Nucl. Acid. Res.
The fidelity of the virus polymerase influences the rate of genetic recombination between viruses coinfecting the same cell. We used cell-based and new, biochemically-defined, assays to demonstrate that the viral polymerase is necessary and sufficient for the strand-transfer event of RNA virus recombination. Furthermore, the fidelity of the polymerase is critical in determining the efficiency with which recombination occurs; low fidelity polymerases exhibit high recombination rates, and vice versa.
The paper is published in Nucleic Acids Research:
Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination
Andrew Woodman; Jamie J. Arnold; Craig E. Cameron; David J. Evans
Nucleic Acids Research 2016; doi: 10.1093/nar/gkw567
Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.
Not as unconnected as you might think. The most numerous photosynthetic organisms on earth – the cyanobacteria – are infected by viruses (cyanhophages). Some of these cyanhophages carry components of the photosynthetic machinery and are thought to contribute to host cell photosynthesis. In a recent study on which we collaborated we show that virus-infected cyanobacteria are inhibited in their ability to fix CO2 (in contrast to uninfected cyanobacteria) whereas photosynthetic electron transport is unaltered. The cyanhophages therefore redirect photosynthesis to support phage development.
These results also have implications for our understanding of global warming. The reduction in CO2 fixation in the marine environment, as a consequence of these cyanophage infections, may be as much as 10%. The global warming calculations are based on assumptions of carbon fixation levels being directly linked to photosynthetic activity. We show that that this is incorrect and that CO2 fixation is likely overestimated in marine environments.
The full abstract of the manuscript “Viruses Inhibit CO2 Fixation in the Most Abundant Phototrophs on Earth” by Puxty et al., is shown below.
Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet. With a global population size of 3.6 × 1027, they are responsible for approximately 10% of global primary production. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters and frequently outnumber their cyanobacterial hosts. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) as well as central carbon metabolism, suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production.
Puxty, R.J., Millard, A.D., Evans, D.J. and Scanlan, D.J. (2016) Current Biology http://dx.doi.org/10.1016/j.cub.2016.04.036
Our snappily-titled manuscript “The Iflaviruses Sacbrood virus and Deformed wing virus evoke different transcriptional responses in the honeybee which may facilitate their horizontal or vertical transmission” has just been published in PeerJ. We analysed changes in the transcriptome following infection with deformed wing virus (DWV) and sacbrood virus, or DWV alone. We propose that the difference in expression we observed of the honeybee immune genes induced by SBV and DWV may be an evolutionary adaptation to the different predominant transmission routes used by these viruses .
We have recently submitted a paper to PeerJ on gene expression changes resulting from deformed wing virus and sacbrood virus infection. A pre-print of this manuscript can be viewed on the PeerJ website.
Manuscript authors and title
2015) Evolutionarily related Sacbrood virus and Deformed wing virus evoke different transcriptional responses in the honeybee which may facilitate horizontal or vertical transmission of these viruses. PeerJ PrePrints 3:e1749
Sacbrood virus (SBV) and deformed wing virus (DWV) are evolutionarily related positive-strand RNA viruses, members of the Iflavirus group, which infect the honeybee Apis mellifera, but have strikingly different levels of virulence when transmitted orally. Honeybee larvae orally infected with SBV usually accumulate high levels of the virus, which halts larval development and causes insect death. In contrast, oral DWV infection at the larval stage usually causes asymptomatic infection with low levels of the virus, although high doses of ingested DWV could lead to DWV replicating to high levels. We investigated effects of DWV and SBV infection on the transcriptome of honeybee larvae and pupae using global RNA-Seq and real-time PCR analysis. This showed that high levels of SBV replication resulted in down-regulation of the genes involved in cuticle and muscle development, together with changes in expression of putative immune-related genes. In particular, honeybee larvae with high levels of SBV replication, with and without high levels of DWV replication, showed concerted up-regulated expression of antimicrobial peptides (AMPs), and down-regulated expression of the prophenoloxidase activating enzyme (PPAE) together with up-regulation of the expression of a putative serpin, which could lead to the suppression of the melanisation pathway. The effects of high SBV levels on expression of these immune genes were unlikely to be a consequence of SBV-induced developmental changes, because similar effects were observed in the honeybee pupae infected by injection. We suggest that the effects of SBV infection on the honeybee immunity could be an adaptation to horizontal transmission of the virus. Up-regulation of the expression of AMP genes in the SBV-infected brood may contribute to protection of the SBV virus particles in dead larvae from bacterial degradation. Suppression of the melanisation may also reduce the loss of infectivity of SBV in the larvae. Therefore it is possible that activation of AMP expression and suppression of melanisation could increase ability of SBV to be transmitted horizontally via cannibalization route. We observed no changes of AMPs and the melanisation pathway genes expression in the orally infected larvae with high levels of DWV replication alone. In the injected pupae, high levels of DWV alone did not alter expression of the tested melanisation pathway genes, but resulted in up-regulation of the AMPs, which could be contributed to the effect of DWV on the regulation of AMP expression in response to wounding. We suggest that the effects of single DWV infection on the expression of these immune-related genes could reflect evolutionary adaptations of DWV to vertical transmission. Up-regulation of AMPs is costly and suppression of melanisation may increase susceptibility to infections, therefore these changes may have negative impact on honeybee survival and, consequently, of the survival of DWV.
Wired.co.uk has also covered the recent PLoS Pathogens honeybee virus paper.
The BBSRC covers our recent studies of honeybee viruses with the subject line Bloodsucking mite threatens UK honeybees.
BBSRC press release
NERC, a co-funder of our Insect Pollinators Initiative research on deformed wing virus, has featured our recent paper in PLoS Pathogens on their Planet Earth Online website.
Planet Earth Online
Our paper on the identification of a virulent strain of deformed wing virus makes the ‘Featured research’ page of PLoS Pathogens this week. Further work from colleagues at Warwick is also contained in the Pearls article on Zoonotic Pathogens on the same page.
Featured in PLoS Pathogens
Recently accepted for publication
Wood et al., (2014) MosaicSolver: a tool for determining recombinants of viral genomes from pileup data. Nucleic Acids Research (in press)
Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, changes in tropism and possibly transmission across species barriers. Determining whether recombination has occurred and the specific recombination points is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method, using siRNAs and sequence data sampling the viral genome population (cDNA library). Matlab software (MosaicSolver) is available.