Human immunodeficiency virus type-1 (HIV-1) encodes a single RNA genome, but it also produces a striking diversity of subgenomic RNA molecules, which allows the expression of all nine viral proteins. Production of this multitude of unspliced and more than 40 spliced RNA transcripts calls for strict coordination of the alternative splicing possibilities. Next to these sense RNAs, also antisense transcripts have been suggested to play a role in virus replication. Recently, the implementation of next-generation sequencing (NGS) methods has led to the description of several other HIV-derived RNAs, among which small RNA molecules that are processed from HIV-1 RNA precursors.
HIV-1 RNA CAPPING AND POLYADENYLATION
Reverse transcription of a retroviral RNA genome and integration into the host genome leads to establishment of the double-stranded DNA provirus. The host biosynthetic machinery is subsequently used for viral gene expression, but viral proteins modulate these processes. Figure 1 illustrates the diverse HIV-1 RNAs produced in infected cells. Panel A shows the DNA provirus integrated into the host cell genome. Transcription is initiated by RNA polymerase II (RNAP II) at the 5′ long terminal repeat (LTR) promoter and runs into the 3′ LTR where an active polyadenylation signal is recognized (Fig. 1b). HIV-1 transcription is activated by the viral Tat protein. We will discuss the regulation of transcription in more detail later as small RNAs are produced during this step. The nascent RNAs are co-transcriptionally capped on the 5′ end (marked as • in Fig. 1). Capping is an early RNA modification that can affect subsequent phases of RNA biology: splicing, cytoplasmic transport, translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNAs. A subset of the cellular RNAs, including small nuclear RNA (snRNAs), small nucleolar RNA (snoRNA) and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. It was recently reported that some unspliced and partially spliced HIV-1 RNAs are also TMG-capped . TMG capping of these Rev-responsive element (RRE)-containing RNAs may represent a new regulatory mechanism for selective gene expression.
Polyadenylation of HIV-1 RNA presents a puzzle, as the active signal in the 3′ LTR is also transcribed from the 5’ LTR, where this signal should be suppressed. Retroviruses have evolved different means to solve this LTR redundancy problem. HIV-1 uses multiple mechanisms to suppress the 5′ signal and to activate the 3′ signal . This complex scenario is schematically depicted in Fig. 1b and the polyA-tail is marked with ◃. At the 5′ end, the promoter-proximal position is not optimal for polyadenylation. On top of that, a local RNA hairpin structure suppresses recognition of the AAUAAA signal by cleavage and polyadenylation specificity factor (CPSF) that initiates the polyadenylation process [3,4]. U1snRNP binding to the nearby splice donor signal also suppresses polyadenylation [5,6]. At the 3′ end, suppression by promoter-proximity and U1snRNP is not possible. Local RNA structure is likely also suppressive here, but this is overcome by CPSF binding to the upstream stimulatory element (USE) that is uniquely present at the 3′ location . Both HIV-1 RNA capping and polyadenylation present possible targets for therapeutic intervention and future drug development .
HIV-1 RNA SPLICING
Simple retroviruses produce a singly spliced Env mRNA from the full-length precursor RNA to encode the viral glycoproteins. Complex retroviruses like HIV-1 are characterized by the production of several multiply spliced RNA species to encode the essential proteins Tat and Rev and the accessory proteins Vif, Vpr, Vpu and Nef. Importantly, splicing of all retroviruses is incomplete: they must maintain and export a fraction of the unspliced viral RNA precursor, which serves both as the major mRNA for the Gag and Pol proteins and as genomic RNA that is packaged in assembling virions . We will discuss new findings concerning HIV-1 RNA splicing, but one should realize that additional mechanisms control the fate of these RNA molecules. For instance, the zinc-finger antiviral protein, which was originally identified as a host factor that inhibits moloney murine leukemia virus, blocks HIV-1 by promoting specific degradation of multiply spliced viral RNAs via recruitment of the mRNA degradation machinery .
The posttranscriptional export of spliced and unspliced HIV-1 RNAs from the nucleus to the cytoplasm is a complex process, as eukaryotic cells normally retain unspliced RNAs in the nucleus, thus actively preventing their exit into the cytoplasm and promoting splicing. HIV-1 has evolved the Rev protein that participates in the nuclear export of unspliced and partially spliced RNAs through recognition of the RRE (Fig. 1c). A recent study indicated that Rev is recruited to HIV-1 transcription sites where an export complex is formed with the cellular export factor CRM1 and RRE-containing RNAs [11▪▪]. The co-transcriptional formation of a stable export complex ensures the efficient export of unspliced and partially spliced RNAs. Multiple additional cellular factors have been implicated in this process, including the pleiotropic protein PSF that connects at the site of viral transcription and the nuclear matrix protein MATR3 that joins at a subsequent step . Adding to the complexity, it was proposed that Rev-mediated nuclear export enhances the subsequent packaging of RRE-containing RNAs in newly assembled virions . This also affects the partially spliced HIV-1 RNAs, which indeed are encapsidated to some extent in HIV-1 virions .
SPLICE ENHANCERS, SPLICE SILENCERS AND RNA STRUCTURE
This complex scenario with multiple RNA species sets the stage for coordinated and multilevel splicing regulation. For instance, it seems that transcription from the viral 5′ LTR promoter produces a transcription complex that is optimally equipped for splicing regulation, which is lost upon promoter exchange . Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive and negative regulation occur through the binding of cellular factors to cis-acting splicing regulatory elements that act together to produce the optimal level of each mRNA to support virus replication [16,17]. New protein factors and RNA sequence elements that regulate HIV-1 RNA splicing have recently been identified. This includes multiple heterogenous nuclear ribonucleoproteins (hnRNPs) [18–20] and members of the Transformer 2 (Trn2) protein family . New regulatory splice signals in HIV-1 RNA include the intronic G run that controls the proper level of Vif and Vpr expression, the former being critical to optimally counteract APOBEC3G-mediated restriction . It is also becoming increasingly clear that HIV-1 is frequently using RNA secondary structure to modulate some of the splice signals [23–25,26▪].
CELL TYPE DIFFERENCES IN HIV-1 RNA SPLICING
HIV-1 RNA biosynthesis requires many cellular factors that control RNA transcription, splicing, intracellular localization, stability and translation. Changes in the level of splicing factors will affect the balance of alternatively spliced viral RNAs . HIV-1 may also influence this balance by changing the cellular milieu. For example, HIV-1 influences the phosphatidylinositol-3-kinase (PI3K) pathway to prevent apoptosis, but this pathway also affects phosphorylation of the serine-arginine, a family of splicing regulatory factors that influence splice site selection . A recent NGS study confirmed that HIV-1 RNA populations differ between cell types . Levels of the viral Tat protein may also fluctuate among cell types and affect the splicing process independent from its transcriptional function .
ADDITIONAL HIV-1 RNA SPLICING PRODUCTS
The complexity of HIV-1 RNA splicing is even greater than initially described, yielding much more than the originally proposed 40 mRNA species . Whereas most previous studies relied on in-vitro infections, an analysis of spliced transcripts generated in peripheral blood mononuclear cells of HIV-infected individuals revealed several novel splicing events [32,33]. HIV-1 splicing has been analyzed mostly for subtype B isolates, whereas subtype C is the most prevalent clade in the pandemic. A study on three primary subtype C isolates revealed the usage of several new splice sites, including two 3′ss for Rev expression and a new 5′ss . Furthermore, integration of HIV-1 DNA into transcribed cellular genes can trigger the expression of chimeric cell-HIV transcripts that are either polyadenylated in the 5′ LTR or include additional viral sequences (Fig. 1d). New transcripts are formed when splicing occurs at a combination of cellular and viral splice sites, which can be constitutive or cryptic signals . Thus, a multitude of HIV-1 transcripts can be produced in HIV-1-infected cells. NGS sequencing, combined with single-molecule enrichment, provided evidence for at least 109 different spliced RNAs, including a previously unappreciated 1-kb class .
RNAP II can initiate transcription efficiently in the absence of the HIV-1 Tat protein, but transcription will pause after formation and folding of the transacting responsive (TAR) element at the 5′ end of nascent transcripts, yielding short TAR-containing transcripts (Fig. 1e) of ∼60-nt in size [36,37]. Several mechanisms of pausing have been proposed, including the TAR RNA hairpin acting as terminator [37,38], recruitment of negative elongation factor (NELF) [39,40▪], or targeting by microprocessor (DGCR8/Drosha) . Recruitment of microprocessor could label TAR as a substrate for the RNA interference (RNAi) pathway, even though the hairpin does not have the usual pre-miRNA hairpin characteristics [42▪▪]. However, microprocessor seems to be involved in regulation of transcriptional elongation of cellular genes , independent of its cleavage function [43▪]. However, there is a growing body of evidence that 5′ promoter proximal hairpins can encode miRNAs in a microprocessor-independent, but Dicer-dependent manner [44▪,45]. The HIV-1 TAR hairpin has also been suggested to encode an miRNA in several studies (Fig. 1e) [46–48]. This TAR-derived miRNA may target genes involved in apoptosis and cell survival [49▪].
If RNA viruses want to express a miRNA, they have to evolve a strategy to avoid cleavage of the viral RNA genome by the RNAi machinery [41,50,51]. Retroviruses have developed unique strategies to evade such problems (Fig. 2). The retroviruses bovine leukemia virus and bovine foamy virus use an intragenomic RNAP III promoter to produce subgenomic RNA transcripts that are processed into mature miRNAs in a microprocessor-independent manner [52–54]. The 3′ end of the pre-miRNAs is created by RNAP III termination, thus avoiding the need for microprocessor-mediated processing, which would trigger cleavage and degradation of full-length viral RNAs. We recently discovered that HIV-1 can use the ∼60-nt short TAR transcripts to produce TAR miRNAs (Fig. 1e). The 3′ end of the TAR pre-miRNA coincides with the 3′ end of these short transcripts, hereby avoiding the need for microprocessor-mediated processing and consequently HIV-1 genome inactivation (Harwig et al., unpublished observation).
To resume HIV-1 transcription, RNAP II pausing needs to be overcome by recruitment of transcription factors, including the viral Tat protein, to the TAR-paused transcription complexes. HIV-1 thus uses TAR not only to pause RNAP II, but also to recruit positive transcription factors, hereby providing a platform to regulate transcriptional elongation of HIV-1 [55,56]. The Tat protein recruits the positive transcription elongation factor-b (P-TEFb) from the inactive 7SK small nuclear ribonucleoprotein (snRNP) complex bound to the HIV-1 promoter [57–59]. The P-TEFb complex will subsequently hyper-phosphorylate the RNAP II C-terminal domain and NELFs, thus removing all roadblocks for synthesis of full-length transcripts.
ANTISENSE HIV-1 RNA TRANSCRIPTS
Inspired by findings for the other human retrovirus, the human T-cell leukemia virus type 1 (HTLV-1), early RT-PCR experiments documented that antisense transcripts are generated from the HIV-1 provirus [60,61]. Multiple initiation sites were found in the LTR and an antisense polyadenylation signal was described inside the HIV-1 genome (Fig. 1f). The viral Tat protein was reported to be a modulator of these antisense transcripts  and the nuclear factor-κB motifs in the 3′ LTR were also shown to be involved . The production of antisense HIV-1 transcripts was confirmed by high-throughput sequencing analysis [48,63]. The Schopman study proposed that these RNAs may also originate from strong cellular promoters in the flanking chromosomal sequences . Production of both sense and antisense transcripts allows the formation of RNA duplexes that could modulate the effective synthesis of HIV-1 sense transcripts (Fig. 1f). Antisense-mediated suppression of HIV-1 gene expression and replication was proposed to self-limit virus replication , possibly via epigenetic effects on the 5′ LTR promoter . The antisense-sense RNA duplexes can also be recognized by the cellular RNAi machinery to cause silencing of HIV-1 gene expression , thus providing an additional molecular mechanism for viral latency (Fig. 1f).
In addition to a regulatory function, some of the antisense transcripts seem to have the capacity to encode a protein [61,65–67]. CD8 T-cell responses against cryptic epitopes derived from such putative proteins have been detected in chronically infected individuals, which supports translation of the antisense RNA in vivo. Nevertheless, it remained difficult to detect these proteins in HIV-1-expressing cells and codon-optimized constructs and protein-tagging was needed to demonstrate their presence . Based on these studies, a polyadenylated HIV-1 antisense transcript was predicted to encode the antisense protein (ASP), a highly hydrophobic protein that is localized to the plasma membrane . In a subsequent ASP protein localization study, a cytoplasmic distribution in a punctate manner was observed, reminiscent of autophagosomes . It was proposed that ASP induces autophagy, thus explaining the difficulties to detect this protein in infected cells . ASP protein translation is suppressed by the presence of six short upstream open-reading frames, but alternative splicing can remove most of them, thus pointing to a complex regulatory mechanism for ASP synthesis . Using a reporter virus, antisense transcription activity was found to be significantly more abundant in monocyte-derived cells than activated T-lymphocytes .
New findings in the field of RNA biology have recently been made at an accelerated pace because of the development of new powerful techniques like NGS. These discoveries provide new information about some old RNA dogmas. For instance, RNAi was for years considered to be a straightforward mechanism in which a structured RNA is cleaved in the nucleus by microprocessor, exported to the cytoplasm and cleaved by Dicer to produce a miRNA. Recently, numerous exceptions on this pathway have been reported, including microprocessor-independent and Dicer-independent pathways, yielding an intricate network of different pathways to fine-tune miRNA production [42▪▪,70]. Retroviruses can thus expand their RNA arsenal and produce miRNAs via the microprocessor-independent pathway, thereby avoiding cleavage of their RNA genome [52,54] (Harwig et al., unpublished observation). NGS also revealed a novel 1-kb class of spliced mRNAs . It has yet to be confirmed whether these 1-kb mRNAs have a function or whether they result from infrequent usage of cryptic splice sites in the HIV-1 RNA genome.
Much mechanistic detail of HIV-1 transcription is available, but certain questions remain. A vital step in HIV-1 transcription is the recruitment of P-TEFb from inactive 7SK snRNP complexes by Tat, which overcomes roadblocks exerted by negative factors, RNA structure and microprocessor [41,43▪,71]. Characterization of this novel regulatory mechanism of transcription elongation could lead to a more comprehensive understanding of HIV-1 latency . Most HIV-1 latency studies focus exclusively on the mechanism of transcriptional initiation, but latency is likely to be much more complex. Not only mechanistic insight on the synthesis of all HIV-1 RNA classes, including the TAR-encoded miRNA, antisense RNA and siRNAs, but also knowledge about their function remains important. This may also facilitate the development of novel therapeutic strategies to purge latent HIV-1 proviruses. Such strategies should shrink the viral reservoir and may eventually trigger virus clearance and thus provide a cure for the patient.
HIV-1 RNA studies identified novel therapeutic options. For instance, splicing has been proposed as target for the development of new therapeutic antiviral agents . This includes splicing inhibitors, but excessive HIV-1 RNA splicing or a disruption of the balance between the different RNA forms is also detrimental to virus replication [27,74]. Thus, basic HIV-1 RNA studies remain important to fuel the translational pipeline toward antiviral therapies.
Financial support and sponsorship
This work was supported by the Netherlands Organisation for Scientific Research (Chemical Sciences Division; NWO-CW; Top grant).
Conflicts of interest
There are no conflicts of interest.
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