Transfection of haloarchaea by the DNAs of spindle and round haloviruses and the use of transposon mutagenesis to identify non-essential regions.
Spindle-shaped halovirus His2 and spherical halovirus SH1 represent ecologically dominant virus morphotypes in high-salt environments. Both have linear dsDNA genomes with inverted terminal repeat sequences and terminal proteins, and probably replicate using protein priming. As a first step towards conventional genetic analyses on these viruses, we show that purified viral DNAs can transfect host cells. Intact terminal proteins were essential for this process. Despite the narrow host ranges of these viruses, at least under laboratory conditions, their DNAs were able to transfect a wide range of haloarchaeal species, demonstrating that the cytoplasms of diverse haloarchaea possess all the factors necessary for viral DNA synthesis and virion assembly. Transposon mutagenesis of viral DNAs was then used in conjunction with transfection to produce recombinant viruses, and to then map the insertion sites to identify non-essential genes. The inserts in 34 His2 mutants were mapped precisely, and most clustered in a few, specific regions, particularly in the inverted terminal repeats and near the ends of ORFs. The results are consistent with the small genome size and densely packed, often overlapping ORFs that are transcribed as long operons. This study is the first demonstration of transfection and transposon mutagenesis in protein-primed archaeal viruses.
Porter
Kate
K
Biota Holdings Ltd., 10/585 Blackburn Road, Notting Hill, Victoria 3168, Australia.
Dyall-Smith
Michael L
ML
D004251:DNA Transposable Elements; D004267:DNA Viruses / Q000235:genetics* / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D016679:Genome, Viral; D006216:Halobacteriaceae / Q000235:genetics / Q000821:virology*; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication
D004251:DNA Transposable Elements; D004279:DNA, Viral; D014764:Viral Proteins