HIV-1: To Splice or Not to Splice, That Is the Question

Abstract

The transcription of the HIV-1 provirus results in only one type of transcript-full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

Keywords: HIV-1; HIV-1 latency; HIV-1 oversplicing; HIV-1 splicing.

Figure 1

Splicing in HIV-1. Most HIV-1 transcripts remain unspliced. When splicing occurs, it always splices from D1 to one of the downstream acceptors, removing a section under a colored arc. Once having spliced from D1, the RNA may splice again to remove the sequence between D4 and A7 containing the vpu/env genes and the RRE (Rev Response Element; the region removed is sometimes called the env intron). Transcripts that undergo this additional D4 to A7 splice (needed to create tat, rev, and nef mRNAs, and short mRNAs for vif and vpr) comprise the group of fully spliced transcripts. Transcripts that splice only from D1 but not from D4 to A7 (vif, vpr, vpu/env mRNAs, and a long transcript isoform of tat mRNA) make up the partially/incompletely spliced transcripts. Two small exons (shown in green) can be included or skipped in mRNAs other than vif. Possible splice patterns that include the first small exon are shown when D1 splices to A1 and then splicing occurs from D2 to a downstream acceptor (shown by the gray arcs below the line). The second small exon can be included by a similar mechanism when splicing from D1 to A2 (or D2 to A2) is followed by splicing from D3 (not shown).

Figure 2

Exons in HIV-1. (Upper Panel) The first exon is defined by the cap and D1. The final exon is defined by A7 and the poly A site. The two small alternative exons are defined by A1 and D2, and A2 and D3 (grey). The exon defined by D4 is constitutive, but can use A3, A4, or A5 for the acceptor to define the exon. (Lower Panel) D4 can also use A1 or A2 to define the vif or vpr exons respectively, independently of D2 and D3.

Figure 3

A possible model for oversplicing when a distant suppressor is lost. The 5′ UTR can fold into one of two conformations that exist in equilibrium—dimerized or with D1 open for splicing. (Top) If splicing is suppressed, then a small proportion of the transcripts exposing D1 will splice. (Bottom) If a suppressor is removed, almost all transcripts with open D1 will splice to an unsuppressed acceptor. This drives the dimerized/D1 open equilibrium to the right and the unspliced (dimerized) full-length transcripts are lost.