Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: Construction and testing of viral shuttle vectors

Directed open reading frame (ORF) disruption and a serial selection techniq ue in Escherichia coli and the extremely thermophilic archaeon Sulfolobus s olfataricus allowed the identification of otherwise cryptic crucial and non crucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15.5-kbp viral genome can incorporate a 2.96-kbp insert ion without loss of viral function and package this DNA properly into infec tious virus particles. The selection technique, based on the preferential b inding of ethidium bromide to relaxed DNA and the resulting inhibition of e ndonuclease cleavage to generate a pool of mostly singly cut molecules, sho uld be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irr adiation, it forms infectious virus particles, and it is stable at high cop y number in both S. solfataricus and E. coli. The classification of otherwi se unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic syst ems for Crenarchaeota.