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The transcription programme of the protein-primed halovirus SH1

¹ Biota Holdings Ltd, 10/585 Blackburn Road, Notting Hill, Victoria 3168, Australia
² Department of Microbiology and Immunology, University of Melbourne, 3052, Australia
³ Max-Planck Institute for Biochemistry, Department of Membrane Biochemistry, Martinsried 82152, Germany

Correspondence
Kate Porter
K.Porter{at}biota.com.au

SH1 is the only reported isolate of a spherical halovirus, a dominant morphotype in hypersaline lakes. The virus lytically infects the haloarchaeon Haloarcula hispanica, and carries a 30.9 kb linear dsDNA genome that, in a previous study, was proposed to contain 56 protein-coding genes, probably organized into between four and eight operons. In the present study, these predictions were directly tested by determining the orientations and lengths of virus transcripts using systematic RT-PCR and primer extension. Seven major transcripts were observed that together covered most of the genome. Six transcripts were synthesized from early in infection (1 h post-infection; p.i.) onwards, while transcript T6 was only detected late in infection (5–6 h p.i.). No transcripts were detected in the inverted terminal repeat sequences or at the extreme right end of the genome (ORFs 55–56). Start points for the major transcripts were mapped by primer extension and corresponded closely to the 5' termini determined by RT-PCR. Between 1 and 4 h p.i., transcripts usually terminated not far beyond the end of their last coding ORF, but late in infection, transcripts from the same promoters often terminated at more distal points, resulting in much of the genome being transcribed from both strands. Since many of these transcripts are complementary, RNA–RNA interactions are likely, and may play a role in regulating viral gene expression. Puromycin blockage of post-infection protein synthesis significantly altered the levels of certain virus transcripts, indicating that de novo protein synthesis is essential for the correct regulation of SH1 gene expression.

Two supplementary tables listing the oligonucleotide primers used in this study and the positions of the first and last ORFs relative to the inverted terminal repeats in protein-primed replication systems, and a supplementary figure showing the nucleotide sequence of the reference ladder used in the primer extension assays, are available with the online version of this paper.