Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements

Abstract

Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 59 splice site (59ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 59ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication. Human immunodeficiency virus type 1 (HIV-1) produces a single primary RNA transcript. The full-length transcript functions as RNA genome that is packaged into virions and as mRNA for translation of the Gag and Pol proteins. HIV-1 RNA contains several splice donor (SD) [59 splice site (59ss)] and splice acceptor [39 splice site (39ss)] sites. Differential usage of these sites results in the production of a variety of spliced RNAs that encode the other viral proteins. Splicing has to be strictly regulated for the balanced production of all viral RNAs and proteins.Formation of the spliceosome complex is initiated by binding of U1 small nuclear ribonucleoprotein (snRNP) to the 59ss, which is mediated by the complementarity between the 11 single-stranded nucleotides at the 59 end of the U1 small nuclear RNA (snRNA) component and the 59ss sequence (Freund et al., 2003). The major 59ss located in the 59 untranslated leader of HIV-1 transcripts is used for the production of all spliced mRNAs. As both spliced and unspliced transcripts are required for a productive replication cycle, splicing at this 59ss has to be controlled to prevent complete processing. Previous studies indicated that both RNA structure and sequence elements are involved in this control. The 59ss region can fold a stemloop structure, the splice donor (SD) hairpin, that partially occludes the U1 annealing site and reduces splicing frequency (Abbink & Berkhout, 2008;Mueller et al., 2014). Splicing is also influenced by binding of several SR proteins to splice regulatory elements (SREs) in the SD region (Asang et al., 2012;Mueller et al., 2015). The SD hairpin structure may also influence binding of these SR proteins. In addition, the extent of sequence complementarity between the 59ss and U1 may influence snRNP binding and splicing efficiency.

A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal

“…For example, the frequently observed double variation C287A/U288C opens two bps (bp 5 and 6: C 287 –G 294 and U 288 –A 293 ) but results in the formation of two alternative bps (A 287 –U 295 and C 288 –G 294 ). Some virus isolates hold multiple nt differences in this leader RNA domain, but as previously shown [ 15 , 17 ] most isolates can fold a similar SD hairpin structure. Only a minority of the variations does not allow Watson-Crick bp formation.…”

“…Mutation-reversion analysis of the lower stem of the TAR hairpin indeed indicated that maintenance of the structure is more critical than the sequence requirement [ 12 ]. Conservation of the SD sequence is likely related to the presence of the U1 snRNA and SR protein binding sites [ 16 , 17 , 30 , 53 ]. These sequence requirements may have prevented the evolution of more informative sequence variation.…”

“…This forced evolution approach mimics the search for bp co-variations in a phylogenetic survey to identify biologically relevant RNA structures [ 13 ]. HIV-1 mutation studies indicated that the thermodynamic stability of the SD stem-loop structure is important for virus replication [ 14 , 15 , 16 , 17 ]. Stabilization of the hairpin caused a severe virus replication defect and decreased the splicing efficiency.…”

“…To do so, mutants with a destabilized structure were analyzed for the impact on virus replication and splicing efficiency. We observed that destabilization of the wt hairpin does increase the level of HIV-1 splicing [ 15 , 16 , 17 ] , which indicates that HIV-1 RNA splicing is indeed at least partially suppressed by the SD stem-loop structure that encompasses the 5′ splice site (5′ss). Like stabilization, destabilization of the SD hairpin reduced virus replication, suggesting that HIV-1 requires a sub-optimal level of splicing in order to express all viral transcripts and proteins in the amount required for optimal virus replication.…”

“…There are other reports of splicing regulation by RNA structure [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. In addition to local RNA structure, several sequence elements—especially binding sites for SR proteins and U1 small nuclear RNA (snRNA)—are known to regulate HIV-1 splicing [ 16 , 17 , 30 ].…”