An exonic splicing silencer downstream of the 3' splice site A2 is required for efficient human immunodeficiency virus type 1 replication

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3'ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3'ss A2 and 5' splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3'ss A2 splicing is necessary for HIV-1 replication.     

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.     

A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability

Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5'-splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation.     

A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates human immunodeficiency virus type 1 rev, env, vpu, and nef gene expression

The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.     

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

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5′ splice site (5′ss) D1, to the first splice acceptor, 3′ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5′ss D2, which is 50 nucleotides downstream of 3′ss A1; a GGGG silencer motif proximal to 5′ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5′ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3′ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5′ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5′ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.Human immunodeficiency virus type 1 (HIV-1) Vif is a 23-kDa basic protein (4, 9) that is incorporated into virus particles during productive infection (8-10). Replication of HIV-1 in some T-cell lines is dependent on the expression of a functional Vif protein. Replication of Vif-deleted HIV-1 is restricted in these cells, which are termed nonpermissive, because of the presence of several host deaminases, the most important of which for HIV-1 replication is APOBEC3G (A3G) (25, 26). Human A3G is a single-stranded DNA deaminase that inhibits the replication of HIV-1 as well as other types of retroviruses and retrotransposons (5, 12, 17, 25, 32). HIV-1 Vif forms a complex with A3G and other cellular proteins to promote A3G ubiquitination, resulting in proteasomal degradation of A3G (1, 11, 14, 18, 26). Vif-deleted HIV-1 produced in the presence of A3G packages increased levels of A3G compared to those found in the wild type (WT) and has reduced infectivity in nonpermissive T-cell lines. This reduced infectivity in the absence of Vif has been correlated with the dC-to-dU hypermutation of newly synthesized minus-strand viral DNA by A3G (6, 13, 31, 32). However, other studies have shown that A3G is also able to restrict virus replication without hypermutating viral DNA (7, 19).It has previously been shown that the expression of Vif in infected cells is maintained at a relatively low level compared to levels of the other HIV-1 accessory proteins. One mechanism to explain this phenomenon is that Vif is degraded more rapidly than other accessory proteins by the proteasome (3). Another mechanism is that a relatively low level of vif mRNA is produced by alternative splicing (22). Alternative splicing of HIV-1 RNA results in the production of approximately 40 different mRNA species, which include three different mRNA size classes: 1.8-kb, completely spliced RNAs; 4-kb, incompletely spliced RNAs; and 9-kb, unspliced RNAs (Fig. ​(Fig.1A).1A). The 4-kb mRNA class encodes Vif, Vpr, Tat, Vpu, and Env, and the completely spliced, 1.8-kb mRNA class encodes Tat, Rev, and Nef. Unspliced viral RNA is both packaged into virions as genomic RNA and used as mRNA for Gag and Gag-Pol proteins (2, 27). As shown in Fig. ​Fig.1A,1A, four different 5′ splice donor sites (5′ss) and eight different 3′ splice acceptor sites (3′ss), which are highly conserved among group M HIV-1 strains, are used to produce alternatively spliced HIV-1 mRNAs at different levels in infected cells (22). The efficiencies with which these 5′ss and 3′ss are used are dependent on the presence of suboptimal cis splicing elements within the 5′ss and 3′ss themselves and more-distant elements, which include exonic splicing silencers, an intronic splicing silencer, and exonic splicing enhancers (ESE) (2, 15, 27).Open in a separate windowFIG. 1.HIV-1 splicing pattern and elements regulating vif mRNA splicing. (A) The conserved 5′ss (D1 to D4) and 3′ss (A1 to A7) located within the 9-kb HIV-1 genome are shown. Completely and incompletely spliced HIV-1 mRNAs (∼4 kb and ∼1.8 kb) are shown as open boxes. Spliced mRNAs are denoted by the translated open reading frame and by the exon content. The incompletely spliced mRNAs, denoted with an I, are differentiated from completely spliced mRNAs by inclusion of the intron between 5′ss D4 and 3′ss A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) can be differentially included within all 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I) and vpr mRNA, which can include only exon 2 (1.[2].3I). LTR, long terminal repeat. (B) Three elements regulating vif mRNA splicing are shown: positively acting enhancer ESEVif, the 5′ss D2 (underlined), and a negatively acting G4 silencer motif. The locations of noncoding exon 2 and the start site for Vif protein synthesis are also shown. (C) HIV-1 5′ss D2-down mutants used in this study are shown. Sequences were aligned and compared with that of the consensus metazoan 5′ss. The sequence of the ESEVif mutant used in this study is also aligned and compared with the WT sequence. nt, nucleotides.HIV-1 Vif is translated from a low-abundance, incompletely spliced mRNA resulting from splicing of HIV-1 RNA between the major splice donor site (5′ss D1) and 3′ss A1. We have demonstrated that vif mRNA splicing is tightly regulated by the presence of multiple regulatory elements (Fig. ​(Fig.1B).1B). These include a highly conserved suboptimal 5′ss (5′ss D2) 50 nucleotides downstream from 3′ss A1, an SRp75-dependent ESE (ESEVif), and a GGGG silencer element proximal to 5′ss D2 (2). Mutations within the relatively weak 5′ss D2 that increase its homology to a consensus 5′ss result in increased inclusion of the noncoding 50-nucleotide exon defined by 3′ss A1 and 5′ss D2 (exon 2), increased single-spliced vif mRNA levels and Vif expression, and an excessive splicing phenotype in which virion production is reduced to 10 to 25% of that of the WT (16). Conversely, mutations that decrease the homology of 5′ss D2 to a consensus 5′ss inhibit splicing at 3′ss A1 and exon 2 inclusion into both incompletely and completely spliced HIV-1 mRNAs as well as decreased levels of vif mRNA. Virus production, however, is not significantly affected. Mutation of ESEVif resulted in a similar phenotype. We have shown previously that increased or decreased exon 2 inclusion into spliced mRNA does not affect the stability or expression of viral mRNAs (15). Based on these results, we hypothesized that the conserved suboptimal 5′ss D2, which together with 3′ss A1 defines exon 2, and ESEVif are necessary to maintain optimal levels of Gag and Gag-Pol required for HIV-1 replication while producing sufficient Vif to overcome the cellular restriction factor A3G (2). To further test this hypothesis, we examined a panel of HIV-1 mutants producing reduced levels of Vif under permissive and nonpermissive conditions. We also investigated the long-term replication capabilities of these mutant viruses in both permissive and nonpermissive A3G-expressing T-cell lines. Mutant viruses demonstrated increasing sensitivity to A3G, which is inversely proportional to their levels of Vif expression. Our results suggest that the reason 5′ss D2 and ESEVif exist in the HIV-1 genome is to regulate the levels of vif mRNA and Vif protein in infected cells.     

The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3′ splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3′ splice site together act to inhibit splicing at the 3′ splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3′ splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3′ splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.     

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.     

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.     

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

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, serine- and 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.     

ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome

We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.     

Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.     

Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer

The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5′ splice site (ss) or 3′ ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3′ ss selectivity within the 3′ ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5′ ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3′ ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.