Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Poxviruses replicate in the host cytoplasm, encoding most necessary enzymes for their double-stranded DNA (dsDNA) genome synthesis.
• The precise mechanism of poxvirus DNA replication and the location of replication origins have remained largely unknown.
• Previous models suggested a limited replication strategy, lacking definitive evidence for specific origins.

Purpose of the Study:

• To elucidate the mode of poxvirus DNA replication and identify the specific origins of replication.
• To investigate the nature and location of replication origins in vaccinia virus (VACV).

Main Methods:

• Utilized directional deep sequencing of short, single-stranded DNA fragments enriched for RNA-primed nascent strands.
• Isolated these fragments from the cytoplasm of vaccinia virus (VACV)-infected cells.
• Identified replication origins by pinpointing the directional switching points in the sequenced fragments.

Main Results:

• Replication origins were identified as transition points from continuous to discontinuous DNA synthesis.
• A prominent initiation point was mapped to a sequence within the hairpin loop at one end of the VACV genome.
• The same sequence was found at the concatemeric junction of replication intermediates.

Conclusions:

• Findings support a poxvirus genome replication model involving both leading and lagging strand synthesis.
• The identified origins are consistent with the need for primase and ligase activities.
• Results align with prior electron microscopy and biochemical studies suggesting a VACV replication origin at the genome's end.