A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.
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.


CONCLUSIONS
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.
Madsen
Joshua M
JM
Interdisciplinary Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA. joshua-madsen@uiowa.edu
Stoltzfus
C Martin
CM
D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005091:Exons; D016346:Gene Products, vif / Q000276:immunology; D016336:Gene Products, vpr / Q000276:immunology; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014779:Virus Replication; D054320:vif Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D016346:Gene Products, vif; D016336:Gene Products, vpr; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D054320:vif Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus