The HIV-1 genome encodes viral proteins in a single unspliced polycistronic transcript of 9.2 kb (Purcell and Martin, 1993; Bohne et al., 2005; Stoltzfus, 2009). The HIV-1 US vRNA contains at least four 5’ splice donor sites and ten 3’ splice acceptor sites and the host cell spliceosome combines multiple splice donors and acceptors, that facilitates alternative splicing resulting in the production of over 100 HIV-1 vRNA transcripts of 4kb HIV-1 SS vRNA and 2 kb HIV-1 MS vRNA (Purcell and Martin, 1993; Ocwieja et al., 2012; Emery et al., 2017; Pasternak and Berkhout, 2021). HIV-1 US vRNA encodes for the structural precursor proteins Gag and Gag-Pol and serves as genomic RNA. The 4kb SS vRNA and 2kb MS vRNA encode for Env and accessory proteins (Vif, Vpu, Vpr), and Tat, Rev and Nef respectively ( Figure 1 ). Early in the infection, HIV-1 vRNA processing depends fully on the host spliceosome machinery and only the HIV-1 MS vRNAs are exported to the cytoplasm and translated into the early proteins Tat and Rev, while the larger HIV-1 SS and US vRNAs accumulate in the nucleus (Stoltzfus, 2009; Karn and Stoltzfus, 2012). Rev protein then facilitates the cytoplasmic export of the larger HIV-1 SS and US vRNAs (See section 3.2) (Schwartz et al., 1992; Schwartz et al., 1992; Karn and Stoltzfus, 2012). The expression levels of Tat and Rev, which are encoded by the multiply spliced transcripts, determine a critical switch to a positive feedback loop of viral transcription, RNA export and processing, and protein production, which drive the production of viral particles during active infection. Tat binds the TAR RNA secondary structure in the nascent transcript at the 5’ LTR and recruits P-TEFb, leading to phosphorylation of the paused RNA Polymerase II and promotion of transcription elongation (Roy et al., 1990; Mancebo et al., 1997; Sedore et al., 2007). Absence of Tat protein below a critical threshold level dramatically reduces vRNA production and breaks the feedback loop necessary for sufficient transcriptional output necessary to maintain active viral production (Das et al., 2011; Kamori and Ueno, 2017). As Tat and Rev become available, a feedback loop of transcription activation and export of US and SS vRNA allows for efficient viral production.

Splicing of the HIV-1 US vRNA starts by recognition of sequences flanking the introns by the spliceosome. The US vRNA together with the proteins that constitute the spliceosome form an RNP that initiates and regulates splicing (Moore and Sharp, 1993). Regulation of HIV-1 vRNA alternative splicing is mediated by cis and trans-acting elements that allow for control of HIV-1 gene expression [reviewed in (Stoltzfus and Madsen, 2006; Caputi, 2011)]. Cis-acting splicing regulatory elements that control HIV-1 splicing function as exonic or intronic splicing enhancers (ESEs, ISEs) or silencers (ESSs, ISSs). The HIV-1 genome contains a total of 4 ESSs, 1 ISS, 1 ISE and 6 ESEs (Bohne et al., 2005; Stoltzfus, 2009; Karn and Stoltzfus, 2012). The most well-known family that act on exonic splicing enhancers (ESEs) are SR proteins that promote active splicing upon recognition of enhancer sequences by recruiting the splicing complex and have a role in mRNA export, stability and translation (Graveley, 2000; Caputi and Zahler, 2002; Huang and Steitz, 2005; Fukuhara et al., 2006; Stoltzfus, 2009; Jablonski and Caputi, 2009). Negative regulation of splicing depends largely on the activity of hnRNPs, proteins that act on exonic splicing silencers (ESSs) by preventing binding of the spliceosome complex (Caputi et al., 1999; Caputi and Zahler, 2002; Jablonski and Caputi, 2009; Sertznig et al., 2018). Tat and Rev have also been shown to promote or inhibit splicing of vRNAs by direct binding to HIV-1 US vRNA or by regulation of host splicing factors, although the exact contribution of Rev to alternative splicing is still unclear (Kjems et al., 1991; Kjems and Sharp, 1993; Stoltzfus, 2009; Jablonski et al., 2010; Mueller et al., 2018).

Trans-acting elements such as secondary structures present across the HIV-1 US vRNA also regulate HIV-1 splicing by hiding or exposing splicing sites to the splicing machinery (Saliou et al., 2009). Splicing donors and acceptors can be contained within RNA secondary structures that facilitate their auto-regulation. For example, splicing donor D1 is present in a stem loop RNA structure that negatively regulates its accessibility to the splicing machinery and impacts splicing efficiency (Abbink and Berkhout, 2008). As well, splicing acceptors A1 and A3 are known to be contained within a secondary RNA structure shown to be important for promoting HIV-1 US vRNA splicing (Ocwieja et al., 2012).

The 3 classes of HIV-1 vRNA splicing variants, US 9kb vRNA, SS 4kb vRNA and MS 2kb vRNA transcripts, are exported and processed by different mechanisms ( Figure 1 ) (Caputi, 2011; Karn and Stoltzfus, 2012; Stoltzfus, 2009). In the early phases of the infection, MS vRNA transcripts are exported via canonical NXF1/NXT1-mediated export pathways (Cullen, 2003; Kohler and Hurt, 2007). Rev, encoded by the exported MS vRNA, plays a central role in the nucleocytoplasmic export of US and SS vRNA via a CRM1-dependent mechanism (Schwartz et al., 1992; Schwartz et al., 1992). Rev contains a nuclear localization signal (NLS) that facilitates its entry into the nucleus via an interaction with Importin B (Henderson and Percipalle, 1997) where it directly targets HIV-1 US and SS vRNAs for export via interaction with the trans-acting Rev responsive element (RRE), present within the env intron (Malim et al., 1989; Rausch and Le Grice, 2015). Once a sufficient threshold of Rev is achieved in the nucleus, multiple Rev molecules multimerize and bind the RRE (Malim and Cullen, 1991; Rausch and Le Grice, 2015). The resulting Rev-RRE RNP complex recruits host cell protein CRM1 to the nuclear export signal (NES) of Rev, that allows for its export to the cytoplasm (Neville et al., 1997; Askjaer et al., 1998). CRM1 and Rev/RRE form an RNP containing host proteins such as Ran-GTP, components of the nuclear pore complex and other RBPs such as eIF5a (an adaptor for Rev-RRE and CRM1), RNA helicases DDX1 and DDX3 (that facilitate Rev-RRE-Crm1 shuttling) and Sam68 that binds the Rev-RRE-CRM1 RNP promoting its export into the cytoplasm and counteracting inhibiting signals (Ruhl et al., 1993; Yedavalli et al., 2004; Fang et al., 2005; Modem et al., 2005; Modem and Reddy, 2008; Zhou et al., 2009; Monette et al., 2011). Other host proteins [Reviewed in (Luo et al., 2016; Truman et al., 2020)] have also been reported to be required for the regulation and efficient export of Rev-RRE complexes. The HIV-1 US vRNA contains cis-acting AU-rich instability sequences (INS) that regulate mRNA stability, export and translation and are counteracted by Rev (Cochrane et al., 1991; Maldarelli et al., 1991; Schwartz et al., 1992; Olsen et al., 1992; Mikaelian et al., 1996). Although the exact function of INS is yet to be fully understood, they contain AU-rich motifs, a hallmark of unstable RNA elements (Hentze, 1991). Several host proteins such as PABP1, hnRNP A1, PSF and p54 have been found to bind INS and possibly prevent access of the splicing machinery, supporting nuclear retention of INS-RRE containing HIV-1 vRNAs (Black et al., 1996; Afonina et al., 1997; Zolotukhin et al., 2003). Interestingly, recent reports suggest that both US and SS vRNAs can also be exported into the cytoplasm by facilitated diffusion mechanisms irrespective of the transport pathway and that the lack of Rev can be compensated by the overexpression of host proteins such as UPF1 (Ajamian et al., 2015; Chen et al., 2020).

In eukaryotic cells, RNA surveillance mechanisms in the nucleus and cytoplasm identify and degrade aberrant mRNAs and function as a quality control mechanisms that prevent the accumulation of potentially toxic truncated proteins. In the nucleus, the RNA exosome complex regulates mRNA quality control (reviewed in (Kilchert et al., 2016)). Following export to the cytoplasm, aberrant RNAs are degraded via the nonsense-mediated decay (NMD) pathway. Other pathways such as the no-go decay (NGD) pathway, non-stop decay (NSD) and Staufen-Mediated mRNA Decay (SMD) also regulate cytoplasmic RNA quality control. The HIV-1 US vRNA contains large introns, a long UTR and multiple AU-rich instability regions in the 3’UTR (Toro-Ascuy et al., 2016). However, not only does HIV-1 evade RNA quality control, the virus hijacks and recruits host mRNA decay proteins to regulate multiple phases of its own HIV-1 mRNA processing (reviewed in (Toro-Ascuy et al., 2016)). UPF1, the central protein involved in NMD, has been shown to interact with US HIV-1 vRNA, promote in nucleocytoplasmatic transport and translation and is incorporated in virus particles (Ajamian et al., 2008; Ajamian et al., 2015). Knockdown of UPF1 resulted in decreased viral production and a reduction in the infectivity of released particles and overexpression of UPF1 resulted in enhanced viral production (Ajamian et al., 2008). These roles of UPF1 in vRNA metabolism were independent of its roles in NMD and were mediated by its ATP-ase domain. UPF2, SMG6 and UPF3A, other proteins involved in NMD, were found to be negative regulators of HIV-1 gene expression and are excluded from HIV-1 US vRNA RNPs (Ajamian et al., 2015; Rao et al., 2019). Small interference RNA-mediated depletion of UPF2 and SMG6 resulted in enhanced viral replication in primary monocyte-derived macrophages (Rao et al., 2019). Staufen1, involved in SMD, also mediates multiple steps of vRNA processing including vRNA translation, trafficking and assembly (Chatel-Chaix et al., 2004; Dugré-Brisson et al., 2005; Rao et al., 2019).

Viral RNAs, like host cell RNAs, are subjected to epitranscriptomic modifications that regulate their processing, export, and further translation. The most abundant mRNA modification is N6-methyladenosine (m6A), catalyzed by methyltransferase complexes, that occurs mainly in the consensus motif RRACH in the 5 or 3’UTR of the mRNA (Riquelme-Barrios et al., 2018; Jiang et al., 2021). Addition of an N6-methyladenosine is catalyzed by writers METTL3, METTL14 and WTAP and the methyl group can be also removed by erasers such as ALKBH5 and FTO (Riquelme-Barrios et al., 2018; Jiang et al., 2021). The m6A addition to the mRNA are recognized by the reader proteins including the predominantly nuclear YTHDC1 and the cytoplasmic YTHDC2, YTHDF1, YTHDF2 and YTHDF3 proteins that regulate mRNA processing, export, stability and translation of m6a-containing mRNAs (Meyer and Jaffrey, 2014; Jiang et al., 2021). In context of the HIV-1 US vRNA, methylation of nascent vRNA transcripts is a critical step in HIV-1 vRNA metabolism (reviewed in (Riquelme-Barrios et al., 2018)). Lichinchi et al. reported that the viral RNA contains multiple m6a editing sites, including at the RRE, and knockdown of methyl eraser ALKBH5 resulted in increased abundance of methylated HIV-1 US vRNA, enhanced Rev-RRE interaction and vRNA export, and increased viral gene expression (Lichinchi et al., 2016) The role of m6A methylation in Rev-RRE mediated vRNA export is, however, controversial, as other studies failed to detect presence of m6A editing sites at the RRE (Tirumuru et al., 2016; Kennedy et al., 2017; Lu et al., 2018). Besides, N6-adenosine writer (METTL3, METTL14) and reader proteins (YTHDF1-3) have been shown to modulate HIV-1 viral replication by affecting Gag expression (METTL3, METTL14) and decreasing viral genomic RNA and inhibiting reverse transcription (Tirumuru et al., 2016; Lu et al., 2018). The m6A reader YTHDF proteins also influence HIV-1 US vRNA expression, although they are reported to have conflicting effects on HIV-1 viral gene expression that remain to be characterized (Kennedy et al., 2017; Lu et al., 2018). These proteins are also implicated in the degradation of incoming genomic vRNA (Tirumuru et al., 2016). More recently it has been shown that m6A addition to HIV-1 vRNA also influences its splicing and stability and affects gene expression mediated by YTH family proteins YTHDF1 and YTHDF2, which bind and stabilize vRNAs, regulate alternative splicing and overall increase HIV-1 gene expression (Tsai et al., 2021). HIV-1 vRNAs can also be regulated by addition of other modifications such as 5-methylcytosine (m5C). Courtney et al. showed in their recent study that addition of m5C to the HIV-1 US vRNA is mediated by writer NSUN2 and its absence influences alternative splicing, export and translation of Gag-containing transcripts, and leads to decreased gene expression (Courtney et al., 2019). Interestingly, HIV-1 infection also causes changes in the epitranscriptomic profile of host cell RNAs and hence influences their overall downstream processing (Cristinelli et al., 2021; Zhang et al., 2021).

The translation of viral proteins and viral assembly, followed by budding and virion maturation, are the last steps in the HIV-1 life cycle in order to produce infectious viral particles ( Figure 1 ). Similar to host mRNAs, HIV-1 vRNAs contain a 5’ cap structure necessary for translation initiation (Ohlmann et al., 2014; Guerrero et al., 2015). In the case of US vRNA, its fate, whether it serves as genomic vRNA or template for translation initiation, is hypothesized to depend on the presence of a secondary structure in the 5’ UTR that can present two conformations: branched multiple hairpins (BMH) that favors viral RNA encapsidation, or long distance interaction (LDI) that favors translation initiation (Huthoff and Berkhout, 2001; Berkhout et al., 2002; Ooms et al., 2004). The TAR structure in the 5’UTR has been shown to pose a barrier to cap-dependent translation initiation due to the presence of secondary RNA structures. This is why RNA helicases and other host proteins such as such as TRBP, Staufen1 or helicase DDX3 play an important role in preserving an efficient translation of the HIV-1 vRNA. Translation of HIV-1 US vRNA can also be initiated via a cap-independent mechanism that involves an internal ribosome entry site (Monette et al., 2013). The HIV-1 US vRNA contains two IRES sequences, one is present in the 5’UTR, and the second one is present within the Gag region. Translation initiation from IRES sequences is crucial in later stages of the viral cycle, where cap-dependent translation initiation is impaired (Amorim et al., 2014). In addition, HIV-1 restriction factors can act by modulating the synthesis of viral proteins via distinct mechanisms, such as Schalfen protein SLFN11, that prevents vRNA translation by codon usage-based inhibition in response to interferon induction (Li et al., 2012).

Translation of viral proteins is also highly regulated by small non-coding RNAs, such as microRNAs. For example, miR29a targets HIV-1 US vRNA leading to translational repression, miR133b, miR138-5, miR326, miR149-5p and miR92a-3p have been shown to reduce HIV-1 replication and miR125 expression negatively correlates with HIV-1 infection (Houzet et al., 2012; Mantri et al., 2012). MicroRNAs can also negatively control HIV-1 gene expression and indirectly targeting essential co-factors for HIV-1 replication (reviewed in (Balasubramaniam et al., 2018; Sadri Nahand et al., 2020)) As so, there is ongoing discussion whether the HIV-1 genome encodes functional microRNAs (Ouellet et al., 2013; Balasubramaniam et al., 2018; Sadri Nahand et al., 2020). A recent report described the presence of microRNA miR-n367, found in the nef region of the HIV-1 genome, that efficiently targets and inhibit Nef protein synthesis, affecting overall HIV-1 gene expression (Omoto et al., 2004).

Regulation of alternative splicing in context of HIV-1 gene expression mainly depends on the action of host factors such as SR and hnRNP proteins that bind, respectively, to exonic splicing enhancer and silencer motifs on the HIV-1 vRNA and modulate splicing efficiency (Caputi et al., 1999; Jablonski and Caputi, 2009; Ohe and Hagiwara, 2015; Dlamini and Hull, 2017). Although SR and hnRNP proteins are part of the host cell splicing machinery, the specificity of HIV-1 towards certain classes and their essential role in efficient viral production make SR proteins an attractive target in the search of novel HIV-1 RNA processing modulating drugs. Therapeutic targeting of HIV-1 alternative splicing can either pursue release or suppression of HIV-1 molecular blocks. Repurposing inhibitors of HIV-1 splicing for cure research becomes an interesting approach to be considered for “block-and-lock” cure approaches aimed at permanently silencing HIV-1 viral production (Yeh et al., 2020; Moron-Lopez et al., 2021), and it might also be relevant in “shock-and-kill” strategies that emphasize accumulation of intron-containing vRNA, which in turn activates innate immune pathways and selectively sensitizes the cell to apoptosis (Berg et al., 2012; Li et al., 2016; Fong et al., 2017; Rao et al., 2021) (see section 4). As so, current cure approaches aimed at inducing HIV-1 transcription via latency reversal agents may also be combined with therapeutics that promote HIV-1 splicing and accumulation of MS vRNA, that can in turn restore the Tat/Rev feedback loop and hence fuel transcription, export and translation of vRNA species encoding immunogenic structural proteins.

Multiple small molecules have been described over the last few decades that hinder HIV-1 splicing, mostly by targeting host SR proteins and modulating their phosphorylation status, preventing their role in spliceosome assembly, or inhibiting their binding to the nascent RNA (Bakkour et al., 2007; Ohe and Hagiwara, 2015; Dlamini and Hull, 2017). For example, Bakkour and colleagues performed a large scale screening of putative compounds able to inhibit ESE-dependent splicing by targeting specific SR proteins, preventing spliceosome assembly and leading to HIV-1 splicing inhibition (Bakkour et al., 2007). Tetracyclic indole derivative IDC16 and 1C8 showed significant reduction in HIV-1 replication by directly targeting SR protein ASF/SF2 and SRSF10, respectively, and hence negatively affecting alternative splicing of HIV-1 vRNA (Bakkour et al., 2007; Cheung et al., 2016; Shkreta et al., 2017). Because of the putative in vivo adverse effects of these compounds, other derivatives were synthesized that would potentially be more target-specific and have less side effects. The most potent compound was ABX464, that inhibits HIV-1 vRNA splicing but also interferes with cap-binding proteins and Rev-dependent export (Campos et al., 2015; Vautrin et al., 2019), and has recently been shown to temporarily decrease Total HIV-1 DNA in ART-suppressed PLWH (Moron-Lopez et al., 2021). The authors hypothesize that ABX464 could be able to induce aberrant HIV-1 transcripts that can be recognized by the immune system, leading to selective killing of infected cells (Moron-Lopez et al., 2021). More recently, an anti-HIV-1 stilbene quinolone (GPS491) has been shown to modulate the abundance and function of host SR proteins, hence altering the HIV-1 splicing pattern and inhibiting the HIV-1 life cycle (Dahal et al., 2022). Lastly, the design of drug screens coupled with transcriptomic analysis can broaden the range of HIV-1 splicing targeting compounds by multiple other mechanisms. Such was the case of FDA-approved JAK1 inhibitor Filgotinib, that has recently been reported to also suppress HIV-1 splicing and prohibit HIV-1 gene expression in cells obtained from PLWH (Yeh et al., 2020).

Although there has been less research development into the identification of agents able to release HIV-1 splicing blocks, future studies should deepen into discovery and repurposing of agents that act on the host cell alternative splicing mechanisms employed by the virus during vRNA processing. For instance, HIV-1 splicing blocks can potentially be released by altering the function of hnRNP proteins that bind inhibitory exonic splicing silencers (Carabet et al., 2019; Solano-Gonzalez et al., 2021) or inhibit surveillance mechanisms that promote vRNA degradation (Durand et al., 2007; Popp and Maquat, 2015). Another possibility to enhance HIV-1 splicing is by modulating splicing site usage by small molecules or RNA interference therapeutics (see Section 5.3). It is worth noting that changes in the pattern of the tightly-regulated splice site use on the HIV-1 vRNA template can alter the ratios of US/MS vRNAs to suboptimal levels, that will, in fact, result in overall inhibition of HIV-1 gene expression. For example, small molecule Digoxin promotes oversplicing and causes an overall increase in total MS vRNA, but also alters the splicing site usage within the MS vRNA and causes a significant decrease in Rev-containing MS vRNA and Rev protein, that eventually results in inhibition of HIV-1 viral production (Wong et al., 2013). Other compounds, such as 8-azaguanine, are also able to promote oversplicing and cause an increase in MS vRNA, but also target and inhibit Rev nuclear import, which prevents export of Rev-dependent vRNA and production of viral structural proteins (Wong RW et al., 2013).

In conclusion, vRNA alternative splicing is a dynamic process that can be potentially utilized as therapeutic alternative. The design of HIV-1 RNA biology targeting strategies, however, would need to address the the intricate mechanisms that regulate the vRNA/protein equilibrium.

Given its essential role in viral protein translation, vRNA nucleocytoplasmic export also represents an attractive target to modulate HIV-1 vRNA metabolism. Export and translation of HIV-1 US and SS vRNA relies greatly on the interplay between Rev and the RRE motif in the vRNA (Rausch and Le Grice, 2015). Inhibition of Rev by small molecules has been studied for decades as potential therapeutics to inhibit viral production (Wong R et al., 2013). The use of small molecules in cure studies that target viral proteins such as Rev or Rev-RRE interaction is promising because of their potential high specificity and low toxic side effects. In addition, inhibition of Rev-mediated vRNA export has a very strong effect on silencing HIV-1 gene expression and can present a new therapeutic angle in block-and-lock cure approaches (Wong R et al., 2013). Complementary to latency reactivation-based cure interventions, accumulation of viral RNA by inhibiting export and promoting nuclear retention of vRNAs can potentially trigger nuclear innate immune signaling pathways and lead to latency reversal and induced cell death in latently infected cells (see section 4) (Berg et al., 2012; Li et al., 2016; Fong et al., 2017; Rao et al., 2021).

Initially purposed as possible antiretroviral compounds, a variety of drugs have been identified that efficiently alter Rev-RRE binding and lead to inhibition of viral production. Early studies on pharmacological disruption of Rev-RRE interaction by use of small molecules unveiled RNA intercalating drugs such as Pyronin Y, aminoglycoside antibiotics and aromatic heterocyclic compounds that bind to the RRE and prevent Rev-dependent export (Schröder et al., 1993; Nishizono and Nair, 2000; DeJong et al., 2003). In a screen reported by Prado et al., several FDA-approved compounds were discovered to inhibit Rev-RRE binding and alter HIV-1 gene expression, including Clomiphen, an estrogen receptor modulator used for the treatment of infertility in women (Prado et al., 2016). The same group subsequently described another compound, benzofluorenone or Benfluron, that targets Rev-RRE binding by binding to the RRE and inhibits viral gene expression (Prado et al., 2018). Recently, a drug discovery study reported the design of the multi-target small molecule 1,4-substituted terphenyl compounds that inhibit HIV-1 transcription and Rev-dependent export by binding to the RRE element (Medina-Trillo et al., 2020). Nevertheless, while some of these drugs are very effective and sensitive, the relative specificity and possible secondary binding targets (i.e. non-HIV RNAs) of these small compounds is, to our knowledge, poorly described. Ideally, strategies that pursue reservoir elimination and cause limited to no toxicity would benefit from availability of drugs that target vRNA or proteins exclusively.

Complete functionality of Rev depends greatly on its ability to translocate between the cytoplasm and the nucleus, a process mediated by the action of host cell proteins, such as CRM1 and Importin B. The antibiotic leptomycin B was the first compound described to interfere with Rev-CRM1 mediated export of HIV-1 vRNA and inhibit HIV-1 gene expression (Wolff et al., 1997). Later, other compounds called selective inhibitors of exportin-1 (CRM1) nuclear export (SINE) have been synthetized and extensively characterized for their role in suppressing HIV-1 replication, including FDA-approved SINE compound selinexor (Daelemans et al., 2002; Boons et al., 2015; Daelemans et al., 2015). In addition, inhibition of Importin-mediated nuclear import by compound Ivermectin disrupts Rev nuclear import and hence prevents export of Rev-dependent vRNA species, resulting in suppression of HIV-1 replication (Wagstaff et al., 2012). Other compounds, such as 8-azaguanine and 2-quinolone, can also inhibit Rev translocation to the cytoplasm, or lead to decrease in Rev production, resulting in accumulation of intron-containing vRNA in the nucleus (Wong RW et al., 2013). Although some of these drugs, such as Selenixor, are approved by drug administrations, their use in high doses causes reported severe side effects (Grade ≥ 3) in a considerable percentage of participants (Neupane et al., 2021). Their potential use and associated toxicity at lower doses and in combination with other drugs is yet to be studied.

Less is known about therapeutics that enhance Rev-mediated export and promote translation of vRNAs. Promoting vRNA export and viral protein production is of interest in shock-and-kill approaches that aim at reversing post-transcriptional blocks and enhance immune-mediated clearance (see Section 4). Lastly, viral RNA export is influenced by epitranscriptomic modifications of the vRNA. Inhibitors that regulate m6a methylation of viral RNAs would also be interesting to investigate in context of HIV-1 curative approaches since epitranscriptomic modifications influence viral gene expression and modulate antiviral immune responses. For example, activators of the methyl writers could be utilized to promote viral gene expression and be used as latency reversing agents (Selberg et al., 2021).

RNA interference therapeutics are also a promising approach to target and eliminate HIV-1 infected cells via gene therapy strategies (reviewed in (Scarborough and Gatignol, 2017)). Several steps of HIV-1 replication can directly be inhibited by delivery of antiviral RNAs (shRNAs or miRNAs), RNA decoys or aptamers that directly target the LTR, Tat, Rev or Gag proteins, or secondary RNA structures such as TAR or RRE. Alternative splicing of HIV-1 vRNAs can be modulated by blocking splicing sites, causing interference with binding of the spliceosome complex and other co-factors required for efficient splicing. Masking of a specific splicing site can be achieved by complementary antisense RNA sequences (Brosseau et al., 2014). Binding of these sequences to the specific ss-containing motif in the HIV-1 vRNA prevents the access of the splicing machinery leading to splicing inhibition (Brosseau et al., 2014). One example is targeting of the U1 snRNA by U1 interference RNAs. These U1i RNAs are designed to interfere with polyadenylation or enhance splicing of HIV-1 RNA, and they are able to efficiently inhibit HIV-1 replication (Del Corpo et al., 2019). As well, antisense RNA sequences can be found in the form of peptide-nucleotide fused molecules, in which the antisense RNA is fused to a protein involved in splicing regulation or inhibition. A similar approach is the use of bi-functional oligonucleotides that include a target-specific antisense RNA and a second oligonucleotide whose function is to recruit proteins or RNA-protein complexes that interfere with splicing (Brosseau et al., 2014).

Up or downregulation of microRNAs that target specific proteins involved in HIV-1 replication can be used as a potent therapeutic tool for promotion or inhibition of HIV-1 gene expression (Zhang, 2009; Swaminathan et al., 2014). Antisense oligonucleotides that inhibit the effect of miRNA have already been used in context of other diseases, such as Hepatitis C Virus infection in which its replication can be inhibited by targeting mi-R122 (Janssen et al., 2013). However, given their known pleiotropic effects and limitations to the delivery methods available, the use of microRNAs as a therapy is debatable (Swaminathan et al., 2012). Moreover, modulating miRNA as a potential therapy in HIV-1 cure studies has been challenged recently as it has been shown that inhibition of miRNAs previously identified to have a role in latency did not lead to latency reactivation (Lopez-Huertas et al., 2019).