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.     

Evidence for Retroviral Intramolecular Recombinations

As a consequence of being diploid, retroviruses have a high recombination rate. Naturally occurring retroviruses contain two repeat sequences (R regions) flanking either end of their RNA genomes, and recombination between these two R regions occurs at a high rate. We deduced that recombination may occur between two sequences within the same RNA molecule (intramolecular) as well as between sequences present within two separate RNA molecules (intermolecular). Intramolecular recombination would usually result in a deletion within the progeny provirus. In this report, we demonstrate that intramolecular recombination between two identical sequences occurred within a chimeric RNA vector. In addition, high rates of recombination between two identical sequences within the same RNA molecule resulted mostly from intramolecular recombination.     

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.     

The mechanism of RNA recombination in poliovirus

We have investigated RNA recombination among poliovirus genomes by analyzing both intratypic and intertypic recombinant crosses involving the same defined genetic markers. Sequence analysis of the recombinant junctions of 13 nonsibling intertypic recombinants showed that intertypic RNA recombination is not site-specific, nor does it require extensive homology between the recombining parents at the crossover site. To discriminate between breaking-rejoining and copy choice mechanisms of RNA recombination, we have inhibited the replication of the recombining parents independently and found opposite effects on the frequency of genetic recombination in intratypic crosses. The results strongly support a copy choice mechanism for RNA recombination, in which the viral RNA polymerase switches templates during negative strand synthesis.     

Diverse Mechanisms of RNA Recombination

Recombination is widespread among RNA viruses, but many molecular mechanisms of this phenomenon are still poorly understood. It was believed until recently that the only possible mechanism of RNA recombination is replicative template switching, with synthesis of a complementary strand starting on one viral RNA molecule and being completed on another. The newly synthesized RNA is a primary recombinant molecule in this case. Recent studies have revealed other mechanisms of replicative RNA recombination. In addition, recombination between the genomes of RNA viruses can be nonreplicative, resulting from a joining of preexisting parental molecules. Recombination is a potent tool providing for both the variation and conservation of the genome in RNA viruses. Replicative and nonreplicative mechanisms may contribute differently to each of these evolutionary processes. In the form of trans splicing, nonreplicative recombination of cell RNAs plays an important role in at least some organisms. It is conceivable that RNA recombination continues to contribute to the evolution of DNA genomes.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 618–632.Original Russian Text Copyright © 2005 by Gmyl, Agol.     

RNA structures facilitate recombination-mediated gene swapping in HIV-1

Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.     

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.     

Evidence for Multiplicity Reactivation of Ultraviolet Light Irradiated Ribonucleic Acid Isolated from Tobacco Mosaic Virus

Multiplicity reactivation (MR) seems to take place in leaves of Nicotiana glutinosa inoculated with ultraviolet (UV) light irradiated RNA from tobacco mosaic virus (TMV-RNA). A similar phenomenon was not observed with UV-irradiated TMV particles. Considering MR as resulting from genetic recombination between viral genomes, a recombination mechanism, which has been difficult to prove with plant viruses, is proposed as being operative during multiplication of TMV. From the pattern of MR of TMV-RNA, the location of the gene for the RNA replicase within a TMV-RNA strand is discussed.     

The polymerase in its labyrinth: mechanisms and implications of RNA recombination.

The wide variety of RNA viruses, and the diseases associated with them, may result in part from the capacity of RNA genomes to evolve through genetic recombination. Here we address the mechanism of RNA recombination, and ask questions about its prevalence and purpose in nature.     

Natural Recombination among Plant Virus Genomes: Evidence from Tobraviruses

RNA recombination in plants was first identified by the repairin vivoof a deleted genomic RNA of brome mosaic virus. Subsequently, evidence of recombination has been detected not only in experimental systems but also among an increasing number of naturally occurring isolates of plant viruses. This article discusses the different recombinants that have been found among viruses in the genusTobravirusand describes other examples of recombination among plant viruses and between the genomes of viruses and their hosts.     

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.     

An update on chloroplast genomes

Plant cells possess two more genomes besides the central nuclear genome: the mitochondrial genome and the chloroplast genome (or plastome). Compared to the gigantic nuclear genome, these organelle genomes are tiny and are present in high copy number. These genomes are less prone to recombination and, therefore, retain signatures of their age to a much better extent than their nuclear counterparts. Thus, they are valuable phylogenetic tools, giving useful information about the relative age and relatedness of the organisms possessing them. Unlike animal cells, mitochondrial genomes of plant cells are characterized by large size, extensive intramolecular recombination and low nucleotide substitution rates and are of limited phylogenetic utility. Chloroplast genomes, on the other hand, show resemblance to animal mitochondrial genomes in terms of phylogenetic utility and are more relevant and useful in case of plants. Conservation in gene order, content and lack of recombination make the plastome an attractive tool for plant phylogenetic studies. Their importance is reflected in the rapid increase in the availability of complete chloroplast genomes in the public databases. This review aims to summarize the progress in chloroplast genome research since its inception and tries to encompass all related aspects. Starting with a brief historical account, it gives a detailed account of the current status of chloroplast genome sequencing and touches upon RNA editing, ycfs, molecular phylogeny, DNA barcoding as well as gene transfer to the nucleus.     

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.RNA viruses are able to undergo rapid genetic changes in order to adapt to new hosts or environments. Although much of this flexibility is due to the error-prone nature of the RNA-dependent RNA polymerase, which generates an array of different point mutations within the viral population (23), recombination is also a common and important mechanism for generating viral diversity (18, 31, 42, 58). Recombination occurs when the RNA-dependent RNA polymerase switches templates during replication, an event that is favored when both templates share identical or very similar sequences. Three types of RNA recombination have been identified: homologous recombination occurs at sites with exact sequence matches; aberrant homologous recombination requires sequence homology, but crossover occurs either upstream or downstream of the site of homology, resulting in a duplication or deletion; and nonhomologous (or illegitimate) recombination is independent of sequence homology (31, 42).When the same cell is infected by viruses of two different strains, or even different species, recombination between their genomic RNAs can potentially lead to the emergence of new pathogens. A case in point is the emergence of Western equine encephalitis virus, a member of the genus Alphavirus, family Togaviridae, which arose by homologous recombination between Eastern equine encephalitis virus and Sindbis virus (14).Some mammalian RNA viruses can recombine at a frequency that is detectable in experimental settings (1, 2, 55), and phylogenetic analysis of partial or complete genome sequences suggests that RNA recombination is a widespread phenomenon. Naturally occurring recombinant viruses have been identified in almost every family of positive-stranded RNA viruses (31, 58).Flaviviruses are members of the genus Flavivirus, family Flaviviridae, a family that also includes the genera Pestivirus and Hepacivirus. Several of the flaviviruses are important human pathogens, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), the dengue viruses, yellow fever virus, and tick-borne encephalitis virus (TBEV).Although there has never been a report of a pathogenic flavivirus strain arising due to recombination involving attenuated vaccine strains (39), the urgent necessity to develop tetravalent vaccines containing all four serotypes of dengue virus—two such vaccines are currently undergoing clinical testing (45)—has recently brought the recombination issue to the forefront of discussion among researchers, regulators, and vaccine producers (39). It has been suggested that recombination, either between the strains present in a multivalent vaccine or between an attenuated vaccine strain and a wild-type strain, could lead to the emergence of new viruses with unpredictable properties (49).So far, recombination between flavivirus genomes has not been demonstrated directly in the laboratory. However, phylogenetic analysis of partial genome sequences available in the GenBank database has suggested that homologous recombination may have occurred between closely related strains of dengue virus (20, 52, 54, 59). An experimental approach for assessing the ability of flavivirus genomes to recombine is therefore urgently needed.Flavivirus virions are composed of a single-stranded, positive-sense RNA genome that, together with the capsid protein C, forms the viral nucleocapsid. The nucleocapsid is covered by a lipid envelope containing the surface glycoproteins prM and E. These glycoproteins drive budding at the membrane of the endoplasmic reticulum during the assembly stage and mediate entry of the virus into host cells (41). Replicons, defined as self-replicating, noninfectious RNA molecules, can be generated by deleting parts or all of the region coding for the structural proteins C, prM, and E from the viral genome but maintaining all seven of the nonstructural proteins and the flanking noncoding sequences, which are required in cis for RNA replication (25). By providing the missing structural protein components in trans, replicons can be packaged into virus-like particles that are capable of a single round of infection (10, 15, 24, 47).Typically, researchers developing novel replicating vaccines, especially ones that involve multiple components, make an effort to come up with strategies to prevent recombination, for example by “wobbling” codons, i.e., replacing codons in homologous regions with synonymous ones encoding the same amino acid but consisting of a different nucleotide triplet (50, 57). In this study, in order to assess the propensity of flavivirus genomes to recombine, we took an opposite approach, establishing a “recombination trap” that favors the selection and sensitive detection of recombination products. This system takes advantage of the ability of replicon pairs containing deletions in their structural protein genes to complement each other in trans and thus be propagated together in cell culture, and by passage at limiting dilutions, it allows infectious RNA genomes arising by recombination between the two replicons to be preferentially selected.Using the recombination trap, we have now obtained the first direct evidence of recombination between flavivirus genomes in the laboratory. Aberrant homologous recombination was observed twice with JEV replicons, resulting in viruses with unnatural gene arrangements and reduced growth properties compared to those of wild-type JEV. No infectious recombinants of any kind were obtained when TBEV or WNV replicons were used. Interestingly, we never detected a fully infectious wild-type genome arising by homologous recombination in any of these systems. The results of this study show that the propensity of flavivirus genomes to recombine in the region coding for the structural proteins appears to be quite low, suggesting that recombination does not represent a major risk in the use of live, attenuated flavivirus vaccines.     

Subgenomic RNAs: The Possible Building Blocks for Modular Recombination ofClosteroviridaeGenomes

TheClosteroviridaeinclude several plant viruses of considerable economic importance, with unusually large positive-strand genomes of up to 20 kb. Molecular characterization of several of these viruses has now confirmed their coherent and unique position among the elongated plant RNA viruses. Structural comparisons of their genomes suggested a modular composition. The recent finding of multiple species of citrus tristeza virus (CTV) defective-RNAs, which have apparently resulted from the recombination of a subgenomic (sg)RNA, with distant parts from the 5′ end of the CTV genome, suggests thatClosteroviridaeare probably able to utilize sgRNAs and/or their promoter signals as factors for the modular exchange and rearrangement of their genomes.     

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.