Rémy Froissart

Helper component-transcomplementation in the vector transmission of plant viruse.

ABSTRACT Plant viruses are most frequently transmitted from one host plant to another by vectors. In noncirculative vector transmission, the virus does not process through a cycle within the vector body. Instead, upon acquisition by the vector, viruses are retained in the mouth parts or the anterior gut; from there, they will be subsequently released in a new host plant. Two molecular strategies have been described for the virus-vector interaction. In the capsid strategy, the virus coat interacts directly with binding sites in the vector mouth parts, whereas an additional nonstructural protein, designated helper component (HC), is required in the helper strategy. The HC and virus particles can be acquired sequentially, and this property introduces the possibility that an HC acquired first by the vector assists the transmission of virus particles located in the same cell, in other cells, or even in other host plants probed by the vector. Such a phenomenon is here called HC-transcomplementation. Surprisingly, the existing definition of HC does not explicitly include the concept of HC-transcomplementation, and it is generally omitted in the literature in any consideration of the virus biology other than the molecular interaction with the vector. Here we propose an extended definition of HC and emphasize the concept of HC-transcomplementation that distinguishes the helper strategy from any other type of vector transmission and may have consequences at the level of the virus population genetics and evolution.

Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.

Large Bottleneck Size in Cauliflower Mosaic Virus Populations during Host Plant Colonization

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses.

Evolution of virulence in emerging epidemics.

Theory predicts that selection for pathogen virulence and horizontal transmission is highest at the onset of an epidemic but decreases thereafter, as the epidemic depletes the pool of susceptible hosts. We tested this prediction by tracking the competition between the latent bacteriophage λ and its virulent mutant λcI857 throughout experimental epidemics taking place in continuous cultures of Escherichia coli. As expected, the virulent λcI857 is strongly favored in the early stage of the epidemic, but loses competition with the latent virus as prevalence increases. We show that the observed transient selection for virulence and horizontal transmission can be fully explained within the framework of evolutionary epidemiology theory. This experimental validation of our predictions is a key step towards a predictive theory for the evolution of virulence in emerging infectious diseases.

AN EXPERIMENTAL TEST OF THE TRANSMISSION-VIRULENCE TRADE-OFF HYPOTHESIS IN A PLANT VIRUS

The transmission–virulence trade‐off hypothesis is one of the few adaptive explanations of virulence evolution, and assumes that there is an overall positive correlation between parasite transmission and virulence. The shape of the transmission–virulence relationship predicts whether virulence should evolve toward either a maximum or to an intermediate optimum. A positive correlation between each of these traits and within‐host growth is often suggested to underlie the relationship between virulence and transmission. There are few experimental tests of this hypothesis; this study reports on the first empirical test on a plant pathogen. We infected Brassica rapa plants with nine natural isolates of Cauliflower mosaic virus and then estimated three traits: transmission, virulence, and within‐host viral accumulation. As predicted by the trade‐off hypothesis, we observed a positive correlation between transmission and virulence, suggestive of the existence of an intermediate optimum. We discovered the unexpected existence of two groups of within‐host accumulation, differing by at least an order of magnitude. When accumulation groups were not accounted for, within‐host accumulation was correlated neither to virulence nor transmission, although our results suggest that within each group these correlations exist.

Biochemical Characterization of the Helper Component of Cauliflower Mosaic Virus

ABSTRACT The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% α-helical. Most α-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

THE DISTRIBUTION OF MUTATIONAL FITNESS EFFECTS OF PHAGE φX174 ON DIFFERENT HOSTS

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short‐term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host‐specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host‐specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context‐dependent DMFE for multihost pathogens.

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.

Reduction of leaf area and symptom severity as proxies of disease-induced plant mortality: The example of the Cauliflower mosaic virus infecting two Brassicaceae hosts

Disease induced effects on host survival are important to understand the evolution of parasitic virulence and host resistance/tolerance. Unfortunately, experiments evaluating such effects are in most cases logistically demanding justifying the measurement of survival proxies. For plant hosts commonly used proxies are leaf area and the nature and severity of visual qualitative disease symptoms. In this study we tested whether these traits are indeed correlated to the host mortality rate induced by viral infection. We infected Brassica rapa and Arabidopsis thaliana plants with different natural isolates of Cauliflower mosaic virus (CaMV) and estimated over time the development of symptoms and the relative reduction of leaf area compared to healthy plants and followed plant mortality. We observed that the mortality of infected plants was correlated with the relative reduction of leaf area of both B. rapa and A. thaliana. Measures of mortality were also correlated with the severity of visual qualitative symptoms but the magnitude of the correlations and the time frame at which they were significant depended on the host plant: stronger and earlier correlations were observed on A. thaliana.

Chapter 8 – Caulimoviruses

Publisher Summary The best known vectors are insects in the order Homoptera, especially aphids. Based on thorough analysis of the morphology of the homopteran feeding apparatus, it is concluded that virus located past the OS in the intima-lined pharynx of the foregut can no longer be egested and is, therefore, noninoculable. Two subcategories of noncirculative viruses, nonpersistent and semipersistent, are usually differentiated on the basis of the time required for virus acquisition and inoculation, as well as the length of time for which the vector can retain virus in an infectious form. However, in some cases variable results have been obtained for semipersistent viruses and, in particular, for cauliflower mosaic virus (CaMV). There is no conclusive evidence of significant differences between the molecular mechanism of virus–vector interaction in nonpersistent and semipersistent transmission. Recent data has been on the molecular mechanisms of virus–vector interactions as the main criteria for defining two distinct viral strategies, the capsid strategy and the helper strategy. In the capsid strategy, the virus interacts directly with the vector via its coat protein, whereas in the helper strategy the virus–vector interaction is mediated by an additional virus-encoded nonstructural protein, generally designated as helper. The helper strategy is frequently found among noncirculative viruses in the genera Potyvirus, Caulimovirus, Waikavirus, Sequivirus, and presumably Closterovirus and even the nematode-transmitted Tobravirus. The molecular mechanisms of virus–vector interaction have been elucidated by researching the mode of action of the helper. This chapter illustrates that the helpers in different virus groups may not all function similarly by comparing the aphid transmission of caulimoviruses with that of potyviruses.