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A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.
Retrovirology 2006; 3:10R

Abstract

BACKGROUND

Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production.

RESULTS

Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3.

CONCLUSION

These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.

Authors+Show Affiliations

Interdisciplinary Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA. joshua-madsen@uiowa.eduNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

16457729

Citation

Madsen, Joshua M., and C Martin Stoltzfus. "A Suboptimal 5' Splice Site Downstream of HIV-1 Splice Site A1 Is Required for Unspliced Viral mRNA Accumulation and Efficient Virus Replication." Retrovirology, vol. 3, 2006, p. 10.
Madsen JM, Stoltzfus CM. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. Retrovirology. 2006;3:10.
Madsen, J. M., & Stoltzfus, C. M. (2006). A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. Retrovirology, 3, p. 10.
Madsen JM, Stoltzfus CM. A Suboptimal 5' Splice Site Downstream of HIV-1 Splice Site A1 Is Required for Unspliced Viral mRNA Accumulation and Efficient Virus Replication. Retrovirology. 2006 Feb 3;3:10. PubMed PMID: 16457729.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. AU - Madsen,Joshua M, AU - Stoltzfus,C Martin, Y1 - 2006/02/03/ PY - 2005/12/13/received PY - 2006/02/03/accepted PY - 2006/2/7/pubmed PY - 2006/8/15/medline PY - 2006/2/7/entrez SP - 10 EP - 10 JF - Retrovirology JO - Retrovirology VL - 3 N2 - BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication. SN - 1742-4690 UR - https://www.unboundmedicine.com/medline/citation/16457729/A_suboptimal_5'_splice_site_downstream_of_HIV_1_splice_site_A1_is_required_for_unspliced_viral_mRNA_accumulation_and_efficient_virus_replication_ L2 - https://retrovirology.biomedcentral.com/articles/10.1186/1742-4690-3-10 DB - PRIME DP - Unbound Medicine ER -