Incomplete genomes of the parvovirus minute virus of mice: selective conservation of genome termini, including the origin for DNA replication

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Abstract
Deletion mutants of minute virus of mice arising during a single high-multiplicity passage and after serial undiluted passage were isolated and the incomplete viral genomes contained therein were analyzed. The DNA isolated from incomplete virions derived from a single high-multiplicty passage was heterogeneous, ranging in size from 15-70% of the intact viral genome, with an average molecular length of .apprx. 2000 nucleotides. Two distinct types of molecules, designated as type I D-DNA and type II D-DNA, could be distinguished on the basis of their degree of secondary structure and these were present in roughly equal amounts. Type I D-DNAs were predominantly single-stranded, recombinant molecules where the self-complementary sequences derived from both genomic termini were conserved. The 5'' terminus was modified relative to the analogous wild-type structure. Although virtually all of the wild-type genome sequence was seen in the total type I D-DNA population, sequences which map between coordinates 47.3-87.1 were clearly underrepresented. The extent and position of the deletions in individual molecules varied significantly. The shortest molecules in the population lacked between 90-95% of the internal wild-type genome sequence and consisted of sequences derived almost exclusively from within 5.0 map units (250 nucleotides) at both ends of the viral genome. These miniature recombinant molecules were selectively amplified during serial undiluted passage and were therefore believed to contain all of the critical recognition sites necessary for the replication of minute virus of mice viral DNA. Type II D-DNAs were virus-specific, double-stranded hairpin molecules whose complementary strands were covalently continuous at variable sites distal to the 5'' end of the viral minus strand. In sharp contrast to the type I genomes, these hairpin molecules consisted of sequences which mapped entirely at the 5'' end of the viral genome between positions 85.0-100. Type II molecules were gradually lost from the total D-DNA population during serial undiluted passage, suggesting that these molecules are not competent for DNA replication but arise as the result of fatal replication errors. Deletion mutants of the type described here for minute virus of mice should be valuable generally as aids to future studies on parovirus DNA replication, transcription and cell-virus interactions.