Genome replication of hyperthermophilic archaeal viruses – REPVIR

The double-stranded (ds) DNA viruses which infect hyperthermophilic members of the third domain of life, the Archaea, are radically different in their properties from viruses of the Bacteria and the Eucarya. They reveal unique diverse morphotypes and carry genomes with more than 90% of putative genes without recognizable functions and detectable homologs in other viruses or cellular life forms. For classification of slightly more than two dozen of known species of these viruses, seven novel viral families have been introduced, including spindle-shaped Fuselloviridae, filamentous Lipothrixviridae, rod-shaped Rudiviridae, droplet-shaped Guttaviridae, spherical Globuloviridae, two-tailed Bicaudaviridae, and bottle-shaped Ampullaviridae. Knowledge on the biology of the hyperthermophilic archaeal viruses is very limited, mainly due to the unique content of their genomes, and nearly nothing is known on the mechanisms of genome replication and involved proteins. The only exception is the bottle-shaped virus ABV, which carries a gene for a family B DNA-dependent DNA polymerase, presumable primed by a protein. Such situation is dramatically different from that in dsDNA viruses of Bacteria and Eucarya, nearly all of which, with very few exceptions, encode their own DNA-dependent DNA polymerases. An absence of any recognisable DNA polymerase gene on the genomes of hyperthermophilic archaeal viruses, other than ABV, is intriguing, especially considering specific structural features of the viral genomes, and suggests these viruses may rely heavily upon host cell replication machinery, however, should be able to modify or recruit it in some unknown way in order to promote selective replication of its own DNA and probably effect cell cycle. The aim of the project is to reveal and characterize the proteins of hyperthermophilic archaeal viruses involved in DNA replication, and to understand mechanisms of replication, as well as regulations that alter cell replication to promote the synthesis of viral DNA. As models for the studies we have selected three viruses from three different families, for which we have obtained some preliminary data and which apparently exploit different mechanisms and proteins for genome replication. These are the rudivirus SIRV1 infecting the hyperthermophilic Sulfolobus islandicus, and the filamentous lipothrixvirus AFV1 and the bottle-shaped ampullavirus ABV, both infecting species of the hyperthermophilic genera Acidianus. The problems of replication of each of the three selected viruses will be addressed in different ways, depending on the available information and peculiarities of the virus-host systems. Thus, studies on ABV replication will mainly comprise characterization of recombinant DNA polymerase and putative terminal protein attached to the linear genome, which most likely primes the replication. One objective of characterization of the ABV DNA polymerase would be the demonstration of its technological potential for whole genomic DNA amplification and for long-range PCR, suggested by putative high thermostability and high processivity of the enzyme. For viruses SIRV1 and SIFV1, which do not encode any recognizable DNA polymerase, the main objective would be to understand the mode of viral genome replication, and to generate and verify hypothesis on involvement of viral and host proteins in this process. The origins of replication will be elucidated, and based on the obtained results viral proteins responsible for initiation of replication will be postulated. Detection of networks of interacting proteins, viral and host, with known and unknown functions, will help to elucidate a role of each protein in the replication machinery of the two viruses. Based on hypothetical functions, appropriate biochemical assays will be designed, and functions verified. The results will enable to hypothesize on mechanisms of DNA replication of each of the two viruses, which can be confirmed by visualization of replicative intermediates by electron microscopy. Preliminary observations suggest that DNA replication might be taking place at special sites in SIRV1-infected cells. We plan to characterize these sites by electron microscopy, by cryo-fixation under high pressure freezing, and to analyze connection of these sites with DNA replication with immuno-electron microscopy, employing antibodies against postulated replication proteins.