Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

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.     

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.     

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.     

Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains

In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and the env/nef 3' splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3' splice site used to generate tat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3' splice site, which is the most downstream of the three rev 3' splice sites, also serves as an element inhibiting splicing at the env/nef 3' splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a different rev 3' splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3' splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev 3' splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3' splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.     

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.     

HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3′ splice site 2 (A2) and 5′splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.     

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.     

Mutations at alternative 5' splice sites of M1 mRNA negatively affect influenza A virus viability and growth rate

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.     

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.     

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE_vpr) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE_vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE_vpr. Further analyses revealed that ESE_vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.