Influence of the Small Leader Exons 2 and 3 on Human Immunodeficiency Virus Type 1 Gene Expression

Krummheuer, Jörg; Lenz, Christian A.; Kammler, Susanne; Scheid, Andreas; Schaal, Heiner

doi:10.1006/viro.2001.0974

Cited by 22 publications

(15 citation statements)

References 55 publications

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“…The most likely explanation for the decrease in spliced viral mRNA is that the increased inclusion of exon 3 into HIV-1 mRNAs resulted in reduced mRNA stability. The potential influence of HIV-1 exon 3 on viral mRNA stability is consistent with a previous report which showed that the expression of HIV-1 cDNA constructs containing HIV-1 mRNA 5Ј leaders with exon 3 are reduced compared to similar constructs lacking exon 3 (20). An alternate explanation for the observed decrease in spliced viral mRNA is that the mutations we have generated within ESSV may disrupt a stability element.…”

Section: Discussionsupporting

confidence: 91%

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

Madsen¹,

Stoltzfus

2005

J Virol

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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 (3ss) 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 3ss 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 3ss 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, 3ss A2 and 5 splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3ss A2 splicing is necessary for HIV-1 replication.In contrast to the alternative splicing of most cellular mRNA, splicing of retroviral mRNA results in the cytoplasmic accumulation of incompletely spliced and unspliced viral mRNA. Incompletely spliced viral mRNA is necessary for the expression of the Env, Vpu, Vpr, and Vif proteins, and the accumulation of unspliced viral mRNA is necessary for expression of the gag and pol gene products and also serves as the genomic viral mRNA encapsidated within progeny virions. Completely spliced viral mRNA is required for expression of the regulatory viral proteins Tat, Rev, and Nef. More than 40 unique incompletely and completely spliced viral mRNA species are generated through the alternative splicing of the HIV-1 primary transcript within an HIV-1-infected cell (23,26).HIV-1 3Ј splice sites (3Јss) are used with differing efficiencies in part because viral polypyrimidine tracts (PPT) are interspersed with purines (27, 30), leading to decreased affinity for the essential cellular splicing factor U2AF65 (for a review of cellular splicing see reference 12). In addition, the efficiency of HIV-1 splicing is also regulated by both positive and negative cis elements within the viral genome that act to promote or repress splicing. To date, four exonic splicing silencers (ESS) and one intronic splicing silencer (ISS) have been identified within the viral genome (Fig. 1). Utilization of HIV-1 3Јss A2 by the spliceosome is negatively regulated by ESSV, 3Јss A3 by ESSp and ESS2, and 3Јss A7 by the ISS and ESS3 (2,3,5,16,29,31).We have previously characterized ESSV as a 24-nucleotide

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Section: Discussionsupporting

confidence: 91%

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

Madsen¹,

Stoltzfus

2005

J Virol

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“…A common theme there is the stability of the RNA. For example, alternative exons in the 5V UTR of the HIV-1 virus can either promote or inhibit the nuclear degradation of their surrounding mRNA, which regulates HIV-1 gene expression (Krummheuer et al, 2001), and alternative splicing of SC35 regulates its mRNA stability (Sureau et al, 2001). Splice variant transcripts can be targeted to specific subcellular sites, such as axons or dendrites (Meshorer et al, 2004).…”

Section: Influence On Mrna Functionmentioning

confidence: 99%

Function of alternative splicing

Stamm

Ben‐Ari

Rafalska

et al. 2005

Gene

1,015

629

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“…A more recent study using heterologous constructs with different HIV-1 5' leaders has indicated a positive effect of exon 2 and a negative effect of exon 3. It was proposed that the positive effect of exon 2 and the negative effect of exon 3 result from an increase or decrease, respectively, in the stabilities of the nuclear non-polyadenylated mRNA precursors [57]. Whether the small exons affect the stabilities of authentic HIV-1 viral mRNAs in infected cells has not yet been shown.…”

Section: Splicing Of Hiv-1 Mrnamentioning

confidence: 99%

Role of Viral Splicing Elements and Cellular RNA Binding Proteins in Regulation of HIV-1 Alternative RNA Splicing

Stoltzfus

Madsen²

2006

CHR

127

160

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In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

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Influence of the Small Leader Exons 2 and 3 on Human Immunodeficiency Virus Type 1 Gene Expression

Cited by 22 publications

References 55 publications

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

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

Function of alternative splicing

Role of Viral Splicing Elements and Cellular RNA Binding Proteins in Regulation of HIV-1 Alternative RNA Splicing

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