactivation.

“Although there is a strict correlation between the ability of TAR RNA to bind to Tat in vitro and the ability of these sequences to support transactivation (Churcher et al. 1993), mutations in the apical loop of the TAR element that do not interfere with Tat binding also interfere with transactivation (Feng and Holland 1988).”

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Regulatory feedback mechanisms driven by Tat and Rev cause HIV-1 transcription to proceed through distinct phases.

“The regulatory feedback mechanisms driven by HIV-1 Tat and Rev ensure that HIV-1 transcription proceeds through distinct phases.”

The larger incompletely spliced HIV-1 mRNAs encode Env and the accessory proteins Vif, Vpr, and Vpu.

“These encode Env and the HIV-1 accessory genes Vif, Vpr, and Vpu.”

P-TEFb is a critical cofactor for Tat activation of elongation.

“In parallel, the search for the “loop factor” and other cofactors for Tat also pointed to P-TEFb as a critical cofactor for Tat activation of elongation.”

After integration into the host genome, the HIV-1 provirus acts as a transcription template regulated at transcriptional and posttranscriptional levels.

“A fter integration into the host genome, the HIV-1 provirus acts as a transcription template that is regulated at the transcriptional and posttranscriptional levels.”

Tat recognition of TAR requires conformational changes in the RNA structure.

“Detailed analysis of Tat's interactions with TAR RNA by NMR subsequently revealed that Tat recognition of TAR requires conformational changes in the RNA structure (Fig. 1B).”

When cells are not fully activated, low amounts of Tat and Rev strongly prevent premature virus production.

“In cells that are not fully activated, limiting levels of Tat and Rev act as potent blocks to premature virus production.”

Tat is inactive in murine cells because murine CycT1 differs from human CycT1 by a single substitution of cysteine 261 for tyrosine.

“Tat is inactive in murine cells, because the murine CycT1 sequence differs from the human sequence by a single substitution of cysteine 261 for tyrosine.”

TAK, the Tat-associated kinase, binds tightly and specifically to Tat.

“Rice and his colleagues (Herrmann and Rice 1995;Herrmann et al. 1996) showed that a protein kinase complex, which they called TAK (Tat-associated kinase), binds tightly and specifically to Tat.”

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Mutations in the apical loop of TAR that do not interfere with Tat binding also interfere with transactivation.

“Although there is a strict correlation between the ability of TAR RNA to bind to Tat in vitro and the ability of these sequences to support transactivation (Churcher et al. 1993), mutations in the apical loop of the TAR element that do not interfere with Tat binding also interfere with transactivation (Feng and Holland 1988).”

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CycT1 is essential for Tat activity.

“Second, persuasive genetic evidence showed that CycT1 is essential for Tat activity.”

Expression of reporter genes under control of the HIV-1 LTR depends on a transactivating factor named Tat.

“The first evidence that gene expression in HIV-1 also requires viral transacting factors came from experiments by Sodroski et al. (1985a,b) who noted that the expression of reporter genes placed under the control of the viral LTR was dependent on a transactivating factor, which they named Tat.”

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Complex formation between Tat, P-TEFb, and TAR requires both the Tat binding site and the TAR loop sequence.

“Crucially, for a putative “loop factor,” complex formation between Tat, P-TEFb, and TAR requires both the Tat binding site and the loop sequence.”

P-TEFb is required for Tat-mediated transactivation.

“After these seminal biochemical observations, additional genetic and biochemical evidence showed unequivocally that P-TEFb is required for Tat-mediated transactivation.”

Rev is required for nuclear export of unspliced and incompletely spliced HIV-1 mRNAs that encode structural proteins.

“Rev is required for the transport from the nucleus to the cytoplasm of the unspliced and incompletely spliced mRNAs that encode the structural proteins of the virus.”

Tat activity requires the TAR regulatory element located downstream from the transcription initiation site between nucleotides +1 and +59.

“Deletion analysis of the viral LTR showed that Tat activity required the transactivation-responsive region (TAR), a regulatory element located downstream from the initiation site for transcription between nucleotides þ1 and þ59 (Fig. 1A).”

Binding of Tat to P-TEFb induces conformational changes in CDK9 that constitutively activate the enzyme.

“The binding of Tat to P-TEFb induces significant conformational changes in CDK9 that constitutively activate the enzyme (Wei et al. 1998;Isel and Karn 1999;Tahirov et al. 2010).”

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• cites Isel and Karn 1999Not checked yet

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Tat binding to TAR involves displacement of U23 by an arginine side chain in Tat's basic binding domain, creating a binding pocket in the major groove with the adjacent G26:C39 base pair.

“This refolding process involves displacement of the first residue in the bulge (U23) by one of the arginine side chains present in the basic binding domain of the Tat protein creating a binding pocket for the arginine side chain in the major groove together with the adjacent G26:C39 base pair (Puglisi et al. 1992;Aboul-ela et al. 1995;Brodsky and Williamson 1997;Davidson et al. 2009).”

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• cites Aboul-ela et al. 1995Not checked yet

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• cites Brodsky and Williamson 1997Not checked yet

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Rev transports unspliced and incompletely spliced mRNAs encoding structural proteins from the nucleus to the cytoplasm.

“Rev transports the unspliced and incompletely spliced mRNAs encoding the structural proteins from the nucleus to the cytoplasm.”

Tat activates viral transcription by stimulating elongation from the HIV-1 LTR.

“Tat activates viral transcription by stimulating elongation from the viral long terminal repeat (LTR).”

CDK9 protein kinase inhibitors are selective inhibitors of HIV-1 transcription.

“First, a set of novel CDK-9 protein kinase inhibitors were shown to be selective inhibitors of HIV-1 transcription (Mancebo et al. 1997).”

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HIV-1 gene expression is controlled by two viral regulatory proteins, Tat and Rev.

“Control of HIV-1 gene expression depends on two viral regulatory proteins, Tat and Rev.”

The HIV-1 LTR includes multiple upstream DNA regulatory elements that serve as binding sites for cellular transcription initiation factors.

“The HIV-1 LTR includes multiple upstream DNA regulatory elements that serve as binding sites for cellular transcription initiation factors (Rittner et al. 1995).”

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Proviruses assemble an ordered nucleosomal structure surrounding the promoter.

“As originally shown by Verdin et al. (1993), proviruses assemble an ordered nucleosomal structure surrounding the promoter.”

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HIV-1 preferentially integrates into active transcription units that provide a favorable environment for viral transcription.

“When HIV-1 infects cells, it preferentially integrates into active transcription units that provide a favorable environment for viral transcription (Lewinski et al. 2006).”

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Signaling through the viral enhancer is essential to reactivate latent proviruses and support virus replication in primary T-cells, whether stimulated by NF-kB or NFAT.

“Although mutation of the NF-kB sites results in only a modest inhibition of virus growth in most transformed cell lines (Chen et al. 1997), signaling through the viral enhancer is essential to reactivate latent proviruses and support virus replication in primary T-cells, regardless of whether it is stimulated by NF-kB or by NFAT (Alcami et al. 1995;Bosque and Planelles 2008).”

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The transactivation mechanism involves phosphorylation events mediated by Tat-activated P-TEFb that modify both positive and negative cellular elongation factors.

“As described in Figure 2A, the transactivation mechanism involves a complex set of phosphorylation events mediated by the Tat-activated P-TEFb that modify both positive and negative cellular elongation factors.”

Sequestration of P-TEFb in the 7SK RNP complex prevents basal transcriptional activation by Tat-independent recruitment of P-TEFb to the provirus.

“The sequestration of P-TEFb in the 7SK RNP complex effectively prevents any basal transcriptional activation by Tat-independent recruitment of P-TEFb to the provirus.”

In the absence of Tat, HIV-1 transcription elongation is highly restricted by the negative elongation factor NELF.

“In the absence of Tat, HIV-1 transcription elongation is highly restricted by the negative elongation factor NELF (Yamaguchi et al. 1999;Narita et al. 2003;Zhang et al. 2007).”

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The HIV-1 LTR is an extremely efficient promoter capable of supporting higher transcription levels than the adenovirus major late promoter or the CMV immediate early promoter.

“As a result, the HIV-1 LTR is an extremely efficient promoter that is capable of supporting even higher levels of transcription than the adenovirus major late promoter or the CMV immediate early promoter.”

Tat overcomes the 7SK RNP barrier by disrupting the complex through competition with HEXIM for CycT1 binding.

“Tat overcomes this barrier by disrupting the 7SK RNP complex by competing with HEXIM for CycT1 binding (Barboric et al. 2007;Sedore et al. 2007;Krueger et al. 2010).”

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• cites Krueger et al. 2010Not checked yet

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Mutation of the NF-kB sites results in only modest inhibition of virus growth in most transformed cell lines.

“Although mutation of the NF-kB sites results in only a modest inhibition of virus growth in most transformed cell lines (Chen et al. 1997), signaling through the viral enhancer is essential to reactivate latent proviruses and support virus replication in primary T-cells, regardless of whether it is stimulated by NF-kB or by NFAT (Alcami et al. 1995;Bosque and Planelles 2008).”

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Transcription from latent proviruses is typically restricted by high levels of HDACs, deacetylated histones, methylated histones, and DNA methylation.

“Typically, transcription from latent proviruses is restricted by high levels of histone deacetylases (HDACs), deacetylated histones, methylated histones, and DNA methylation (for reviews, see Margolis 2010; Karn 2011).”

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Tat:P-TEFb phosphorylates the RNAP II CTD during elongation, creating a hyperphosphorylated RNA polymerase enriched for phosphorylated Ser2 residues.

“Cell-free transcription studies have shown that Tat:P-TEFb also phosphorylates the RNAP II CTD during elongation (Isel and Karn 1999;Kim et al. 2002).This reaction creates a hyperphosphorylated form of the RNA polymerase that is highly enriched for phosphorylated Ser2 residues in the CTD (Ramanathan et al. 2001;Kim et al. 2002).”

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In actively replicating cells such as HeLa and Jurkat T-cells, P-TEFb activity is tightly regulated and most of the enzyme is sequestered in an inactive 7SK RNP complex comprising 7SK RNA and RNA-binding proteins.

“In actively replicating cells, such as HeLa cells and Jurkat T-cells, P-TEFb activity is tightly regulated and the majority of the enzyme is sequestered into a large inactive 7SK RNP complex comprising 7SK RNA and a series of RNA-binding proteins (Fig. 2B) (Nguyen et al. 2001;Yang et al. 2001).”

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Because NF-kB and NFAT recognition sequences overlap, their binding to the HIV-1 motifs is mutually exclusive.

“Because their recognition sequences overlap, binding of these factors is mutually exclusive (Chen-Park et al. 2002;Giffin et al. 2003).”

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Activation of P-TEFb in primary resting central memory T-cells and monocytes requires multiple steps involving initial assembly of the 7SK RNP complex and its relocalization to nuclear speckles where it becomes accessible to Tat and the transcription machinery.

“Activation of P-TEFb in these cells therefore requires multiple steps involving both the initial assembly of the 7SK RNP complex and its relocalization to nuclear speckles where it becomes accessible to Tat and the rest of the transcription machinery.”

Any model for the stimulatory effects of P-TEFb on HIV-1 transcription must account for ELL2 and possibly additional elongation factors.

“Thus, any model for the stimulatory effects of P-TEFb on HIV-1 transcription now has to take into account the role of ELL2 and possibly several additional elongation factors.”

Binding of NF-kB to the HIV-1 motifs is more efficient than NFAT because it is enhanced by cooperative interactions with Sp1.

“Binding of NF-kB is more efficient than NFAT because it is enhanced by cooperative interactions with Sp1 (Perkins et al. 1993).”

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HIV-1 relies on an enhancer region containing two NF-kB binding motifs.

“In addition to the core promoter, HIV-1 relies on an “enhancer region” that contains two NF-kB binding motifs (Nabel and Baltimore 1987) (see online Movie 2 at www.per spectivesinmedicine.org).”

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Stochastic fluctuations in Tat gene expression can act as a molecular switch.

“Insightful studies by Weinberger et al. (Weinberger et al. 2005;Weinberger and Shenk 2006) and Burnett et al. (2009) have emphasized how stochastic fluctuations in Tat gene expression can act as a molecular switch.”

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Each core promoter element participates in cooperative binding of the initiation factor TFIID and its associated TAF cofactors to the TATA element.

“Each of these elements participates in the cooperative binding of the initiation factor TFIID and its associated TAF cofactors to the TATA element (Rittner et al. 1995).”

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Proteomic studies identified large protein complexes containing Tat, P-TEFb, and the human transcription factors/coactivators AFF4, ENL, AF9, and ELL2.

“Our picture of how Tat and P-TEFb stimulate HIV-1 elongation has recently been refined by two proteomic studies that identified large protein complexes containing Tat P-TEFb and the human transcription factors/coactivators AFF4, ENL, AF9, and ELL2 (Fig. 2B) (He et al. 2010;Sobhian et al. 2010).”

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P-TEFb phosphorylates Spt5, a subunit of DSIF that carries a CTD homologous to the RNAP II CTD.

“In addition to targeting RNAP II, P-TEFb is also able to extensively phosphorylate Spt5, a subunit of the DRB sensitivityinducing factor (DSIF), which carries a CTD homologous to the RNAP II CTD (Ivanov et al. 2000;Bourgeois et al. 2002).”

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HIV-1 and many other retroviruses, including HTLV-1 and HTLV-II, express regulatory factors that facilitate transport of intron-containing viral RNA out of the nucleus.

“To circumvent these surveillance mechanisms, HIV-1 and many other retroviruses, including the human T-cell leukemia viruses HTLV-1 and HTLV-II, express regulatory factors that facilitate the transport of intron-containing viral RNA out of the nucleus.”

A few HIV-1 mRNA isoforms are produced by inclusion of exons flanked by 3' splice site A1 and 5' splice site D2 (exon 2) and/or the exon flanked by 3' splice site A2 and 5' splice site D3 (exon 3).

“Adding to the complexity of the mRNA species present in infected cells, a few viral mRNA isoforms are also produced by inclusion of exons flanked by 3 0 ss A1 and 5 0 ss D2 (exon 2) and/or the exon flanked by 3 0 ss A2 and 5 0 ss D3 (exon 3).”

This RNA helix change lets the Rev arginine-rich domain enter the major groove.

“This change in the structure of the RNA helix allows binding of the Rev ARD to the major groove (Battiste et al. 1996;Daugherty et al. 2008).”

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All HIV-1 mRNAs include the highly structured noncoding exon 1 extending from the 5' cap to 5' splice site D1.

“As a result, all HIV-1 mRNAs include the highly structured noncoding exon 1 that extends from the 5 0 cap to 5 0 ss D1.”

The 9-kb and 4-kb HIV-1 transcripts that encode Gag, Pol, and Env depend on Rev for export and expression.

“Initial studies showed that the 9-kb and 4-kb HIV-1 mRNA species, which encode the structural proteins Gag, Pol, and Env, require Rev for their transport and expression.”

Control of HIV-1 splicing involves exonic splicing enhancers and intronic splicing enhancers that facilitate splice site recognition and are selectively bound by SR proteins.

“Control of splicing in HIV-1 involves exonic splicing enhancers (ESEs) and intronic splicing enhancers (ISEs) which facilitate splice site recognition and are selectively bound by members of the SR (Ser-Arg) protein family.”

Formation of HIV-1 splicing complexes is affected by the strengths of various downstream 5' splice sites, an expected consequence of exon definition.

“Formation of HIV-1 splicing complexes is also affected by the strengths of the various downstream 5 0 ss-an expected consequence of exon definition (Robberson et al. 1990;Hoffman and Grabowski 1992).”

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The distinct export requirements result from removal of the env segment spanning splice sites D4 and A7, including the RRE, during complete splicing.

“This division of transport mechanisms is achieved because the region of the HIV-1 env gene between 5 0 ss D4 and 3 0 ss A7 that contains the RRE is removed in the completely spliced mRNAs (Figs. 3 and 4).”

Rev-dependent export requires Rev to bind the RRE.

“Rev-regulated transport requires Rev binding to the RRE.”

HIV-1 Rev interacts with a highly structured RNA element in the env gene called the Rev-responsive element (RRE).

“The first of these factors to be discovered was the HIV-1 Rev protein which interacts with a highly structure RNA element in the env gene referred to as the Rev-responsive element (RRE) (Sodroski et al. 1986;Malim et al. 1989).”

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The fully spliced 1.8-kb HIV-1 transcripts encoding Tat, Rev, and Nef leave the nucleus without Rev via a cellular export route used by host mRNAs.

“On the other hand, the completely spliced 1.8-kb mRNAs, which encode Tat, Rev, and Nef, are exported to the cytoplasm in the absence of Rev by an endogenous cellular pathway used by cellular mRNAs.”

Analysis of HIV-1 mRNA species in virus-infected cells showed striking differences in the relative abundances of different viral mRNA species.

“Analysis of HIV-1 mRNA species in virusinfected cells showed that there are striking differences in the relative abundances of the different viral mRNA species (Purcell and Martin 1993).”

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Maximum HIV-1 replication requires tight regulation of splicing to balance mRNA and genome RNA production.

“The dramatic effects of these splicing element mutations show that maximum virus replication requires tight regulation of splicing to balance mRNA and genome RNA production.”

Splicing at the relatively weak 3' splice site A1, required for high vif mRNA expression and inclusion of noncoding exon 2, is facilitated by several ESEs within exon 2.

“Splicing at the relatively weak 3 0 ss A1, which is required for high vif mRNA expression and inclusion of the noncoding exon 2, is facilitated by several different ESEs (ESE-Vif, ESE M1, and ESE M2) within exon 2 (Kammler et al. 2006;Exline et al. 2008).”

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GAR ESE activity raises levels of incompletely spliced env/vpu mRNAs and completely spliced nef and rev mRNAs to the point that they become the most abundant spliced mRNA species in the HIV-1-infected cell.

“GAR ESE activity is able to raise the levels of incompletely spliced env/vpu mRNAs and completely spliced nef and rev mRNAs to the point that they become the most abundant spliced mRNA species in the HIV-1-infected cell (Purcell and Martin 1993).”

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Splicing at the weak 3' splice sites A4c, A4a, A4b, and A5 is greatly facilitated by a guanosine-adenosine-rich ESE, GAR, within exon 5.

“By contrast, splicing at the weak 3 0 ss A4c, A4a, A4b, and A5 sites is greatly facilitated by a guanosine-adenosine-rich ESE (GAR) within exon 5.”

HIV-1 primary transcripts undergo extensive and complex alternative splicing in the nucleus of infected cells.

“To produce the full range of mRNAs needed to encode the viral proteins, HIV-1 primary transcripts undergo extensive and complex alternative splicing in the nucleus of infected cells (Fig. 3).”

The excessive splicing phenotype caused by ESSV mutation resulted in decreased virus replication.

“This excessive splicing phenotype resulted in a decrease in virus replication (Madsen and Stoltzfus 2005).”

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Small changes in initiation rates, mimicked by mutations in NF-kB and Sp1 binding sites, reduce Tat availability and disproportionately limit HIV-1 transcription, forcing viruses into latency.

“Small changes in initiation rates, which can be experimentally mimicked by introducing mutations into the NF-kB and Sp1 binding sites, are able to reduce Tat availability and disproportionately limit HIV-1 transcription, forcing viruses into latency (Burnett et al. 2009).”

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Because of reduced Vif levels, replication of virus mutants with decreased U1 snRNP affinity exhibits greater sensitivity than wild-type virus to inhibition by APOBEC3G.

“Because of reduced levels of Vif, the replication of these virus mutants exhibit greater sensitivity than wild-type virus to inhibition by the cellular restriction factor APOBEC3G.”

Several other retroviruses, as originally shown for Mason-Pfizer monkey virus, dispense with a protein factor and encode cis elements called constitutive transport elements that directly interact with cellular RNA export factors.

“Several other retroviruses, as originally shown for Mason-Pfizer monkey virus (MPMV), dispense with a protein factor and simply encode cis elements, referred to as constitutive transport elements or CTEs, that directly interact with cellular RNA export factors (for reviews of HIV-1 and MPMV RNA export, see Pollard and Malim 1998;Cullen 2003).”

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Splicing at 3' splice site A2 is repressed by an ESS element within exon 3, ESSV, resulting in relatively low levels of vpr mRNA.

“Similarly, splicing at 3 0 ss A2 is repressed by an ESS element within exon 3 (ESSV), which results in relatively low levels of vpr mRNA (Bilodeau et al. 2001).”

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HIV-1 remains poised to resume replication in response to triggers that stimulate transcription initiation and restore Tat levels.

“However, the virus remains poised to resume its replication in response to triggers that stimulate transcription initiation and restore Tat levels.”

In each spliced HIV-1 mRNA, the 5' splice site D1 is spliced to one of the 3' splice sites.

“In each of the spliced mRNAs, the 5 0 ss D1 (sometimes referred to as the “major splice donor”) is spliced to one of the 3 0 ss.”

Rev binding at the high-affinity RRE site triggers a conformational change that creates two purine-purine non-Watson-Crick base pairs.

“NMR studies have shown that, as in the case of the Tat-TAR interaction, the Rev binding to the high affinity site induces a conformational change that results in the formation of two purine-purine non-Watson-Crick base pairs (Fig. 5A).”

HIV-1 3' splice sites are generally relatively inefficient compared to constitutive cellular 3' splice sites.

“In general, HIV-1 3 0 splice sites are relatively inefficient in comparison to constitutive cellular 3 0 splice sites (for reviews of HIV-1 splicing, see Stoltzfus and Madsen 2006;Stoltzfus 2009).”

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Most HIV-1 strains use four different 5' splice sites and eight different 3' splice sites to produce more than 40 different spliced mRNA species in infected cells.

“Most HIV-1 strains use four different splice donor or 5 0 splice sites (5 0 ss) and eight different acceptor or 3 0 splice sites (3 0 ss) to produce more than 40 different spliced mRNA species in infected cells.”

Rev first attaches to a high-affinity site at the RRE apex, stem IIB.

“Rev binds initially to a high affinity site located near the apex of the RRE structure (stem IIB) (Daly et al. 1989;Heaphy et al. 1990).”

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The RRE is a 351-nucleotide elongated stem-loop.

“The RRE is an elongated stemloop structure of 351 nt (Malim et al. 1989;Mann et al. 1994;Watts et al. 2009).”

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After the initial high-affinity RRE contact, more Rev monomers join the complex.

“Binding of Rev to the high affinity RRE site is then followed by binding of additional monomers to the complex (Malim and Cullen 1991;Zapp et al. 1991;Mann et al. 1994).”

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The HIV-1 U3 sequence upstream of the 3' processing signal, but not linked to the 5' processing signal, has upstream enhancer elements that help CPSF bind and increase 3'-end polyadenylation.

“First, the HIV-1 U3 sequence, which is upstream of the 3 0 processing signal, but not associated with the 5 0 processing signal, contains upstream enhancer elements (USE) that act to facilitate binding of CPSF and enhance polyadenylation at the 3 0 end of the HIV-1 transcripts (Gilmartin et al. 1995).”

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Which export route is used strongly affects the cytoplasmic fate of unspliced HIV-1 RNA.

“In contrast to cellular mRNAs, the cytoplasmic fate of unspliced HIV-1 RNA appears to be strongly influenced by the choice of RNA export pathway.”

Factors that bind upstream of the AAUAAA site are thought to open the poly(A) hairpin and favor use of the 3' LTR poly(A) site.

“Factors binding to sequences upstream of the AAUAAA site are believed to open up the poly(A) hairpin and allow preferential use of the 3 0 LTR poly(A) processing site (Das et al. 1999).”

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HIV-1 directs processing to the 3' LTR cleavage site using multiple regulatory elements.

“HIV-1 uses multiple regulatory elements to direct processing to the 3 0 LTR cleavage site.”

HIV-1 carries two copies of AAUAAA and GU-rich core elements at the R sequence ends in both LTRs.

“Like most retroviruses, HIV-1 contains a duplicated set of AAUAAA and GU-rich core elements at the ends of the R sequences found in both the 5 0 and 3 0 LTRs.”

With excess Rev, a defined RNP complex containing three dimers bound to the RRE forms.

“When excess Rev is present, a defined RNP complex of three dimers bound to the RRE is formed (Fig. 5C) (Daugherty et al. 2010).”

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Mutations in 5'ss D1 that reduce U1 snRNP binding allow use of the normally silent 5' LTR poly(A) site.

“Mutations of 5 0 ss D1 that weaken binding of U1 snRNP allow the usage of the normally silent 5 0 LTR poly(A) site (Ashe et al. 1995(Ashe et al. , 2000)).”

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HIV-1 RNA exits the nucleus through nuclear pore complexes.

“Transport of HIV-1 RNA out of the nucleus, as with most cellular proteins and RNAs, occurs via the nuclear pore complexes (NPC) (Fig. 5D) (for review see Kohler and Hurt 2007).”

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CRS/INS elements placed in heterologous constructs give Rev regulation to RNAs made from those constructs.

“These elements can be introduced into heterologous expression constructs and confer Rev-regulation to RNAs produced from these constructs (Schwartz et al. 1992;Najera et al. 1999).”

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• cites Najera et al. 1999Not checked yet

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A second protein-protein interface enables Rev oligomers to serve as an adaptor to the export machinery, with RNA on one side and Crm1 on the other.

“A second protein -protein interface permits Rev oligomers to act as an adaptor to the host export machinery, with viral RNA bound to one face and Crm1 to another.”

Some HIV-1 mRNA UTRs have AUGs upstream of the true initiator AUG that can interfere with initiation at the true AUG.

“Furthermore, some of the HIV-1 mRNA UTRs contain AUG sequences upstream of the authentic initiator AUG that can interfere with translation initiation at the authentic AUG.”

Rev returns to the nucleus by binding importin-b.

“Rev then reenters the nucleus by binding to the nuclear import factor, importin-b (Henderson and Percipalle 1997).”

• cites Henderson and Percipalle 1997Not checked yet

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Because HIV-1 exon 1 has several structured elements-TAR, the primer-binding site, the poly(A) hairpin, and packaging signals-ordinary ribosomal scanning for initiation is blocked.

“Because the HIV-1 exon 1 contains multiple highly structured regions including the TAR sequence, the primer binding site, the poly(A) hairpin, and RNA packaging sequences, a typical ribosomal scanning mechanism for translational initiation is precluded.”

RRE-bound Rev contacts Crm1 via a 10-residue leucine-rich nuclear export signal near its carboxyl terminus.

“Rev bound to the RRE interacts with the karyopherin family member Crm1 (also referred to as exportin 1) through an 10 amino acid leucine-rich export nuclear export signal (NES) near the Rev carboxyl terminus.”

U1 snRNP inhibits the 5' LTR 3' processing and poly(A) site by binding adjacent 5'ss D1.

“Finally, the splicing factor U1 snRNP acts to inhibit the 3 0 processing and poly(A) site in the 5 0 LTR by binding to the adjacent 5 0 ss D1.”

The 5' and 3' LTR poly(A) processing sites are embedded in a poly(A) hairpin in exon 1 just downstream of TAR.

“Second, the 5 0 and 3 0 LTR poly(A) processing sites are imbedded in a region of secondary structure called the poly(A) hairpin located in exon 1 immediately downstream from the TAR hairpin structure.”

In human cells, HIV-1 assembly is defective when gag-pol mRNA export relies on the hepatitis B posttranscriptional element.

“Similarly, defective HIV-1 assembly occurred in human cells when export of gag-pol mRNA is dependent on the presence of the hepatitis B posttranscriptional element (PRE) (Jin et al. 2009).”

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Rev regulation depends on accumulation of a nuclear pool of unspliced and incompletely spliced mRNAs.

“Rev regulation requires accumulation of a pool of unspliced and incompletely spliced mRNAs in the nucleus.”

Several cellular RNA-binding proteins, including PABP1, hnRNP A1, and the PSF/p54(nrb) heterodimer, have been linked to CRS/INS-mediated retention.

“A number of different cellular RNA-binding proteins have been implicated in the retention mediated by CRS/INS elements including poly(A)-binding protein 1 (PABP1), heterogeneous ribonuclear protein A1 (hnRNP A1), and the heterodimer of two related proteins polypyrimidine tract binding proteinassociated splicing factor (PSF) and p54(nrb) (Black et al. 1996;Afonina et al. 1997;Najera et al. 1999;Zol”

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• cites Afonina et al. 1997Not checked yet

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• cites Najera et al. 1999Not checked yet

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In the Rev dimer, the arginine-rich RNA-binding helices sit at the ends of a V-shaped assembly.

“In the Rev dimer, the arginine-rich RNAbinding helices are located at the ends of a V-shaped assembly.”

After nuclear export, RanGAP and RanBP1 promote hydrolysis of bound GTP to GDP.

“After export to the cytoplasm through the NPC, the bound GTP is hydrolyzed to GDP facilitated by the proteins RanGAP (Ran GTPase-activating protein) and RanBP1.”

A USE element near the 5' end of Nef binds SR protein 9G8, which recruits CF1m and CPSF.

“Another USE element near the 5 0 end of the Nef gene binds the cellular SR protein 9G8 which recruits the 3 0 processing factor CF1m and CPSF (Valente et al. 2009).”

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One mechanism that skips the vpu ORF and allows efficient translation at the downstream env AUG is 5' cap-dependent ribosome shunting.

“An additional mechanism, which is used to bypass the vpu open reading frame and permit efficient translation at the downstream env AUG, involves 5 0 cap-dependent ribosome shunting in which the scanning ribosome jumps over large regions of the mRNA before recognizing the correct initiation codon (Krummheuer et al. 2007).”

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Pausing by itself seems insufficient for frameshifting, since other translation roadblocks do not support it.

“Pausing alone appears to be insufficient for frameshifting because addition of other roadblocks to translation are unable to support frameshifting (Brierley and Dos Ramos 2006).”

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HIV-1 and other retroviruses have posttranscriptional elements that bind cellular RNA-binding proteins and can enhance translation initiation.

“Additionally, HIV-1 and other retroviruses contain posttranscriptional elements (PCEs) that bind to cellular RNA binding proteins that can act as enhancers to facilitate translation initiation.”

The actions of Tat and Rev, unprecedented when discovered, have revealed previously unknown and unexpected features of transcription elongation, splicing, and RNA export.

“The mechanisms of action of both proteins, which were unprecedented at the time of their discovery, have now illuminated features of transcription elongation control, RNA splicing, and RNA export that were entirely unknown and unexpected.”

Two essential cis-acting elements 200 nt upstream of the Gag stop codon are needed for HIV-1 frameshifting.

“Two essential cis-acting sequence elements located 200 nt upstream of the Gag termination codon are required for frameshifting.”

HIV-1 gene expression is governed by Tat and Rev, RNA-binding proteins that coordinate interactions with host transcription, splicing, and RNA transport machinery.

“As summarized above, gene expression in HIV-1 is controlled by the RNA-binding proteins Tat and Rev, which orchestrate complex interactions with the cellular transcription, RNA splicing, and RNA transport machinery.”

HIV-1, like all retroviruses, uses programmed frameshifting, in which mRNA sequence and structure signals specify a reading-frame change during translation.

“In common with all other retroviruses, HIV-1 has evolved a novel mechanism of programmed frameshifting in which specific sequence and structural signals in the mRNA can specify an mRNA reading frame change during translation (for review, see Brierley and Dos Ramos 2006;Bolinger and Boris-Lawrie 2009).”

• cites Brierley and Dos Ramos 2006Not checked yet

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• cites Bolinger and Boris-Lawrie 2009Not checked yet

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In HIV-1, a -1 reading-frame shift is needed to move from Gag to the pro and pol frame.

“In HIV-1, a 21 shift in the translational reading frame is required to shift from the Gag reading frame to the pro and pol reading frame.”

A growing set of host nuclear RNA-binding proteins bind HIV-1 RNA or Rev and may affect export and cytoplasmic use.

“In addition, there have been a growing number of host cell nuclear RNA binding proteins identified that bind to HIV-1 RNA or to Rev and may influence its export and cytoplasmic utilization (for reviews, see Cochrane et al. 2006;Cochrane 2009;Suhasini and Reddy 2009).”

• cites Cochrane et al. 2006Not checked yet

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• cites Cochrane 2009Not checked yet

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• cites Suhasini and Reddy 2009Not checked yet

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The export route taken by HIV-1 RNA influences viral assembly at the plasma membrane.

“Remarkably, the export pathway used by the RNA affects viral assembly at the plasma membrane (Jin et al. 2009;Sherer et al. 2009), suggesting the site of translation influences subsequent protein function.”

• cites Jin et al. 2009Not checked yet

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• cites Sherer et al. 2009Not checked yet

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The location of translation affects later protein function.

“suggesting the site of translation influences subsequent protein function.”