An exonic splicing silencer downstream of the 3' splice site A2 is required for efficient human immunodeficiency virus type 1 replication.
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.
Madsen
Joshua M
JM
Interdisciplinary Program in Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Stoltzfus
C Martin
CM
D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D012326:RNA Splicing; D037941:Silencer Elements, Transcriptional; D014779:Virus Replication