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

Joshua M Madsen,C Martin Stoltzfus

doi:10.1186/1742-4690-3-10

Abstract

BackgroundInefficient 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.ResultsHere 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.ConclusionThese 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.

Highlights

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
Optimization of HIV-1 5'ss D2 results in increased splicing at 3'ss A1 and increased inclusion of exon 2 We have previously shown that disruption of ESSV within exon 3 results in increased splicing at 3'ss A2 and decreased unspliced mRNA accumulation
In an effort to analyze the effect on HIV-1 replication of increased splicing at HIV-1 3'ss A1, we generated mutations within the downstream 5'ss D2 (NLD2UP) intended to increase the sequence homology to the metazoan 5'ss signal (Fig. 1B)

Summary

Introduction

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). Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Regulation of HIV-1 alternative splicing occurs primarily because of the presence of suboptimal 5'ss and 3'ss, which decrease the recognition by the cellular splicing machinery of the splice signals [35]. We have previously shown that mutations which either disrupt an ESS within exon 3 (ESSV) or optimize the 5'ss D3 splicing signal, result in increased splicing at HIV-1 3'ss A2 [12,15]. Increased splicing at HIV-1 3'ss A2 results in decreased unspliced mRNA accumulation and a reduction in virus replication, which was restored by second site reversions that either inactivate 3'ss A2 or 5'ss D3 [15]

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