Genetic recombination and its role in the development of the HIV-1 pandemic

Introduction Genetic recombination is part of the normal mechanisms of retroviral replication and, as such, plays an important role in the generation of viral diversity. Generation of recombinant retroviruses requires that two viruses infect a single cell, either simultaneously, by a single transmission event, or sequentially, in multiple transmission event, or sequentially, in multiple transmission events. In HIV-1, recombination can occur between different strains of the same subtype (intrasubtype recombination), different or different groups (intergroup recombination). The importance of recombination in shaping HIV-1 global diversity has been increasingly recognized in recent years. Several factors have contributed to this trend, including new surveys carried out in previously unexplored or insufficiently sampled areas, more frequent phylogenetic analysis of multiple genome segments [including protease and reverse transcriptase (RT)] used to detect drug resistance mutations, and growing numbers of full-length HIV-1 genomes analyzed in multiple geographical areas. The cumulating picture emerging from these studies indicates that HIV-1 recombinant forms are much more prevalent, geographically spread, and diverse in the global pandemic than previously known. This applies not only to circulating recombinant forms (CRFs) (recombinant forms indentified in at least three epidemiologically-linked individuals), but also, and even more notoriously, to unique recombinant forms (URFs) (recombinant forms found in a single individual or in a single epidemiologically-linked cluster). The recurrent finding of URFs in high proportions in areas where multiple HIV-1 genetic forms co-circulate attests to the high frequency of dual infections with diverse variants, which can either occur simultaneously or, more probably, sequentially, as suggested by the report of the first well documented case of HIV-1 superinfection in humans [1]. In the present review, basic concepts on the mechanisms of HIV-1 recombination, recent developments in the molecular epidemiology of HIV-1 recombinant forms, and their implications for vaccine development and therapeutic strategies are discussed. Molecular mechanisms of HIV-1 recombination Retroviruses and other RNA viruses exhibit high mutation rates due to the lack of proof-reading activity of the viral polymerases, the short replication times, and the large population sizes. Consequently, they are present in the host organism as mixtures of genetically diverse but related populations termed quasispecies [2]. These represent all possible mutants, as illustrated by the presence of antiretroviral drug resistance-associated mutations in treatment-naïve patients, the so-called ‘natural resistance’ [3,4]. Retroviruses are also known for their high recombinogenic potential [5,6], deriving from the fact that recombination constitutes an intrinsic part of their normal replication cycle. Recombination can mediate the repair of defective retroviral genomes [6–8], can increase viral diversity, and can accelerate the spread of beneficial mutations among viral quasispecies [9]. The increased variation potential mediated by recombination confers on retroviruses the capability to respond rapidly to changing selective pressures, either immunological [10,11] or pharmacological [12,13], through the prompt generation of the fittest variants possessing the adequate set of mutations to elude those pressures [14]. Recombination in retroviruses requires the co-packaging in each virion of two RNA genomes [5,15]. Recombination between genomes packaged in two different virions is possible only by productive infection of a single cell by both virions, which allows for the production of heterozygous particles. In a subsequent cycle of infection, a recombinant genome can be generated through alternate jumps of RT between both co-packaged genomes (Fig. 1).Fig. 1.: Generation of recombinant retroviruses. Two virions with genetically dissimilar genomes infect a single cell, either sequentially or simultaneously. Both viral genomes undergo reverse transcription and the proviruses become integrated in the cell chromosome. Heterodimers of genomic transcripts derived from both integrated proviruses are packaged in viral particles. In a second replication cycle, heterozygous viruses produce recombinant proviral genomes by alternate template jumps during reverse transcription.Synthesis of the double-stranded proviral DNA by RT involves two obligatory template jumps, necessary for long terminal repeat duplication at both genome ends (reviewed in [16]) (Fig. 2). In the first jump, the minustrand, strong-stop DNA (ssDNA) intermediate, initiated at the tRNA annealed to the primer binding site, is translocated from the 5’ end to the 3’ end of the genome. This jump is possible by the presence of repeated sequences at both extremities of the genome, and can be intermolecular or intramolecular. The second jump involves the transfer of the plus-strand ssDNA, initiated at the 3’ polypurine tract, from the 3’ end to the 5’ end of the genome. This jump is mediated by base pairing of the primer binding site of plus-strand ssDNA (generated by copying the 3’ end of the tRNA attached covalently to the minus-strand DNA) to its complementary sequence at the minus-strand DNA, and is almost always intramolecular.Fig. 2.: Generation of a recombinant double-stranded provirus during reverse transcription. (a) Proviral DNA synthesis starts at the primer binding site (PBS), near the 5’ end of the genome, of one of the co-packaged strands, primed by the 3’ segment of a cellular tRNA. Polymerization proceeds until reaching the 5’ end of the genome, generating the minus-strand ssDNA. (b) This DNA intermediate anneals to the 3’ end of the same or the other co-packaged strand thanks to the presence of repeated (R) sequences at both ends of the genome. (c) RT resumes minus-strand DNA polymerization, while the RNAse H carried by RT degrades the RNA hybridized to the growing DNA chain. Alternate template jumps along polymerization of minus-strand DNA generates a recombinant genome. (d) An RNA oligonucleotide located 5’ of the U3 segment, resistant to RNAse H cleavage, the polypurine tract (PPT), primes the synthesis of the plus-strand DNA, which continues through the end of the minus-strand DNA copying 18 nucleotides of the covalently attached tRNA, thus regenerating the PBS. (e) This DNA intermediate, termed the plus-strand ssDNA, undergoes a second template jump, which is almost always intramolecular, by annealing of the PBS to its complementary sequence in the minus-strand DNA. (f) The synthesis of both strands is completed using each other as template. In HIV-1 and other lentiviruses, a second PPT located at the center of the genome, the central PPT, is also used as primer for plus-strand DNA synthesis (omitted from the figure). LTR, Long Terminal Repeat.Besides the obligatory strong stop DNA jumps, strand switches may occur along internal genome segments [5,17,18], which in heterozygous virions would generate a recombinant genome. In HIV-1, it has been reported that an average of two to three jumps occurs during each replication cycle [19,20]. Experimental evidence supports internal interstrand jumps occuring predominantly, if not exclusively, during minus-strand DNA synthesis [15,17,19–21]. In the forced copy-choice model, breaks in the RNA would force RT to switch templates, thus restoring the continuity in the genome to generate a viable progeny [22]. A related model proposes that the low processivity of retroviral RT would result in pauses along RNA-dependent DNA polymerization, which would promote template switching without the need of breaks [23]. In agreement with this model, in vitro studies indicate that pauses in polymerization enhance template switching [24,25]. During pauses, which may be favored by secondary structures [26], nucleotide misincorporations [27], or low dNTP concentrations [24,28,29], the extent of RNAse H cleavage of the original (donor) template beneath the stalled RT is increased [27]. This process generates a longer single-stranded DNA segment, which would become free to anneal to the opposite (acceptor) strand, positioning it near the polymerase site, thus facilitating template switching [28,29]. In contraposition to this model, other authors postulate that pause-independent strand transfers might be predominant [30], which might be enhanced by interstrand proximity mediated by secondary structure interactions [32]. The frequency of recombination may also be highly dependent on sequence homology [33]. In a recent study, for a sequence difference of 25% or more, HIV-1 recombination directed by sequence homology was not more frequent than that which was homology independent [33]. Multiple methods are available for the analysis of recombinant sequences. Those that have been used more frequently in HIV-1 are similarity [34,35] or diversity [36,37] plots and bootscanning [38]. More precise mapping of breakpoints can be achieved by informative site analysis [39]. An HIV-1 intersubtype recombinant form is identified when its phylogentic relationships with different subtypes switches along the genome. Detection of intrasubtype recombination is difficult unless both parental viruses are identified or they group in distinct recognized phylogenetic clusters. For a complete characterization of the mosaic structure, full genome analysis is required. A more practical alternative for a large number of samples is to analyze discrete segments in separate regions of the genome. Commonly, segments of gag and env (most frequently, the V3 loop region) or of pol and env have been used for phylogenetic analysis. HIV-1 recombinant forms in the global pandemic Although recombination in an HIV-1 virus isolated from an infected individual was suggested as early as 1988 [40], corresponding to the African isolate MAL, the recognition of the importance of recombination in shaping HIV-1 diversity in the global pandemic is a relatively recent development. The classification of HIV-1 in distinct genetic subtypes was originally based on clustering in phylogenetic trees of env and sag sequences [41–43], with five of the nine presently recognized subtypes having been indentified in these early studies The first case of intersubtype recombination in infected individuals was reported in 1994 in Brazil in two sexual partners harboring a BF recombinant virus [44]. One year later, a high frequency (10%) of HIV-1 intersubtype recombinant virses was reported to be found in sequence databases [45]. In 1996, the full genome analysis of isolates from a genetic form circulating in Thailand and Central Africa indicated that it was an intersubtype recombinant form, later designated CRF01_AE [36,46]. Its recombinant nature had been suggested previously by analysis of partial sequences [46]. In 1998, the second CRF (an AG recombinant) was identified by analysis of full genome sequences from Nigeria and Djibouti [47]. This CRF (currently designated CRF01_AG) has recently been shown to be the most common HIV-1 variant circulating in West Africa and some West-Central African Countries [48,49]. The current nomenclature of CRF and the criteria for their definition were adopted in 1999 [50]. 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