Christophe Brugidou

Mass Spectrometry of an Intact Virus

The simultaneous measurement of the time-of-flight and the number of charges on each electrosprayed ion provides a new way to determine the mass of a whole intact virus. The weights of an icosahedral virus (rice yellow mottle virus) consisting of a single-stranded RNA surrounded by a homogeneous protein shell with a mass of 6.5×106 Da, and a rod-shaped RNA virus (tobacco mosaic virus) with a mass of 40.5×106 Da were measured with this technique.

Mass spectrometry and viral analysis.

BACKGROUND Electrospray ionization (ESI) mass spectrometry is a powerful new approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry; it is not clear, however, whether the native conformation of the biomolecules is maintained throughout the ionization and analysis process. We set out to address this question using live viruses. RESULTS Viral ions have been generated in the gas phase using electrospray ionization mass spectrometry. These ions have been collected, following ion filtering through the mass analyzer, and then analyzed by transmission electron microscopy. Transmission electron microscopy revealed that rice yellow mottle virus and tobacco mosaic virus retained their respective spherical and rod-like ultrastructure. The viability of the isolated tobacco mosaic virus was confirmed by inoculation and infection of tobacco plants. CONCLUSIONS These results demonstrate the utility of electrospray for supramolecular complexes with molecular weights of over 40 million Da and offer conclusive evidence that native biomolecular structures can be conserved through the electrospray process.

Inferring the Evolutionary History of Rice Yellow Mottle Virus from Genomic, Phylogenetic, and Phylogeographic Studies

ABSTRACT Fourteen isolates of Rice yellow mottle virus (RYMV) were selected as representative of the genetic variability of the virus in Africa from a total set of 320 isolates serologically typed or partially sequenced. The 14 isolates were fully sequenced and analyzed together with two previously reported sequences. RYMV had a genomic organization similar to that of Cocksfoot mottle sobemovirus. The average nucleotide diversity among the 16 isolates of RYMV was 7%, and the maximum diversity between any two isolates was 10%. A strong conservative selection was apparent on both synonymous and nonsynonymous substitutions, through the amino acid replacement pattern, on the genome size, and through the limited number of indel events. Furthermore, there was a lack of positive selection on single amino acid sites and no evidence of recombination events. RYMV diversity had a pronounced and characteristic geographic structure. The branching order of the clades correlated with the geographic origin of the isolates along an east-to-west transect across Africa, and there was a marked decrease in nucleotide diversity moving westward across the continent. The insertion-deletion polymorphism was related to virus phylogeny. There was a partial phylogenetic incongruence between the coat protein gene and the rest of the genome. Overall, our results support the hypothesis that RYMV originated in East Africa and then dispersed and differentiated gradually from the east to the west of the continent.

3D Domain Swapping Modulates the Stability of Members of an Icosahedral Virus Group

BACKGROUND Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Rice genomics: Present and future

A review of the present and future of rice genomics is presented. Rice is a model species for cereals as well as a very important crop. Its genome has been the focus of many mapping experiments associated with QTL localization. These genetic maps now serve as a background for physical mapping, genome sequencing and gene discovery. Recent progress are reviewed. The next step in rice genomics is functional genomics with the determination of the function of the genes. The most straightforward approaches are discussed.

Distribution and Characterization of Rice yellow mottle virus: A Threat to African Farmers

Africa produces only 2.7% of the world’s rice and is the second-largest riceimporting region in the world (6.5 Mt in 2003). This amount represents about 25% of the world rice importation (40). With an average of 2 t/ha, excluding Egypt, rice production in Africa remains significantly below the world average (Asia 3.8 t/ha, Latin America 3.0 t/ha, United States 7.0 t/ha; Food and Agriculture Organization of the United Nations [FAO], published online). Insects and diseases are the two major constraints limiting rice production in Africa and Asia. Of all the rice diseases, the one caused by the Rice yellow mottle virus (RYMV), discovered in Kenya in 1970, is one of the most damaging in Africa. RYMV has been reported in many countries in East and West Africa, where in some cases whole fields have been devastated. RYMV has only been reported from the African continent and is endemic in every country where it has been reported. RYMV is a member of the genus Sobemovirus and possesses all the characteristic biophysical and biological properties of the members of the genus. The major insect vectors are chrysomelids, which play an essential role in primary infection, while secondary infection is due mainly to plant-to-plant contact. The virus particles are stable in infected dried leaves. Limitations to the spread of the virus include restricted mobility of the insect vectors, a limited host range, and absence of seed transmission. Partially resistant and highly resistant varieties have been identified, but currently the disease is not controlled adequately, and its incidence is increasing significantly in Africa. Due to the importance of rice as a staple food, the scientific community is active in studying the virus and the resistant mechanisms in rice. At the Institut de Recherche pour le Developpement (IRD) and the International Laboratory for Tropical Agricultural Biotechnology (ILTAB), different tools, such as RYMV polyclonal and monoclonal antibodies, have been produced for RYMV detection; and a series of studies has been initiated for a better understanding of the virus–host interactions. The synthesis of an infectious clone, the sequencing of many isolates of the virus, and structural studies of virus particles permitted a study of the function of the different viral proteins and an increased understanding of the virus cycle. African virologists also have initiated studies on the epidemiology of the RYMV, mainly the virulence and variability of virus strains collected throughout Africa (36,45). The relationships between genetic variability of the virus and geographical distances have been studied (19). Currently, phytopathologists are working in the field to better assess the agronomic impact of the virus (36,61,62). Breeders and geneticists at IRD and West African Rice Development Association (WARDA) in Cote d’Ivoire are working together to identify and isolate natural resistance genes (6,41). The International Institute for Tropical Agriculture (IITA) in Nigeria undertook breeding programs in Africa. Molecular biologists are offering new ways of controlling the disease through production of transgenic rice plants (37,52). This article is intended to provide a review of the disease and give an update on recent research results. The disease’s impact on rice production in Africa, its distribution, symptomatology, and epidemiology, the physical and genetic characteristics of the virus, and resistance mechanisms and breeding efforts for resistance will be discussed.

Proteome analysis of plant-virus interactome: comprehensive data for virus multiplication inside their hosts

Known host-parasite molecular interactions are widespread among parasite families, but these interactions have to be particularly large considering that viruses generally encode few proteins. Although some particular virus-host interactions are well described, no global study has yet shown multiple and simultaneous interactions in a host-parasite biological system. To prove that these multiple interactions occur in biological conditions, the complexes formed by a plant virus (rice yellow mottle virus) and the proteins of its natural host (rice) were extracted and purified from infected tissue sample. Remarkably mass spectrometry permitted the identification of a large number of proteins from the complexes that are involved in different functions not encoded by the virus but probably essential for its biological life cycle. This recruiting of proteins was strongly confirmed by the repetition of experiments using different pairs of virus-host and the use of high salt concentration to extract the complexes. We mainly identified proteins involved in plant defense, metabolism, translation, and protein synthesis and some proteins involved in transport. This study demonstrates that viruses are able to recruit many proteins from their hosts to ensure their development. Among different pairs of virus-host, similar protein functions were identified suggesting a particular importance of these proteins for viruses. The identification of particular paralog proteins among multigenic families suggests the high specificity of the recruiting for some protein functions.

Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus andmutagenesis of the coat protein

A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus

Summary. Isolates of Rice yellow mottle virus (RYMV) were typed at the molecular level through the sequences of the open reading frame (ORF) 4 (coding for the coat protein) and ORF1 (coding for the movement protein), and serologically by means of polyclonal and monoclonal antibodies. The overall patterns of diversity shown by molecular and serological analyses were similar: East-African isolates differed from West-African ones, and the West-African isolates from forest differed from the savannah ones. Each major strain had a different serological profile. However, molecular typing was more discriminating than immunological typing since several sequence variants belonged to the same serotype. In rare instances, there were explainable discrepancies between molecular and serological typing. Two amino acids at positions 115 (alanine vs threonine) and 191 (valine vs threonine) consistently discriminated between the major serotypes. These positions were located in antigenic sites as revealed by Spot-scan method and were recognised by discriminating monoclonal antibodies. One shared epitope, lying within a conserved region, may be responsible for the cross-reactivity between RYMV isolates. A rationale for the correlation between molecular and immunological typing of RYMV and other sobemoviruses is proposed.

The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing.

In plants RNA silencing is a host defense mechanism against viral infection, in which double-strand RNA is processed into 21-24-nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence-specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single-strand and double-strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21- and 24-nt siRNA accumulation, respectively. Unexpectedly, cell-to-cell movement and systemic propagation of silencing were enhanced in P1-expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4-dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4-dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter-defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity.