Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2

ABSTRACT 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 withintat exon 2 (ESS2) and a suboptimal 3′ splice site together act to inhibit splicing at the 3′ splice site flanking tatexon 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 oftat mRNA splicing mediated by ESS2 and ESS3.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4606 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4606-4615 ◽

Cited By ~ 98

Author(s):

B A Amendt ◽

Z H Si ◽

C M Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splice Site ◽

Exon 2 ◽

Exon Splicing ◽

Cellular Factors ◽

Immunodeficiency Virus ◽

Hiv 1

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.

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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

Journal of Virology ◽

10.1128/jvi.73.12.9764-9772.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9764-9772 ◽

Cited By ~ 15

Author(s):

Patricia S. Bilodeau ◽

Jeffrey K. Domsic ◽

C. Martin Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Splice Site ◽

Regulatory Elements ◽

Splice Sites ◽

Exon 2 ◽

Immunodeficiency Virus ◽

Splicing Regulatory Elements ◽

Hiv 1

ABSTRACT 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 theenv/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 generatetat 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 differentrev 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 rev3′ 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.

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Repair of a Rev-Minus Human Immunodeficiency Virus Type 1 Mutant by Activation of a Cryptic Splice Site

Journal of Virology ◽

10.1128/jvi.75.7.3495-3500.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3495-3500 ◽

Cited By ~ 3

Author(s):

Koen Verhoef ◽

Patricia S. Bilodeau ◽

Jeroen L. B. van Wamel ◽

Jørgen Kjems ◽

C. Martin Stoltzfus ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splice Site ◽

Site Mutation ◽

Reading Frame ◽

Splicing Silencer ◽

Immunodeficiency Virus ◽

Revertant Virus

ABSTRACT We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5′ splice site (ss) that, when used in conjunction with the regular HIV 3′ ss #5, fuses the tat and revreading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5′ ss by mutational inactivation of an adjacent exon splicing silencer element.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.6480 ◽

1995 ◽

Vol 15 (11) ◽

pp. 6480-6480 ◽

Cited By ~ 9

Author(s):

B A Amendt ◽

Z H Si ◽

C M Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Exon 2 ◽

Exon Splicing ◽

Cellular Factors ◽

Immunodeficiency Virus

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Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4597 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4597-4605 ◽

Cited By ~ 102

Author(s):

A Staffa ◽

A Cochrane

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splice Site ◽

Regulatory Elements ◽

Control Element ◽

Exon Splicing ◽

Immunodeficiency Virus

The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.

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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

Journal of Virology ◽

10.1128/jvi.79.16.10478-10486.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10478-10486 ◽

Cited By ~ 41

Author(s):

Joshua M. Madsen ◽

C. Martin Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splice Site ◽

Viral Mrna ◽

Splice Sites ◽

Splicing Silencer ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT 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.

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Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression

Journal of Virology ◽

10.1128/jvi.01558-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 3921-3931 ◽

Cited By ~ 37

Author(s):

C. M. Exline ◽

Z. Feng ◽

C. M. Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Regulatory Elements ◽

Mrna Splicing ◽

Viral Mrnas ◽

Exon 2 ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

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A Second Exon Splicing Silencer within Human Immunodeficiency Virus Type 1tatExon 2 Represses Splicing of Tat mRNA and Binds Protein hnRNP H

Journal of Biological Chemistry ◽

10.1074/jbc.m104070200 ◽

2001 ◽

Vol 276 (44) ◽

pp. 40464-40475 ◽

Cited By ~ 82

Author(s):

Sandrine Jacquenet ◽

Agnès Méreau ◽

Patricia S. Bilodeau ◽

Laurence Damier ◽

C. Martin Stoltzfus ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Splicing Silencer ◽

Exon Splicing ◽

Immunodeficiency Virus

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RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element

Journal of Virology ◽

10.1128/jvi.75.18.8487-8497.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8487-8497 ◽

Cited By ~ 79

Author(s):

Patricia S. Bilodeau ◽

Jeffrey K. Domsic ◽

Akila Mayeda ◽

Adrian R. Krainer ◽

C. Martin Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Splice Site ◽

Rna Splicing ◽

Consensus Sequence ◽

Mrna Levels ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Hiv 1

ABSTRACT The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

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