An Exonic Splicing Silencer Downstream of the 3′ Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication

Madsen, Joshua; Stoltzfus, C. Martin

doi:10.1128/jvi.79.16.10478-10486.2005

Cited by 59 publications

(118 citation statements)

References 40 publications

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“…In addition, we observed that the deletion in exon 3 increased splicing efficiency (Fig. 4B), probably due to deletion of previously described splicing inhibitory sequences (10,17). In contrast, the deletion in intron 3 reduced splicing efficiency (Fig.…”

Section: Inhibition Of the 5ј-splice Site Of Exon 3 By Srp55 And Srp7mentioning

confidence: 58%

“…These authors speculate that ASF/SF2 enhances splicing to the 3Ј-splice site of exon 3, either through a yet unidentified splicing enhancer, or by inhibiting a previously identified splicing silencer in exon 3 named ESSV (10,17). This splicing silencer interacts with heterogeneous nuclear ribonucleoprotein A/B (10,17,41), and ASF/SF2 is known to counteract heterogeneous nuclear ribonucleoprotein A1, another member of the heterogeneous nuclear ribonucleoprotein family (42,43). However, ASF/SF2 binds to the GAR enhancer and is necessary for (37,38).…”

Section: Discussionmentioning

confidence: 99%

“…There are several enhancers and silencers on HIV-1 pre-mRNA (16). A silencer in exon 3 called ESSV inhibits the vpr splice acceptor SA3 (10,17). Two silencers in exon 4, named ESS2 and ESS2p, inhibit splicing into exon 4 thereby inhibiting the production of tat mRNA (18,19).…”

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confidence: 99%

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Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Tranell

Fenyõ

Schwartz

2010

Journal of Biological Chemistry

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HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine-and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serineand arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.To produce all the mRNAs that are needed for HIV-1 to be infectious it uses alternative splicing. The full-length 9-kb transcript contains several splice sites, including four 5Ј-splice sites, also called splice donors (SD1-4) 2 and eight 3Ј-splice sites, also called splice acceptors (SA2-3, -4a-c, -5, and -7) (supplemental Fig. 1A), which can all be used in different combinations to create more than 35 differently spliced mRNAs (supplemental Fig. 1, B and C) (1-6). Cloning and expression of individual HIV-1 mRNAs have revealed that mRNAs spliced to SA3 produce Vpr, those spliced to SA4 produce Tat, those spliced to SA4a and -4b produce Rev and Nef, and those spliced to SA5 produce Nef (1,2,7,8). mRNAs spliced to SA2 are believed to produce Vif (5, 7). Two additional mRNAs that are believed to produce Rev and Nef are spliced to SA4c or to SA7, respectively (4). To produce the correct amounts of all mRNAs, the splice acceptors in HIV-1 are sub-optimal because of short and interrupted polypyrimidine tracts, non-canonical branch points, and inhibitory sequences that down-regulate the usage of several splice sites (6, 9 -13).Enhancer or silencer sequences regulate alternative splicing by up-regulating or down-regulating the usage of a splice site (14). Generally, the family of serine-and arginine-rich proteins (SR proteins) target enhancer sequences, and silencer sequences are targeted by heterogeneous nuclear ribonucleoproteins (15). There are several enhancers and silencers on HIV-1 pre-mRNA (16). A silencer in exon 3 called ESSV inhibits the vpr splice acceptor SA3 (10, 17). Two silencers in exon 4, named ESS2 and ESS2p, inhibit splicing into exon 4 thereby inhibiting the production of tat mRNA (18,19). SA7 is used by all mRNAs in the 2-kb class. In exon 7 a silencer called ESS3 blocks U2 small nuclear ribonucleoprotein, thereby suppressing SA7 (19 -21). An intronic silencer that blocks SA7 is located in the intron, directly upstream of exon 7 (22). An enhancer element called ESE3 that activates SA7 is located close to ESS3 in exon 7 (9, 21, 23). An enhancer in exon 4 has been named ESE2, because it attenuates ESS2 and thereby enhances splicing into SA4 (24)...

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Section: Inhibition Of the 5ј-splice Site Of Exon 3 By Srp55 And Srp7mentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

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Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Tranell

Fenyõ

Schwartz

2010

Journal of Biological Chemistry

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“…1A) (13,23,24). Mutations of 5Јss D2 that increase its binding affinity for U1 snRNP result in increased splicing at 3Јss A1 and increased vif mRNA, whereas mutations which lower the affinity to U1 snRNP result in decreased splicing at 3Јss A1 and reduced vif mRNA (13,25).…”

mentioning

confidence: 99%

Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs

Mandal

Feng

Stoltzfus

2010

J Virol

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HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3 splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5 splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3 splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.The HIV-1 primary RNA transcript undergoes a complex splicing pathway leading to the production of over 40 different spliced mRNA species as well as unspliced (US) RNA (30). Unspliced HIV-1 mRNA, which is transported into the cytoplasm by a Rev-dependent process, encodes Gag and Gag-Pol structural proteins. Unspliced viral RNA is also packaged into the virus particles as genome RNA. Spliced HIV-1 mRNAs are either incompletely spliced (IS) and serve as mRNAs for viral Env, Vpu, Vpr, and Vif proteins or completely spliced (CS) to serve as mRNAs for Tat, Rev, or Nef protein. Splicing is a two-step catalytic reaction that occurs within a large complex called the spliceosome. One of the earliest steps in spliceosome assembly is the recognition of 5Ј splice donor sites (5Јss) by cellular U1 snRNP (28, 39). The extent of base pairing between the 5Јss and the 5Ј-terminal sequence of U1 snRNA determines the strength of the 5Јss and thus contributes to the recognition and frequency of U1 snRNP usage (17, 32). In retroviruses, 3Ј splice acceptor sites (3Јss) are weak, and recognition of these sites is assisted by the binding of cellular splicing regulatory proteins which regulate alternative splicing and gene expression. These regulatory proteins (SR proteins and hnRNPs) bind to exonic splicing enhancers (ESE) or exonic or intronic splicing silencers (ESS or ISS) within the viral genome (10, 38). U1 snRNP bound at the 5Јss also activates splicing at the upstream 3Јss by a mechanism referred to "exon definition" or "exon bridging" (18, 31).Our laboratory has shown that splicing of the mRNAs encoding HIV-1 accessory proteins Vif and Vpr is regulated by suboptimal 5Јss D2 and D3 downstream of 3Јss A1 and A2, respectively (Fig. 1A) (13,23,24). Mutations of 5Јss D2 that increa...

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“…Thus, the introns are inefficiently removed by the splicing machinery, which in turn, results in the accumulation of mRNAs with retained introns. Various other positive and negative cis-acting elements within the viral genome can also regulate the levels of intron-retaining viral mRNAs in the nucleus by regulating splicing (146).…”

Section: Nuclear Export Of Viral Mrnas and The Rev-rre Pathwaymentioning

confidence: 99%

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

Sherpa¹

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HIV-1 is a retrovirus of global health interest as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). It is also used as a molecular tool to study various eukaryotic cellular processes. For instance, major discoveries on the Crm1 cellular nuclear export were made using the HIV Rev protein and its RNA binding partner, the Rev Response Element (RRE). The RRE is a cis-acting RNA element with multiple stem-loops present in all intron-retaining HIV mRNAs. Binding of the Rev protein to the primary binding site, and Rev multimerization on other regions of the RRE, is required for the nucleo-cytoplasmic export of these mRNAs. This is an essential step in HIV replication. However, the precise secondary structure of the HIV-1 RRE remains controversial. Studies have reported that the RRE has either 4 or 5 stem-loops, which differ only in the rearrangement of regions that lie outside of the primary Rev binding site. To understand the role played by these regions, we have examined the relationship between these two structures and Rev-RRE activity.In vitro transcribed NL4-3 RRE was found to migrate as a "doublet" band on a native polyacrylamide gel. Using in-gel Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE), we found that one of these bands consisted of an RRE with a 5 stem-loop structure, whereas the other was a 4 stem-loop structure. Thus, our data demonstrate, for the first time, that the NL4-3 RRE exists in two alternative structures.To study the significance of these alternative structures, we made RRE mutants predicted to allow only one or the other of the structures to form. The predictions were confirmed using SHAPE. We then compared the activity of the two forms to each other and to the wt RRE. Analysis of the complexes that each RRE formed with purified Rev protein in vitro showed no significant difference in Rev binding affinity between the two structures. However, we observed differences in the migration rates of these complexes on native gels, suggesting structural differences. The RREs were also tested for their abilities to promote viral replication, by inserting each RRE into the Nef region of an RRE-defective provirus which contained RRE mutations that do not change the Env protein.Growth kinetics and competition assays showed that the virus with the 5 stemloop RRE had a higher replicative fitness than the virus with either the wt or the 4 stem-loop RRE. Between the wt and the 4 stem-loop RRE-containing viruses, the virus with the wt RRE appeared more fit. These results suggest that HIV may use two alternative RRE secondary structures to modulate replication, potentially allowing adaptation to environmental demands in time and/or space, analogous to the use of riboswitches in bacteria.

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An Exonic Splicing Silencer Downstream of the 3′ Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication

Cited by 59 publications

References 40 publications

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

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