Variable mechanisms of RNA-recombination

Recombination is widespread among RNA viruses but underlying mechanisms remain poorly understood. Until recently, replicative template switching was considered the only possible mechanism of RNA recombination but new evidence suggests that other variants of replicative mechanisms may also exist. In addition, nonreplicative recombination (i.e., joining of preexisting molecules) of genomes of RNA viruses is possible. Recombination is an efficient tool contributing to both variability and stability of the viral RNA genomes. Nonreplicative joining of RNA pieces in the form of trans-splicing is an important physiological mechanism in at least certain organisms. It is conceivable that RNA-recombination has contributed, and perhaps is still contributing, to the evolution of DNA genomes.     

Nonreplicative RNA Recombination in Poliovirus

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.     

Genome Instability in Picornaviruses

Picornaviruses are small animal RNA viruses and include etiological agents of poliomyelitis, foot and mouth disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.     

Genomic instability in picornaviruses

Picornaviruses are small animal RNA viruses and include wtiological agents of poliomyelitis, foot and mouse disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.     

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.     

Imprecise recombinant viruses evolve via a fitness-driven,iterative process of polymerase template-switching events

Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population.     

Whole‐genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine‐scale recombination rate variation in birds

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine‐scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200 kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage‐specific manner, indicating differences in the extent of linked selection between species. We detected 400–3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine‐scale variation in recombination rate may be associated with the evolution of the epigenetic landscape.     

Global Patterns of Recombination across Human Viruses

Viral recombination is a major evolutionary mechanism driving adaptation processes, such as the ability of host-switching. Understanding global patterns of recombination could help to identify underlying mechanisms and to evaluate the potential risks of rapid adaptation. Conventional approaches (e.g., those based on linkage disequilibrium) are computationally demanding or even intractable when sequence alignments include hundreds of sequences, common in viral data sets. We present a comprehensive analysis of recombination across 30 genomic alignments from viruses infecting humans. In order to scale the analysis and avoid the computational limitations of conventional approaches, we apply newly developed topological data analysis methods able to infer recombination rates for large data sets. We show that viruses, such as ZEBOV and MARV, consistently displayed low levels of recombination, whereas high levels of recombination were observed in Sarbecoviruses, HBV, HEV, Rhinovirus A, and HIV. We observe that recombination is more common in positive single-stranded RNA viruses than in negatively single-stranded RNA ones. Interestingly, the comparison across multiple viruses suggests an inverse correlation between genome length and recombination rate. Positional analyses of recombination breakpoints along viral genomes, combined with our approach, detected at least 39 nonuniform patterns of recombination (i.e., cold or hotspots) in 18 viral groups. Among these, noteworthy hotspots are found in MERS-CoV and Sarbecoviruses (at spike, Nucleocapsid and ORF8). In summary, we have developed a fast pipeline to measure recombination that, combined with other approaches, has allowed us to find both common and lineage-specific patterns of recombination among viruses with potential relevance in viral adaptation.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

Detection of Putative Recombination Events in the 16S Ribosomal RNA Gene of Some Members of Candidatus phytoplasma

Recombination plays a major evolutionary role by creating genetic diversity and provides the potential to find rapid adaptation to new environmental conditions. We sought the occurrence of possible recombination events in the 16S ribosomal RNA gene of 60 accessions belonging to the group 16SrI of Candidatus phytoplasma (aster yellows phytoplasma). Three bioinformatic programs were used (TOPALI v2.5, RECCO and RDP package). All the three programs indicated the presence of putative recombination signals in aligned sequences. Recombination events located in the 16S ribosomal RNA gene revealed the presence of four recombining accessions gathering sugarcane grassy shoot phytoplasmas (JF928001, DQ459439, EF614269 and JN223446).     

Complete nucleotide sequence analysis of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> Indian isolate: further evidence for natural recombination among potexviruses

The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV) was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96–97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2) analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent, and a Taiwan CymMV (AY571289) as the minor parent.     

Recombination in the Plant Genome and its Application in Biotechnology

Genetic variation within and between species is based on recombination of DNA molecules. Recombination also plays a very important role in the repair of damaged DNA. Clarity about the mechanism by which recombination occurs is of profound interest not only to understand how this process assures the maintenance of genome integrity and at the same time is the driving force of evolution, but also for its application in biotechnology. The isolation of genes involved in recombination and the elucidation of the role of many of the corresponding gene products in Escherichia coli and Saccharomyces cerevisiae has formed the basis for comparative analysis in other, more complex eukaryotic systems. The identification of homologous genes from different organisms, including plants, suggests a conservation of the general mechanisms of recombination. Transgenes introduced in an organism may be incorporated in the genome by either homologous or nonhomologous recombination (end joining). The preferred pathway differs strongly between organisms. In plants there is a preference for random integration of the introduced DNA by nonhomologous recombination, which might lead to the accidental inactivation of important genes and to variable and unpredictable expression of the transgene itself. Therefore, there is an urgent need for the development and improvement of techniques for the directed integration of transgenes at specific locations in the genome. The integration of transgenes by homologous recombination would allow specific modification or disruption of endogenous genes, providing a tool for more detailed analysis of gene function. In combination with the recent introduction of site-specific recombination systems from E. coli or yeast into plants, this may lead to the development of versatile systems for modification of the plant genome.     

Somatic recombination in adult tissues: What is there to learn?

Somatic recombination is essential to protect genomes of somatic cells from DNA damage but it also has important clinical implications, as it is a driving force of tumorigenesis leading to inactivation of tumor suppressor genes. Despite this importance, our knowledge about somatic recombination in adult tissues remains very limited. Our recent work, using the Drosophila adult midgut has demonstrated that spontaneous events of mitotic recombination accumulate in aging adult intestinal stem cells and result in frequent loss of heterozygosity (LOH). In this Extra View article, we provide further data supporting long-track chromosome LOH and discuss potential mechanisms involved in the process. In addition, we further discuss relevant questions surrounding somatic recombination and how the mechanisms and factors influencing somatic recombination in adult tissues can be explored using the Drosophila midgut model.     

How chromosomal rearrangements shape adaptation and speciation: Case studies in Drosophila pseudoobscura and its sibling species Drosophila persimilis

The gene arrangements of Drosophila have played a prominent role in the history of evolutionary biology from the original quantification of genetic diversity to current studies of the mechanisms for the origin and establishment of new inversion mutations within populations and their subsequent fixation between species supporting reproductive barriers. This review examines the genetic causes and consequences of inversions as recombination suppressors and the role that recombination suppression plays in establishing inversions in populations as they are involved in adaptation within heterogeneous environments. This often results in the formation of clines of gene arrangement frequencies among populations. Recombination suppression leads to the differentiation of the gene arrangements which may accelerate the accumulation of fixed genetic differences among populations. If these fixed mutations cause incompatibilities, then inversions pose important reproductive barriers between species. This review uses the evolution of inversions in Drosophila pseudoobscura and D. persimilis as a case study for how inversions originate, establish and contribute to the evolution of reproductive isolation.     

Divided genomes and intrinsic noise

Summary Segmental genomes (i.e., genomes in which the genetic information is dispersed between two or more discrete molecules) are abundant in RNA viruses, but virtually absent in DNA viruses. It has been suggested that the division of information in RNA viruses expands the pool of variation available to natural selection by providing for the reassortment of modular RNAs from different genetic sources. This explanation is based on the apparent inability of related RNA molecules to undergo the kinds of physical recombination that generate variation among related DNA molecules. In this paper we propose a radically different hypothesis. Self-replicating RNA genomes have an error rate of about 10^–3–10^–4 substitutions per base per generation, whereas for DNA genomes the corresponding figure is 10^–9–10^–11. Thus the level of noise in the RNA copier process is five to eight orders of magnitude higher than that in the DNA process. Since a small module of information has a higher chance of passing undamaged through a noisy channel than does a large one, the division of RNA viral information among separate small units increases its overall chances of survival. The selective advantage of genome segmentation is most easily modelled for modular RNAs wrapped up in separate viral coats. If modular RNAs are brought together in a common viral coat, segmentation is advantageous only when interactions among the modular RNAs are selective enought to provide some degree of discrimination against miscopied sequences. This requirement is most clearly met by the reoviruses.     

Why do RNA viruses recombine?

Recombination occurs in many RNA viruses and can be of major evolutionary significance. However, rates of recombination vary dramatically among RNA viruses, which can range from clonal to highly recombinogenic. Here, we review the factors that might explain this variation in recombination frequency and show that there is little evidence that recombination is favoured by natural selection to create advantageous genotypes or purge deleterious mutations, as predicted if recombination functions as a form of sexual reproduction. Rather, recombination rates seemingly reflect larger-scale patterns of viral genome organization, such that recombination may be a mechanistic by-product of the evolutionary pressures acting on other aspects of virus biology.     

Recombination rate variation in mice from an isolated island

Recombination rate is a heritable trait that varies among individuals. Despite the major impact of recombination rate on patterns of genetic diversity and the efficacy of selection, natural variation in this phenotype remains poorly characterized. We present a comparison of genetic maps, sampling 1212 meioses, from a unique population of wild house mice (Mus musculus domesticus) that recently colonized remote Gough Island. Crosses to a mainland reference strain (WSB/EiJ) reveal pervasive variation in recombination rate among Gough Island mice, including subchromosomal intervals spanning up to 28% of the genome. In spite of this high level of polymorphism, the genomewide recombination rate does not significantly vary. In general, we find that recombination rate varies more when measured in smaller genomic intervals. Using the current standard genetic map of the laboratory mouse to polarize intervals with divergent recombination rates, we infer that the majority of evolutionary change occurred in one of the two tested lines of Gough Island mice. Our results confirm that natural populations harbour a high level of recombination rate polymorphism and highlight the disparities in recombination rate evolution across genomic scales.