The virus family Fuselloviridae infects the hyperthermophilic and acidophilic Crenarchaeon genus Sulfolobus and has been isolated from terrestrial hotsprings worldwide. Two previously uncharacterized Fuselloviruses, SSV-13 and SSV-L1, were isolated and sequenced and are compared to the five fully sequenced viruses presently in the public databases. Conserved promoters in all viruses and similar putative origins of replication suggest that these viruses use a transcriptional and genomic replication program similar to the relatively well-characterized SSV1. Pairwise comparisons of conserved genes in the seven virus genomes show that, like its host Sulfolobus, these viruses' genetic divergence correlates with geographic separation. Genome rearrangements, horizontal movement of genes between Fuselloviruses, other Crenarchaeal viruses, and other hosts are also discussed.

The development of a novel gene knockout system (LIPCR) for these viruses is presented with detailed methods. Use of this knockout system is demonstrated with two viral vectors that have fully and partially deleted integrase genes. The complete integrase deletion does not prevent virus replication but appears to prevent integration of the virus into the host genome and appears to decrease the relative fitness of the virus compared to a virus with a complete integrase gene. The partial integrase deletion removes the catalytic residues demonstrated to be necessary for enzymatic function but leaves the attachment site located within the integrase gene. Interestingly, this mutant appears to be still capable of integration in our lab host, Sulfolobus solfataricus. Possible reasons for this are discussed.