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Analysis of competing HIV-1 splice donor sites uncovers a tight cluster of splicing regulatory elements within exon 2/2b.

Analysis of competing HIV-1 splice donor sites uncovers a tight cluster of splicing regulatory elements within exon 2/2b.
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Brillen AL, Walotka L, Hillebrand F, Müller L, Widera M, Theiss S, Schaal H,


Brillen AL, Walotka L, Hillebrand F, Müller L, Widera M, Theiss S, Schaal H, (click to view)

Brillen AL, Walotka L, Hillebrand F, Müller L, Widera M, Theiss S, Schaal H,

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Journal of virology 2017 04 26() pii 10.1128/JVI.00389-17

Abstract

The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Non-coding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For the vif mRNA, intron 1 must be removed, while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented.Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion into viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impact of these SREs on the viral splicing pattern and protein expression was exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by usage of locked nucleic acids (LNAs) impaired Vif expression and viral replication.Importance Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNP proteins binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

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