First insights into the entry process of hyperthermophilic archaeal viruses.

A decisive step in a virus infection cycle is the recognition of a specific receptor present on the host cell surface, subsequently leading to the delivery of the viral genome into the cell interior. Until now, the early stages of infection have not been thoroughly investigated for any virus infecting hyperthermophilic archaea. Here, we present the first study focusing on the primary interactions between the archaeal rod-shaped virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) (family Rudiviridae) and its hyperthermoacidophilic host, S. islandicus. We show that SIRV2 adsorption is very rapid, with the majority of virions being irreversibly bound to the host cell within 1 min. We utilized transmission electron microscopy and whole-cell electron cryotomography to demonstrate that SIRV2 virions specifically recognize the tips of pilus-like filaments, which are highly abundant on the host cell surface. Following the initial binding, the viral particles are found attached to the sides of the filaments, suggesting a movement along these appendages toward the cell surface. Finally, we also show that SIRV2 establishes superinfection exclusion, a phenomenon not previously described for archaeal viruses.

Simple and elegant design of a virion egress structure in Archaea.

Some viruses of Archaea use an unusual egress mechanism that involves the formation of virus-associated pyramids (VAPs) on the host cell surface. At the end of the infection cycle, these structures open outward and create apertures through which mature virions escape from the cell. Here we describe in detail the structure and composition of VAPs formed by the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in cells of its hyperthermophilic archaeal host. We show that the VAPs are stable and autonomous assemblies that can be isolated from membranes of infected cells and purified without affecting their structure. The purified VAPs are heterogeneous in size, reflecting the dynamics of VAP development in a population of infected cells; however, they have a uniform geometry, consisting of seven isosceles triangular faces forming a baseless pyramid. Biochemical and immunoelectron microscopy analyses revealed that the 10-kDa P98 protein encoded by the SIRV2 virus is the sole component of the VAPs. The VAPs were produced in Sulfolobus acidocaldarius and Escherichia coli by heterologous expression of the SIRV2-P98 gene. The results confirm that P98 is the only constituent of the VAPs and demonstrate that no other viral protein is involved in the assembly of pyramids. P98 was able to produce stable structures under conditions ranging from moderate to extremely high temperatures (80 °C) and from neutral to extremely acidic pH (pH 2), demonstrating another remarkable property of this exceptional viral protein.

Diversity of virus-host systems in hypersaline Lake Retba, Senegal.

Remarkable morphological diversity of virus-like particles was observed by transmission electron microscopy in a hypersaline water sample from Lake Retba, Senegal. The majority of particles morphologically resembled hyperthermophilic archaeal DNA viruses isolated from extreme geothermal environments. Some hypersaline viral morphotypes have not been previously observed in nature, and less than 1% of observed particles had a head-and-tail morphology, which is typical for bacterial DNA viruses. Culture-independent analysis of the microbial diversity in the sample suggested the dominance of extremely halophilic archaea. Few of the 16S sequences corresponded to known archeal genera (Haloquadratum, Halorubrum and Natronomonas), whereas the majority represented novel archaeal clades. Three sequences corresponded to a new basal lineage of the haloarchaea. Bacteria belonged to four major phyla, consistent with the known diversity in saline environments. Metagenomic sequencing of DNA from the purified virus-like particles revealed very few similarities to the NCBI non-redundant database at either the nucleotide or amino acid level. Some of the identifiable virus sequences were most similar to previously described haloarchaeal viruses, but no sequence similarities were found to archaeal viruses from extreme geothermal environments. A large proportion of the sequences had similarity to previously sequenced viral metagenomes from solar salterns.

The Sulfolobus rod-shaped virus 2 encodes a prominent structural component of the unique virion release system in Archaea.

Recently a unique mechanism of virion release was discovered in Archaea, different from lysis and egress systems of bacterial and eukaryotic viruses. It involves formation of pyramidal structures on the host cell surface that rupture the S-layer and by opening outwards, create apertures through which mature virions escape the cell. Here we present results of a protein analysis of Sulfolobus islandicus cells infected with the rudivirus SIRV2, which enable us to postulate SIRV2-encoded protein P98 as the major constituent of these exceptional cellular ultrastructures.

Viruses in acidic geothermal environments of the Kamchatka Peninsula.

Screening for viruses in samples taken from acidic hot springs of Kamchatka (Russia) revealed a collection of morphotypes, including linear, spherical and complex fusiform shapes, which show partial similarity to those found in acidic geothermal environments in other geographical locations. One of the viruses, Acidianus filamentous virus 9, AFV9, was isolated and its structure and genome were studied in detail.

Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.