Understanding superinfection exclusion by complex populations of Citrus tristeza virus.

Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection restricts a secondary infection with the same or closely related virus. Previously we showed that SIE by Citrus tristeza virus (CTV) occurs only between isolates of the same virus genotype. This work, however, was done using single genotype-containing isolates, while most field citrus trees harbor complex populations composed of different virus genotypes. Here we examined SIE in plants simultaneously infected with several CTV genotypes. The experiments showed that exclusion of a secondary infection by a CTV variant was triggered by the presence of another variant of the same genotype in the primary population, even under the conditions of its low-level accumulation, and was not affected by co-occurrence of additional heterologous genotypes. The same rule appeared to be in effect when SIE by mixed populations was tested in a series of different citrus varieties.

Simultaneous visualization of two Citrus tristeza virus genotypes provides new insights into the structure of multi-component virus populations in a host.

Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.

A 5'-proximal region of the Citrus tristeza virus genome encoding two leader proteases is involved in virus superinfection exclusion.

Superinfection exclusion (SIE), a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus, has been observed with various viruses. Earlier we demonstrated that SIE by Citrus tristeza virus (CTV) requires viral p33 protein. In this work we show that p33 alone is not sufficient for virus exclusion. To define the additional viral components that are involved in this phenomenon, we engineered a hybrid virus in which a 5'-proximal region in the genome of the T36 isolate containing coding sequences for the two leader proteases L1 and L2 has been substituted with a corresponding region from the genome of a heterologous T68-1 isolate. Sequential inoculation of plants pre-infected with the CTV L1L2T68 hybrid with T36 CTV resulted in superinfection with the challenge virus, which indicated that the substitution of the L1-L2 coding region affected SIE ability of the virus.

A viral protein mediates superinfection exclusion at the whole-organism level but is not required for exclusion at the cellular level.

UNLABELLED: Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels--the cellular and the whole-organism levels--by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE: Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.

Genetic Diversity, Reassortment, and Recombination in Alfalfa mosaic virus Population in Spain.

The variability and genetic structure of Alfalfa mosaic virus (AMV) in Spain was evaluated through the molecular characterization of 60 isolates collected from different hosts and different geographic areas. Analysis of nucleotide sequences in four coding regions--P1, P2, movement protein (MP), and coat protein (CP)--revealed a low genetic diversity and different restrictions to variation operating on each coding region. Phylogenetic analysis of Spanish isolates along with previously reported AMV sequences showed consistent clustering into types I and II for P1 and types I, IIA, and IIB for MP and CP regions. No clustering was observed for the P2 region. According to restriction fragment length polymorphism analysis, the Spanish AMV population consisted of seven haplotypes, including two haplotypes generated by reassortment and one involving recombination. The most frequent haplotypes (types for P1, MP, and CP regions, respectively) were I-I-I (37%), II-IIB-IIB (30%), and one of the reassortants, II-I-I (17%). Distribution of haplotypes was not uniform, indicating that AMV population was structured according to the geographic origin of isolates. Our results suggest that agroecological factors are involved in the maintenance of AMV genetic types, including the reassortant one, and in their geographic distribution.

Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis.

BACKGROUND: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans. RESULTS: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli. GENERAL SIGNIFICANCE: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities. CONCLUSION: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.

Biosynthesis of isoprenoids in plants: structure of the 2C-methyl-D-erithrytol 2,4-cyclodiphosphate synthase from Arabidopsis thaliana. Comparison with the bacterial enzymes.

The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 A resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.