viruses Review HIV-1: To Splice or Not to Splice, That Is the Question Ann Emery 1 and Ronald Swanstrom 1,2,3,* 1 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; [email protected] 2 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA 3 Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27599, USA * Correspondence: [email protected] 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. Citation: Emery, A.; Swanstrom, R. HIV-1: To Splice or Not to Splice, That Keywords: HIV-1; HIV-1 splicing; HIV-1 oversplicing; HIV-1 latency Is the Question. Viruses 2021, 13, 181. https://doi.org/10.3390/v13020181 Academic Editors: Karen Beemon 1. How Splicing Works Generally for Cellular mRNAs and for HIV-1 and Marie-Louise Hammarskjold 1.1. HIV-1 Splicing Overview Received: 5 January 2021 RNA synthesis from the HIV-1 provirus results in only full-length transcripts, and Accepted: 22 January 2021 most avoid splicing to remain full length at approximately 9.2 kb [1–3]. These unspliced Published: 26 January 2021 RNAs are the mRNAs encoding the gag/pro/pol genes and function as the genomic RNA that is packaged into new virions. All other viral RNA products are the result of splicing Publisher’s Note: MDPI stays neutral (Figure1). Transcripts that splice always use splice donor D1 in the 5 0 UTR and splice to with regard to jurisdictional claims in one of the downstream acceptors A1 through A5. The area under the arc gets spliced out published maps and institutional affil- and the sequence upstream of D1 is juxtaposed to the next ORF downstream of whichever iations. acceptor site was used. Thus, splices to A1 make a vif transcript, to A2 a vpr transcript, and so on. This is the first step of splicing. Once splicing from D1 has occurred, the transcript may or may not splice out the sequence from D4 to A7. D4 to A7 contains the coding sequences for vpu and env, and the Copyright: © 2021 by the authors. Rev Response Element (RRE). This D4 to A7 splice happens only if D1 has already been Licensee MDPI, Basel, Switzerland. used. Transcripts that splice from D1 but do not splice out D4 to A7 are longer and are This article is an open access article collectively called the 4 kb size class or partially/incompletely spliced, since they retain the distributed under the terms and “env” intron. Those RNAs that splice from D1 and splice out the D4-A7 segment are the conditions of the Creative Commons shorter 1.8 kb size class of viral mRNAs, also called completely spliced. In general, cellular Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ transcripts that are not completely spliced are not exported from the nucleus and so HIV-1 4.0/). unspliced and partially spliced transcripts (which retain the D4-A7 sequence with the Viruses 2021, 13, 181. https://doi.org/10.3390/v13020181 https://www.mdpi.com/journal/viruses Viruses 2021, 13, x FOR PEER REVIEW 2 of 15 Viruses 2021, 13, 181 2 of 14 so HIV-1 unspliced and partially spliced transcripts (which retain the D4-A7 sequence with the RRE) cannot be exported by the normal cellular mRNA export pathway [4]. To circumvent thisRRE) restriction, cannot be the exported viral Re byv the protein normal binds cellular the mRNA RRE’s exportcomplex pathway secondary [4]. To circumvent structure (in thethis retained restriction, env intron) the viral and Rev links protein the unspliced/partially binds the RRE’s complex spliced secondary transcript structure to (in the the alternate CRM1-mediatedretained env intron) nuclear and linksexport the pa unspliced/partiallythway. It is also possible, spliced though transcript infre- to the alternate quent, to spliceCRM1-mediated directly from D1 nuclear to A7, exportwhich pathway.creates a transcript It is also possible,isoform of though nef mRNA. infrequent, to splice directly from D1 to A7, which creates a transcript isoform of nef mRNA. Splicing Step 1: From D1 to Acceptor Splicing Step 2: Two Additional Donors Make Two Alternative Small Exons From D4 to A7 D1 D2 D3 D4 RRE gag/pro/pol vif vpr tat env/rev env/nef A1 A2A3 A4 A5 A7 Figure 1. SplicingFigure in 1. HIV-1. Splicing Most in HIV-1. HIV-1 transcriptsMost HIV-1 remain transcripts unspliced. remain When unspliced. splicing When occurs, splicing it always occurs, splices it from D1 to one of the downstreamalways splices acceptors, from D1 removing to one of athe section downstream under a acceptors, colored arc. removing Once having a section spliced under from a colored D1, the RNA may splice again toarc. remove Once thehaving sequence spliced between from D1, D4 the and RNA A7 containing may splice the againvpu /toenv removegenes the and sequence the RRE (Revbetween Response D4 Element; the region removedand A7 iscontaining sometimes the called vpu/ theenv envgenesintron). and the Transcripts RRE (Rev that Response undergo Element; this additional the region D4 toremoved A7 splice is (needed to create tat, rev,sometimes and nef mRNAs, called the and env short intron). mRNAs Transcripts for vif and thatvpr undergo) comprise this the additional group of fully D4 to spliced A7 splice transcripts. (needed Transcripts that splice onlyto create from D1 tat but, rev not, and from nef mRNAs, D4 to A7 and (vif, vprshort, vpu mRNAs/env mRNAs, for vif and and vpr a long) comprise transcript the isoformgroup of of fullytat mRNA) make up the partially/incompletelyspliced transcripts. spliced Transcript transcripts.s that splice Two small only exonsfrom D1 (shown but not in green)from D4 can tobe A7 included (vif, vpr, orvpu skipped/env in mRNAs mRNAs, and a long transcript isoform of tat mRNA) make up the partially/incompletely spliced other than vif. Possible splice patterns that include the first small exon are shown when D1 splices to A1 and then splicing transcripts. Two small exons (shown in green) can be included or skipped in mRNAs other than occurs from D2 to a downstream acceptor (shown by the gray arcs below the line). The second small exon can be included vif. Possible splice patterns that include the first small exon are shown when D1 splices to A1 and by a similar mechanismthen splicing when occurs splicing from D2 from to D1a downstream to A2 (or D2 acceptor to A2) is (shown followed by by the splicing gray arcs from below D3 (notthe line). shown). The second small exon can be included by a similar mechanism when splicing from D1 to A2 (or D2 to A2) is followedAn by unusualsplicing from feature D3 of(not HIV-1 shown). splicing is the optional inclusion of two small noncoding exons. Two additional splice donors, D2 and D3, can be used to make these small exons, An unusualshown feature in green of HIV-1 in Figure splicing1. Either is the or optional both small inclusion exons can of two be included small noncod- in a transcript, with ing exons. Twothe additional example in splice Figure donors,1 for inclusion D2 and of D3, the can A1-D2 be used small to exon. make Collectively these small these permuta- exons, showntions in green of unspliced, in Figure partially 1. Either spliced or both and small completely exons can spliced, be included combined in a with tran- either, both, or script, with thenone example of the smallin Figure exons 1 for give inclusion rise to over of the 50 wellA1-D2 characterized small exon. final Collectively viral RNA transcripts. these permutationsGenerally, of unspliced, splicing partially is a sp fastliced and and efficient completely cotranscriptional spliced, combined process with but in HIV-1 it either, both, ormust none be of highly the small suppressed exons give to makerise to full over length 50 well or partiallycharacterized spliced final transcripts. viral In these cases, introns are retained, which is something that typically does not happen with cellular RNA transcripts. mRNA transcripts. Many studies have asked “what controls the inclusion or skipping of Generally, splicing is a fast and efficient cotranscriptional process but in HIV-1 it the small exons”, and “how does splicing from D1 modulate acceptor site usage between must be highly suppressed to make full length or partially spliced transcripts.