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Title: Identifying the Important HIV-1 Recombination Breakpoints}
Article:
Abstract:
Recombinant HIV-1 genomes contribute significantly to the diversity of variants within the HIV/AIDS pandemic. It is assumed that some of these mosaic genomes may have novel properties that have led to their prevalence, particularly in the case of the circulating recombinant forms( CRFs). In regions of the HIV-1 genome where recombination has a tendency to convey a selective advantage to the virus, we predict that the distribution of breakpoints—the identifiable boundaries that delimit the mosaic structure—will deviate from the underlying null distribution. To test this hypothesis, we generate a probabilistic model of HIV-1 copy-choice recombination and compare the predicted breakpoint distribution to the distribution from the HIV/AIDS pandemic. Across much of the HIV-1 genome, we find that the observed frequencies of inter-subtype recombination are predicted accurately by our model. This observation strongly indicates that in these regions a probabilistic model, dependent on local sequence identity, is sufficient to explain breakpoint locations. In regions where there is a significant over-( either side of the env gene) or under-( short regions within gag, pol, and most of env) representation of breakpoints, we infer natural selection to be influencing the recombination pattern. The paucity of recombination breakpoints within most of the envelope gene indicates that recombinants generated in this region are less likely to be successful. The breakpoints at a higher frequency than predicted by our model are approximately at either side of env, indicating increased selection for these recombinants as a consequence of this region, or at least part of it, having a tendency to be recombined as an entire unit. Our findings thus provide the first clear indication of the existence of a specific portion of the genome that deviates from a probabilistic null model for recombination. This suggests that, despite the wide diversity of recombinant forms seen in the viral population, only a minority of recombination events appear to be of significance to the evolution of HIV-1.
Introduction:
The causative agent of AIDS, HIV, exhibits a high rate of evolution as a direct result of the error-prone nature of reverse transcriptase and its tendency to switch between RNA templates[1],[2]. These mutational events, combined with high levels of viral turnover[3],[4]—and diversifying selection due to the action of the immune response[5]–[7]—generate the extreme diversity observed within infected individuals and in the pandemic as a whole[8]. As a consequence of the epidemiological history of the HIV-1 pandemic, specifically the key role of founder effects[9],[10], the global diversity is partitioned into distinct phylogenetic clusters, termed subtypes[11]. If dual infection or superinfection with viruses from different subtypes occurs, recombination can generate an inter-subtype recombinant[12]–[15]. When an inter-subtype recombinant is transmitted between multiple individuals, i. e., has the potential to be of epidemiological significance, it is termed a Circulating Recombinant Form( CRF)[11]. As with the subtypes, these form distinct clusters in phylogenetic trees and some( CRF01 and 02 in particular) contribute disproportionately to the pandemic, as do certain subtypes( particularly C). Superinfection and thus recombination also occurs between viruses from the same subtype or CRF[15]–[18], but these are harder to detect by phylogenetic analyses due to the lack of phylogenetic substructure within subtypes and CRFs. It is widely assumed that the HIV recombinants have novel properties that led directly to their prevalence, particularly in the case of the CRFs[19]. As a result, enormous effort is expended on characterising CRFs both geographically and in terms of the precise location of the strand-switches, the recombination breakpoints that delimit their mosaic structure. We hypothesise that only a subset of recombination breakpoints will convey any selective advantage. Given the already noted propensity of reverse transcriptase to switch RNA templates, the null hypothesis is that the majority of recombination breakpoints are selectively neutral with limited biological significance, i. e., recombination patterns are adequately explained by strand-switching and have limited impact on viral fitness and evolution. Factors known to promote strand-switching, and hence recombination, include sequence identity[20],[21] and additional features of the RNA such as homopolymeric runs[22] and secondary RNA structure[23],[24]. Here we are particularly interested in the role of sequence identity and the propensity for recombination to occur. It has been established that high sequence identity between the two RNAs[1],[2], and particularly local sequence identity, is important for efficient strand-switching[21]. This is because strand-switching results from the transfer of the nascent DNA from one RNA( the donor) onto the other( the acceptor)[25]. After this transfer, synthesis must be resumed on the acceptor RNA. Discordant residues between donor and acceptor RNAs result in mismatches in the heteroduplex formed by the nascent DNA and the acceptor RNA, and destabilise it. An unstable heteroduplex near the 3′-OH of the nascent DNA does not constitute a suitable structure for priming reverse transcription on the acceptor RNA and, by making resumption of reverse transcription on the acceptor RNA less efficient, decreases the probability of successful template switching. To test our hypothesis regarding the importance of breakpoints, we generate a probabilistic model for the copy-choice recombination process( Figure 1). This takes into account the local sequence identity between the co-packaged RNA genomes and produces an expected null distribution of breakpoints across two parental sequences. The model is based on an analysis of 162 inter-subtype recombinant forms generated in the laboratory in an experimental setting where no selection is applied to the recombinant products[21]. As we have the exact parental sequences, the location of the breakpoints can be determined accurately, i. e., to the nearest mismatch either side of the identical region in which the switch has occurred. This permits a detailed understanding of the influence of sequence identity on template switching. We use this model to generate a null distribution for the observed recombinant breakpoints from the global HIV-1/AIDS pandemic. Regions that deviate from this expected distribution we infer to include the breakpoints that are of greater importance as a consequence of the mosaic structures they have generated. Our results strongly indicate that it is approximately on either side of the envelope gene, or at least gp120, that most of the recombination of significance is occurring, possibly as a result of this region's major involvement in immune evasion.
Discussion:
Our analysis confirms that local sequence identity between the genomic RNAs has a major influence on the probability of a strand-transfer event[19], with significantly fewer breakpoints than expected by chance found within zones of five nucleotides or less of a residue discordant between the two genomic RNAs( Figure 2, inset). This tendency for a reduced probability of template switching permits us to accurately model the expected distribution of recombination breakpoints for any given pair of parental sequences( Figure 3). Applying this model to recombinant sequences from the HIV-1 pandemic( Figure 4) we find that( i) the distribution of recombinant breakpoints across HIV-1's genome is, for the most part, adequately accounted for by a simple model of recombination dependent on sequence identity, and( ii) genomic regions can be identified where there are either more or fewer breakpoints than predicted. This validates our hypothesis that only a subset of recombination events should be considered important, i. e., to convey any selective advantage to the virus. We consider these important recombination breakpoints to be those over-represented with respect to the distribution predicted by our model. The most evident case is constituted by breakpoints at either side of env, which indicates a tendency for the shuttling of the entire envelope gene, or at least the coding region for extra-cellular gp120( Figure 4). Whether this is a result of coincidental or sequential recombination events, it indicates that selection is frequently promoting env's transfer from one genetic background into another. This tendency for most of env to be recombined effectively as an integral unit must be directly related to the envelope protein's functional significance in relation to viral fitness determinants, and in particular its propensity to be subject to high levels of positive selection as a direct result of the action of the immune response on HIV's envelope gene[6],[7],[26],[27]. The paucity of recombination breakpoints within the envelope gene itself( but also in parts of gag and pol) indicates that recombinants with breakpoints in these regions have a tendency to be selected against. This is presumably due to constraints arising from inter-dependencies within gene and genomic regions[28] as a consequence of the maintenance of protein structural and functional integrity in the context of high viral diversity. Such inter-dependencies are probably related to co-variation of sites[29], for example, purifying selection acting to maintain protein folds[30] or, in env, to maintain glycosylation patterns[31] critical for the evasion of neutralising antibodies[32]. Recombination patterns thus have the potential to provide insight into key dependencies between intra- and inter-genic regions. Our results emphasise that detailed mapping of individual HIV-1 recombinant structures should be considered in the context of a probabilistic expectation generated by the process of template switching during reverse transcription. This underlines the importance of determining which recombination breakpoints are the most important in the maintenance of a persistent infection. Individual recombination breakpoints, analogous to point mutations, will have varying consequences for viral persistence in infected individuals and populations. Further fine-scale mapping of recombination distributions is required to understand more precisely the significance of recombination breakpoints, for example, related to escape from immune control[18] and epistatic interactions[33],[34]. We would also expect to find recombination hotspots in data associated with drug resistance[35]–[37]. In conclusion, our findings provide a clear indication that the majority of recombinant breakpoints detected in the HIV-1 pandemic provide limited selective advantage, with the exception being specific genomic regions in which recombination events have a higher probability of being important to viral evolution.
Multiple variants of HIV can infect the same cell , and because each viral particle contains two copies of the viral genomic RNA , RNAs from different viruses can occasionally be incorporated together within a viral particle . When this virus subsequently infects another cell , genetic exchange ( recombination ) may occur between these two divergent copies of genomic RNA as a result of a switch between the RNA molecules while they are copied into DNA . This process is very important to understand as it contributes to the generation of new HIV variants . In this study , we have analysed a set of recombinant HIV genomes generated in the laboratory to construct a probabilistic model of the propensity for the switch to take place in specific regions of the genome , dependent on the local similarity of the parental viral sequences . This model allows us to predict the locations where recombination should occur more frequently . By comparing these predictions to the patterns of recombination observed in the HIV-1 pandemic , we identify the genomic regions in which recombination has been more important , in that it has provided an evolutionary advantage to the virus .