Regulation of
            <i>vif</i>
            mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5′ Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G

Mandal, Dibyakanti; Exline, Colin M.; Feng, Zhanlian; Stoltzfus, C. Martin

doi:10.1128/jvi.02231-08

Cited by 18 publications

(29 citation statements)

References 33 publications

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“…Recently it was shown that splicing to exon 2A, another incompletely spliced HIV-1 exon that encodes the vif gene, appears to be positively regulated by SRp75 through the interaction of SRp75 with an enhancer in this region (46,47). We show that SRp75 can induce production of vpr mRNA by inhibiting the 5Ј-splice site of exon 3.…”

Section: Discussionmentioning

confidence: 81%

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: Discussionmentioning

confidence: 81%

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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“…Increased splicing at 3Јss A1, on the other hand, results in an increased level of Vif, a 10-fold inhibition of virus production, and also, surprisingly, a defect of Gag processing (24,26). We showed in those studies that the strength of U1 snRNP binding at 5Јss D2 is important for regulation of splicing at 3Јss A1 but that splicing at 5Јss D2 is not required for this regulatory effect (25). Similarly, mutations of 5Јss D3 that change it to a consensus 5Јss result in increased splicing at 3Јss A2 (9).…”

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confidence: 90%

“…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). Decreased splicing at 3Јss A1 reduces the level of Vif and inhibits HIV-1 replication in cells that are expressing elevated levels of the restriction factor APOBEC3G (25).…”

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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 5=ss D2 has to be rendered splicing incompetent, even though binding of U1 snRNP to this 5=ss is a prerequisite for efficient recognition of the upstream 3=ss A1 (22,23). In general, 5=ss splice donor repression may be a common requirement for the generation of all spliced but intron-containing HIV-1 mRNAs, e.g., the env mRNAs (24).…”

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confidence: 99%

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confidence: 99%

An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

Widera

Erkelenz

Hillebrand

et al. 2013

J Virol

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Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3= splice site (3=ss) A1 but lack splicing at 5=ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3=ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3=ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5=ss D2. Here we show that an intronic G run (G I2 -1) represses the use of a second 5=ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G I2 -1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vifto-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. R eplication of human immunodeficiency virus type 1 (HIV-1) is counteracted by four major classes of host-encoded restriction factors: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), TRIM5␣ (tripartite motif 5␣), tetherin (BST-2, CD317, or HM1.24), and SAMHD1 (1-4). A3G (5) belongs to a family of cytidine deaminases that includes seven members (A3A to A3D and A3F to A3H) located in a gene cluster on chromosome 22 (6-8). It is encapsidated into newly assembled virions and introduces C-to-U substitutions during minus-strand synthesis, resulting in G-to-A hypermutations and aberrant DNA ends in the HIV-1 genome. Furthermore, A3G, independent of its deaminase activity, inhibits reverse transcriptase-mediated minus-strand elongation by direct binding to the viral RNA (9). This leads to massive impairment of viral replication (10). However, the HIV-1-encoded accessory protein Vif counteracts A3G by direct interaction, by inducing proteasomal degradation, and by repression of mRNA synthesis (10). Whereas HIV-1 is able to replicate efficiently in A3G-expressing cells, Vifdeficient virus strains are completely suppressed (5). Nevertheless, a narrowly restricted level of Vif is crucial for optimal HIV-1 replication since proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site is inhibited by high levels of Vif (11).During the course of infection, the HIV-1 9-kb single-sense pre-mRNA is processed into more than 40 alternatively spliced m...

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Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5′ Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G

Cited by 18 publications

References 33 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

An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

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