Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and ...mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis.
Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses.
This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages.
This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section.
It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses ...in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.
Viruses are notorious for rapidly exchanging genetic information between close relatives and with the host cells they infect. This exchange has profound effects on the nature and rapidity of virus ...and host evolution. Recombination between dsDNA viruses is common, as is genetic exchange between dsDNA viruses or retroviruses and host genomes. Recombination between RNA virus genomes is also well known. In contrast, genetic exchange across viral kingdoms, for instance between nonretroviral RNA viruses or ssDNA viruses and host genomes or between RNA and DNA viruses, was previously thought to be practically nonexistent. However, there is now growing evidence for both RNA and ssDNA viruses recombining with host dsDNA genomes and, more surprisingly, RNA virus genes recombining with ssDNA virus genomes. Mechanisms are still unclear, but this deep recombination greatly expands the breadth of virus evolution and confounds virus taxonomy.
The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding ...single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family
The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through
assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses.
Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.
Archaeosine (G
) is a structurally complex modified nucleoside found quasi-universally in the tRNA of
and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside ...the
G
is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G
at position 15, coupled with its novel molecular structure, led to a hypothesis that G
stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G
contributes to tRNA stability and define the biological role of G
, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G
into the genome of the hyperthermophilic archaeon
and the mesophilic archaeon
, resulting in modification of the tRNA with the G
precursor 7-cyano-7-deazaguansine (preQ
) (deletion of
) or no modification at position 15 (deletion of
). Assays of tRNA stability from
-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the
mutant strains, demonstrate that G
at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of
Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in
results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in
Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.
Viruses infecting the
harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family
, where >90% of the viral genes do not have detectable ...homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects
in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the
The major capsid gene
is essential for SSV1 infectivity. However, the universally conserved minor capsid gene
could be deleted without a loss in infectivity and results in virions with abnormal morphology.
Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses,
, allows the production of infectious viruses. However, viruses lacking
have abnormal shapes, indicating that the
gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses.
Summary
Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. Although a number of in vitro systems have been ...established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile. In this work, we have developed a stable and selectable shuttle vector based on the virus SSV1 of Sulfolobus shibatae. We have introduced pUC18 for propagation in Escherichia coli and the genes pyrEF coding for orotidine‐5′‐monophosphate pyrophosphorylase and orotidine‐5′‐monophosphate decarboxylase of Sulfolobus solfataricus as selectable marker to complement pyrimidine auxotrophic mutants. Furthermore, the beta‐galactosidase gene (lacS) was introduced into this vector as a reporter under the control of the strong and heat‐inducible promoter of the Sulfolobus chaperonin (thermosome). After transformation of a S. solfataricus pyrEF/lacS double mutant, the vector was found to reside as a single‐copy vector, stably integrated into the host chromosome via the site‐specific recombination system of SSV1. Specific beta‐galactosidase activities in transformants were found to be fourfold higher than in wild‐type S. solfataricus cells, and increased to more than 10‐fold after heat shock. Greatly increased levels of lacS mRNA were detected in Northern analyses, demonstrating that this reporter gene system is suitable for the study of regulated promoters in Sulfolobus and that the vector can also be used for the high‐level expression of genes from hyperthermophilic archaea.
PDF
