Complex recombination patterns arising during geminivirus coinfections preserve and demarcate biologically important intra-genome interaction networks

Genetic recombination is an important process during the evolution of many virus species and occurs particularly frequently amongst begomoviruses in the single stranded DNA virus family, Geminiviridae. As in many other recombining viruses it is apparent that non-random recombination breakpoint distributions observable within begomovirus genomes sampled from nature are the product of variations both in basal recombination rates across genomes and in the over-all viability of different recombinant genomes. Whereas factors influencing basal recombination rates might include local degrees of sequence similarity between recombining genomes, nucleic acid secondary structures and genomic sensitivity to nuclease attack or breakage, the viability of recombinant genomes could be influenced by the degree to which their co-evolved protein-protein and protein-nucleotide and nucleotide-nucleotide interactions are disreputable by recombination. Here we investigate patterns of recombination that occur over 120 day long experimental infections of tomato plants with the begomoviruses Tomato yellow leaf curl virus and Tomato leaf curl Comoros virus. We show that patterns of sequence exchange between these viruses can be extraordinarily complex and present clear evidence that factors such as local degrees of sequence similarity but not genomic secondary structure strongly influence where recombination breakpoints occur. It is also apparent from our experiment that over-all patterns of recombination are strongly influenced by selection against individual recombinants displaying disrupted intra-genomic interactions such as those required for proper protein and nucleic acid folding. Crucially, we find that selection favoring the preservation of co-evolved longer-range protein-protein and protein DNA interactions is so strong that its imprint can even be used to identify the exact sequence tracts involved in these interactions.     

Extensive intrasubtype recombination in South African human immunodeficiency virus type 1 subtype C infections

Recombinant human immunodeficiency virus type 1 (HIV-1) strains containing sequences from different viral genetic subtypes (intersubtype) and different lineages from within the same subtype (intrasubtype) have been observed. A consequence of recombination can be the distortion of the phylogenetic signal. Several intersubtype recombinants have been identified; however, less is known about the frequency of intrasubtype recombination. For this study, near-full-length HIV-1 subtype C genomes from 270 individuals were evaluated for the presence of intrasubtype recombination. A sliding window schema (window, 2 kb; step, 385 bp) was used to partition the aligned sequences. The Shimodaira-Hasegawa test detected significant topological incongruence in 99.6% of the comparisons of the maximum-likelihood trees generated from each sequence partition, a result that could be explained by recombination. Using RECOMBINE, we detected significant levels of recombination using five random subsets of the sequences. With a set of 23 topologically consistent sequences used as references, bootscanning followed by the interactive informative site test defined recombination breakpoints. Using two multiple-comparison correction methods, 47% of the sequences showed significant evidence of recombination in both analyses. Estimated evolutionary rates were revised from 0.51%/year (95% confidence interval [CI], 0.39 to 0.53%) with all sequences to 0.46%/year (95% CI, 0.38 to 0.48%) with the putative recombinants removed. The timing of the subtype C epidemic origin was revised from 1961 (95% CI, 1947 to 1962) with all sequences to 1958 (95% CI, 1949 to 1960) with the putative recombinants removed. Thus, intrasubtype recombinants are common within the subtype C epidemic and these impact analyses of HIV-1 evolution.     

Productive Homologous and Non-homologous Recombination of Hepatitis C Virus in Cell Culture

Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.     

High-frequency RNA recombination of murine coronaviruses.

The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.     

Evidence of recombination among enteroviruses.

Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5' noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.     

Avoidance of protein fold disruption in natural virus recombinants

With the development of reliable recombination detection tools and an increasing number of available genome sequences, many studies have reported evidence of recombination in a wide range of virus genera. Recombination is apparently a major mechanism in virus evolution, allowing viruses to evolve more quickly by providing immediate direct access to many more areas of a sequence space than are accessible by mutation alone. Recombination has been widely described amongst the insect-transmitted plant viruses in the genus Begomovirus (family Geminiviridae), with potential recombination hot- and cold-spots also having been identified. Nevertheless, because very little is understood about either the biochemical predispositions of different genomic regions to recombine or what makes some recombinants more viable than others, the sources of the evolutionary and biochemical forces shaping distinctive recombination patterns observed in nature remain obscure. Here we present a detailed analysis of unique recombination events detectable in the DNA-A and DNA-A-like genome components of bipartite and monopartite begomoviruses. We demonstrate both that recombination breakpoint hot- and cold-spots are conserved between the two groups of viruses, and that patterns of sequence exchange amongst the genomes are obviously non-random. Using a computational technique designed to predict structural perturbations in chimaeric proteins, we demonstrate that observed recombination events tend to be less disruptive than sets of simulated ones. Purifying selection acting against natural recombinants expressing improperly folded chimaeric proteins is therefore a major determinant of natural recombination patterns in begomoviruses.     

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection

Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment—based on data on the timing of coat protein detection—the per base and replication cycle recombination rate was on the order of 2 × 10⁻⁵ to 4 × 10⁻⁵. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.     

An exploratory algorithm to identify intra-host recombinant viral sequences

Since recombination leads to the generation of mosaic genomes that violate the assumption of traditional phylogenetic methods that sequence evolution can be accurately described by a single tree, results and conclusions based on phylogenetic analysis of data sets including recombinant sequences can be severely misleading. Many methods are able to adequately detect recombination between diverse sequences, for example between different HIV-1 subtypes. More problematic is the identification of recombinants among closely related sequences such as a viral population within a host. We describe a simple algorithmic procedure that enables detection of intra-host recombinants based on split-decomposition networks and a robust statistical test for recombination. By applying this algorithm to several published HIV-1 data sets we conclude that intra-host recombination was significantly underestimated in previous studies and that up to one-third of the env sequences longitudinally sampled from a given subject can be of recombinant origin. The results show that our procedure can be a valuable exploratory tool for detection of recombinant sequences before phylogenetic analysis, and also suggest that HIV-1 recombination in vivo is far more frequent and significant than previously thought.     

Homologous recombination between different genotypes of hepatitis B virus

Phylogenetic analysis was used to examine the evolutionary relationships among 99 complete HBV sequences. Analysis revealed nine viral genomes clustered with different genotypes depending on genome region analyzed. This discordance indicated that recombination events occurred during HBV history. The putative breakpoints between genomes of different genotypes have been mapped. Six mosaic genomes representing B/C hybrids were isolated in East Asia and three A/D hybrids in Italy. At least some recombinant strains appear to be fully viable and possess high evolutionary potential. As a result, B/C recombinants overspread through the East Asia region. They were found among the isolates from Japan, China and Indonesia. Our results suggest that recombination is a significant and relatively frequent event in the evolution of HBV genome. A possible mechanism and the implications of recombination for the natural history of HBV, clinically important properties, and phylogenetic reconstruction are discussed.     

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.     

Human immunodeficiency virus type 1 genetic recombination is more frequent than that of Moloney murine leukemia virus despite similar template switching rates

Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.     

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×10⁶ in BHK-21 (vertebrate) cells and ∼1.05×10⁵ in C₇10 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Recombination in hepatitis C virus: identification of four novel naturally occurring inter-subtype recombinants

Recombination in Hepatitis C virus (HCV) is considered to be rare. In this study, we performed a phylogenetic analysis of 1278 full-length HCV genome sequences to identify potential recombination events. Nine inter-genotype recombinants were identified, all of which have been previously reported. This confirms the rarity of inter-genotype HCV recombinants. The analysis also identified five inter-subtype recombinants, four of which are documented for the first time (EU246930, EU246931, EU246932, and EU246937). Specifically, the latter represent four different novel recombination types (6a/6o, 6e/6o, 6e/6h, and 6n/6o), and this was well supported by seven independent methods embedded in RDP. The breakpoints of the four novel HCV recombinants are located within the NS5B coding region and were different from all previously reported breakpoints. While the locations of the breakpoints identified by RDP were not identical, they are very close. Our study suggests that while recombination in HCV is rare, this warrants further investigation.     

Distribution of the phenotypic effects of random homologous recombination between two virus species

Recombination has an evident impact on virus evolution and emergence of new pathotypes, and has generated an immense literature. However, the distribution of phenotypic effects caused by genome-wide random homologous recombination has never been formally investigated. Previous data on the subject have promoted the implicit view that most viral recombinant genomes are likely to be deleterious or lethal if the nucleotide identity of parental sequences is below 90%. We decided to challenge this view by creating a bank of near-random recombinants between two viral species of the genus Begomovirus (Family Geminiviridae) exhibiting 82% nucleotide identity, and by testing infectivity and in planta accumulation of recombinant clones randomly extracted from this bank. The bank was created by DNA-shuffling-a technology initially applied to the random shuffling of individual genes, and here implemented for the first time to shuffle full-length viral genomes. Together with our previously described system allowing the direct cloning of full-length infectious geminivirus genomes, it provided a unique opportunity to generate hundreds of "mosaic" virus genomes, directly testable for infectivity. A subset of 47 randomly chosen recombinants was sequenced, individually inoculated into tomato plants, and compared with the parental viruses. Surprisingly, our results showed that all recombinants were infectious and accumulated at levels comparable or intermediate to that of the parental clones. This indicates that, in our experimental system, despite the fact that the parental genomes differ by nearly 20%, lethal and/or large deleterious effects of recombination are very rare, in striking contrast to the common view that has emerged from previous studies published on other viruses.     

Identifying the important HIV-1 recombination breakpoints

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.     

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.     

Characterization of reticulate networks based on the coalescent with recombination

Phylogenetic networks aim to represent the evolutionary history of taxa. Within these, reticulate networks are explicitly able to accommodate evolutionary events like recombination, hybridization, or lateral gene transfer. Although several metrics exist to compare phylogenetic networks, they make several assumptions regarding the nature of the networks that are not likely to be fulfilled by the evolutionary process. In order to characterize the potential disagreement between the algorithms and the biology, we have used the coalescent with recombination to build the type of networks produced by reticulate evolution and classified them as regular, tree sibling, tree child, or galled trees. We show that, as expected, the complexity of these reticulate networks is a function of the population recombination rate. At small recombination rates, most of the networks produced are already more complex than regular or tree sibling networks, whereas with moderate and large recombination rates, no network fit into any of the standard classes. We conclude that new metrics still need to be devised in order to properly compare two phylogenetic networks that have arisen from reticulating evolutionary process.     

Phylogenomic networks

Phylogenomics is aimed at studying functional and evolutionary aspects of genome biology using phylogenetic analysis of whole genomes. Current approaches to genome phylogenies are commonly founded in terms of phylogenetic trees. However, several evolutionary processes are non tree-like in nature, including recombination and lateral gene transfer (LGT). Phylogenomic networks are a special type of phylogenetic network reconstructed from fully sequenced genomes. The network model, comprising genomes connected by pairwise evolutionary relations, enables the reconstruction of both vertical and LGT events. Modeling genome evolution in the form of a network enables the use of an extensive toolbox developed for network research. The structural properties of phylogenomic networks open up fundamentally new insights into genome evolution.