Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA

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Abstract
The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies and electron microscopy. RF DNA is a linear, double-stranded molecule comparable in lengh to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. Newly synthesized ssDNA is probably immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt.