On the generation of the MSD-Ѱ class of defective HIV proviruses
Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are replication-incompetent due to a variety of genetic defects. Most defective proviruses (around 90%) contain large internal deletions or are G-to-A hypermutated, resulting in destruction of most if not all viral open reading frames, which is consistent with the idea that cytotoxic T cells (CTLs) effectively remove cells that produce viral antigens. An intriguing subclass of defective proviruses (around 10%) that are consistently detected in such patients carry a small deletion or a point mutation in a relatively precise and well conserved region near the 5ʹ end of the HIV genome, in the area that encodes the major splice donor (MSD) site and the packaging signal Ѱ in the viral RNA genome. Why this subclass of proviruses is defective has never been properly understood. We now propose a mechanistic scenario for how these MSD-Ѱ mutations can prevent viral protein expression. Based on ample results in literature, we argue that MSD inactivation triggers the activity of the 5ʹ-polyadenylation site, resulting in the production of ultra-short non-protein-coding HIV transcripts.
KeywordsHIV Reservoir Defective genomes Splicing Polyadenylation
cytotoxic T lymphocyte
major splice donor
- polyA or pA
cleavage and polyadenylation specificity factor
upstream splicing enhancer
Defective HIV proviruses are produced in large quantities during natural infection due to mutations introduced during the error-prone process of HIV reverse transcription and APOBEC-induced hypermutation [1, 2]. In untreated patients, this process is counterweighted by the unhindered production of new intact proviruses by virus replication, but in patients on suppressive antiretroviral therapy (ART), defective proviruses accumulate to very high levels . Bruner et al.  showed that even in patients who started ART during early infection, 93% of all proviruses were defective, and if HIV replication was blocked by ART during chronic infection, this percentage of defective HIV genomes reached 98%. Similar percentages of defective proviruses have been reported by other groups [5, 6]. It is thought that ART selects for defective proviruses due to continuous cytotoxic T cell (CTL)-mediated surveillance for cells that produce foreign viral antigens, which in ART-treated patients is not counterweighted by virus replication [7, 8]. CTL pressure does decrease after initiation of ART due to decreased antigen exposure, but does not disappear completely .
Although defective HIV proviruses are considered by many clinically irrelevant, they do frustrate the accurate measurement of the clinically relevant reservoir of intact HIV genomes that forms a major barrier to curing infected individuals. Furthermore, defective proviruses can be expressed and recognized by the host immune system, which may “distract” CTLs from eliminating the latent reservoir [7, 8, 10] and contribute to the increased levels of immune activation and inflammation on ART [11, 12]. It is therefore important to analyse the pool of defective HIV genomes in greater detail .
The persistence of the MSD-Ѱ mutated proviruses during ART suggests an inability to produce viral proteins, but no explanation for such a production defect was yet presented. In fact, MSD inactivation was shown to induce alternative RNA splicing events that can give rise to the expression of viral proteins, e.g. Tat and Rev, or aberrant proteins . Although the level of gene expression can be reduced for these HIV genomes, e.g. due to reduced Tat levels, the corresponding host cells will still be recognized and cleared by CTLs. This MSD-Ѱ class thus far could not be fully understood. Based on extensive literature findings in the field of HIV molecular biology that thus far were ignored, we report an attractive, yet simple explanation for the protein production defect of MSD-Ѱ mutated HIV genomes.
We started by a sequence alignment of the previously reported MSD-Ѱ mutants to identify the critical motifs that were consistently affected. For instance, Fig. 1 shows the deletions reported in the study by Ho et al. . All deletions include the MSD motif, whereas Ѱ sequences do frequently remain present, arguing for a functional role of the MSD motif that controls HIV-1 RNA splicing. In addition, also proviruses with point mutations were reported in the MSD region, e.g. affecting the critical intronic GU dinucleotide of the splice donor site (UG-GU mutated to UG-GG) . Importantly, it was demonstrated that such a point mutant can exhibit a severe replication defect in reconstructed viruses .
With this mechanistic background, it can easily be understood that inactivation of MSD by mutation or deletion can trigger an effective shutdown of HIV transcription through activation of the 5ʹ pA site. Thus, it follows that a small characteristic HIV transcript of 97 nucleotides plus polyA tail will be synthesized in the cells that carry a MSD-mutated provirus, as illustrated in Fig. 2b. This non-coding HIV transcript that encompasses the TAR motif was indeed reported by the Proudfoot laboratory back in 1995 . This short TAR transcript is polyadenylated at the 5ʹ-pA site and was confirmed in studies on the regulatory role of the polyA hairpin structure [25, 26, 27]. As this short transcript encodes the complete TAR element it may be processed into the TAR miRNA, of which the precise role has not been determined yet [28, 29].
Although removal of all HIV coding capacity by a large internal deletion is also very effective in preventing HIV gene expression, MSD inactivation is arguably the most elegant way to produce non-expressing proviruses, the host cells of which will survive under massive CTL pressure. This 5ʹ pA activation model seems much more relevant to explain the loss of HIV protein production than the proposed model of alternative usage of splice sites, which at best could reduce and not interrupt HIV protein expression. Although the inactivation of HIV splice sites can indeed trigger the usage of new splice sites [30, 31, 32, 33], this does not prevent protein translation and consequently CTL recognition.
Consistent with the mechanistic model presented in Fig. 2a, short TAR-containing HIV transcripts are produced in treated patients at a level at least 10-fold higher than extended HIV transcripts [34, 35]. These authors did assume that short TAR-containing transcripts represent abortive transcripts. Our model predicts that short transcripts that are polyadenylated at the 5ʹ-pA site may significantly contribute to this small RNA pool.
One could argue that the same end result, that is activation of the 5ʹ-pA site, could be achieved by weakening or opening of the local polyA hairpin structure that suppresses CPSF binding [17, 25]. However, this would require surgical precision for the provirus mutation as the sequence elements that control the polyadenylation process should not be affected. These include the canonical AAUAAA signal and the actual cleavage site that are embedded in the polyA hairpin [24, 36]. This may explain why 5ʹ pA-activation by hairpin destabilization is not observed, at least not frequently.
The 5ʹ pA activation model does not only apply to the relatively minor MSD-Ѱ class of defective HIV proviruses, it will also relate to those members of the two major classes of defective proviruses with large deletions or hypermutated genomes in which the MSD is destroyed. The latter two classes were supposed to be defective by inactivation of one or multiple open reading frames, but 5ʹ pA activation provides a dominant mechanism to abort any viral protein expression. This new mechanism may therefore also be very relevant for scenario’s dealing with the relevance of ongoing viral protein expression [6, 7].
The generation of variant HIV genomes is the result of two independent processes: mutation and subsequent selection of the most fit virus. In this case, host cells carrying a HIV provirus with a protein production defect will survive preferentially under intense CTL pressure that has been built in infected individuals during months or years of unsuppressed virus replication. The mechanistic MSD-pA scenario that we propose suggests that the cells with proviruses carrying MSD-inactivating mutations are selected because of their non-protein-expressing phenotype. Although not likely to be of decisive influence, the presence of hotspots of viral recombination may also influence the type of MSD deletions that occur (Fig. 1). In particular, this MSD-Ѱ part of the viral RNA genome is highly structured and can cause the viral Reverse Transcriptase to pause , which can induce recombination and MSD deletion. In any case, the subsequent selection of cells that do not express viral proteins is the key event.
A complete understanding of the pool of defective HIV proviruses remains of critical importance for accurate measurement of the latent virus reservoir. There may be multiple ways to inactivate HIV and we here describe that—besides prominent deletions and hypermutations—more subtle changes like MSD mutations can also destroy HIV expression.
Supported by AIDS Fonds Grant 2012025 (AOP) and NIH RO1 Grant 1R01AI145045IH (BB).
BB initiated this analysis based on discussions with AOP and ATD, BB wrote the first version of the manuscript, which was corrected by AOP and ATD. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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