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Background: Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult. Results: We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a "Core" region conserved in sequence. We show that it is followed by a long extension, which we termed "Iceberg" region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup. Conclusions: Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme. [ABSTRACT FROM AUTHOR]

Cowpea chlorotic mottle virus (CCMV, family Bromoviridae) is found worldwide and has been used as a model virus for a long time, but no data is available about the genetic diversity of field isolates. Recently, two new field isolates (Car1 and Car2) of CCMV obtained from cowpea showed distinct phenotypic symptoms when inoculated to cowpea. CCMV-Car1 induced severe mosaic and interveinal chlorosis, while CCMV-Car2 produced mild mottling and leaf rolling. Both isolates produced asymptomatic infection in Nicotiana benthamiana. The complete genome of both isolates was amplified by reverse transcription-polymerase chain reaction using specific primers against the CCMV sequences available in the GenBank database, cloned and sequenced. Both nucleotide and amino acid sequences were compared between the newly sequenced CCMV isolates and the three previously characterized CCMV strains (T, M1, and R). Phylogenetic analysis of the RNA 1 sequence showed that CCMV-Car1 was in a separate branch from the rest of the CCMV isolates while CCMV-Car2 grouped together with CCMV-R. On the basis of RNA 2 and RNA 3 sequences, two major groupings were obtained. One group included CCMV-Car1 and CCMV-Car2 isolates while the other contained CCMV-T, CCMV-M1, and CCMV-R strains. Recombination programs detected a potential recombination event in the RNA 1 sequence of CCMV-Car2 isolate but not in RNA 2 and RNA 3 sequences. The results showed that both mutations and recombination have played an important role in the genetic diversity of these two new isolates of CCMV. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae is invaluable for understanding fundamental cellular processes and disease states of relevance to higher eukaryotes. Plant viruses are intracellular parasites that take advantage of resources of the host cell, and a simple eukaryotic cell, such as yeast, can provide all or most of the functions for successful plant virus replication. Thus, yeast has been used as a model to unravel the interactions of plant viruses with their hosts. Indeed, genome-wide and proteomics studies using yeast as a model host with bromoviruses and tombusviruses have facilitated the identification of replication-associated factors that affect host-virus interactions, virus pathology, virus evolution, and host range. Many of the host genes that affect the replication of the two viruses, which belong to two dissimilar virus families, are distinct, suggesting that plant viruses have developed different ways to utilize the resources of host cells. In addition, a surprisingly large number of yeast genes have been shown to affect RNA-RNA recombination in tombusviruses; this opens an opportunity to study the role of the host in virus evolution. The knowledge gained about host-virus interactions likely will lead to the development of new antiviral methods and applications in biotechnology and nanotechnology, as well as new insights into cellular functions of individual genes and the basic biology of the host cell. [ABSTRACT FROM AUTHOR]