Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b

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. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For 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 in 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 HEXplorerguided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use 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 hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.KEYWORDS HEXplorer score, exon recognition, host restriction factor, human immunodeficiency virus, pre-mRNA processing, splicing regulatory elements D uring long terminal repeat (LTR)-driven transcription, over 50 mRNA isoforms emerge by alternative splicing of the HIV-1 precursor mRNA (1, 2). According to their distinct sizes, mRNA isoforms can be divided into three different classes: 2-kb mRNAs (intronless), encoding Tat, Rev, and Nef; intron-containing 4-kb mRNAs, encoding Vif, Vpr, Vpu, and Env; and 9-kb unspliced mRNAs, encoding Gag and Gag-Pol (3). Viral gene expression follows a strict chronological order (4-6). In the early phase, only intronless mRNAs are transported out of the nucleus and translated, whereas intron-

Production of HIV-1 vif mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region

“…Of note, twelve effective nSNVs were clustered in SLSA1, suggesting a possible involvement of SLSA1 (sequence and/or structure) in modulation of vif production. Another important point to be mentioned in Figure 5 is that six nSNVs (V234gtg, P238ccg, K240aaa, L242ctt, A248gcg, and V249gtg) are located at distinct sites from elements reported previously ( Kammler et al, 2006 ; Exline et al, 2008 ; Mandal et al, 2009 ; Widera et al, 2013 ; Brillen et al, 2017 ). This finding suggests a potential existence of novel splicing regulatory elements so far unidentified.…”

“…Numbers below the sequences represent amino acid positions of HIV-1 NL4-3 Pol-integrase. SA1/SD2 sites and reported splicing regulatory elements (ESEVif, ESE-M1, ESE-M2, G4 motif, ESS2b, ESE2b, and G I2 -1 in Kammler et al, 2006 ; Exline et al, 2008 ; Mandal et al, 2009 ; Widera et al, 2013 ; Brillen et al, 2017 ) are shown. A blue line indicates the SLSA1 region.…”

“…Several splicing regulatory elements that affect vif mRNA production and/or usage of SA1 and SD2 sites have been reported. These elements include, from 5′ to 3′, exonic splicing enhancer Vif (ESEVif), ESE-M1/M2, G4 motif, exonic splicing silencer 2b (ESS2b), ESE2b, and G I2 -1 ( Kammler et al, 2006 ; Exline et al, 2008 ; Mandal et al, 2009 ; Widera et al, 2013 ; Brillen et al, 2017 ). As presented in Figure 5 , we here mapped twenty nSNVs that alter vif expression level identified by us so far (results in this study and in Nomaguchi et al, 2016 ) to SA1D2prox along with above splicing regulatory elements.…”

“…For naturally occurring viruses, multiple mutations may have accumulated over time in the entire SA1D2prox region including SLSA1, and thereby have stabilized the whole RNA structure surrounding and containing SA1D2prox. The nSNVs in SA1D2prox may primarily affect vif production via various regulatory elements and factors ( Figure 5 ) ( Kammler et al, 2006 ; Exline et al, 2008 ; Mandal et al, 2009 ; Widera et al, 2013 ; Brillen et al, 2017 ). Consequently, we could expect that the importance of SLSA1 structure and SA1D2prox sequence for vif production became obvious only for proviral clones carrying natural SA1D2prox sequences.…”

Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays

“…Designer exon splicing reporters were generated on the basis of an FGB minigene containing only neutral sequences as described before (Brillen et al 2017). Two SRSF7 binding sites were introduced by substituting the Bsu36I/NotI fragment with the PCR product using the primer pair #4705/#2620.…”