Genomic clones of bovine parvovirus: construction and effect of deletions and terminal sequence inversions on infectivity

Abstract

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytopathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. DNA isolated from progeny virions arising from transfected infectious clones was found to be indistinguishable from wild-type DNA by restriction enzyme analysis. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogenous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Analysis of low-molecular-weight DNA isolated from lysates of cells transfected with these clones demonstrated that rescue and replication of BPV DNA could be detected 3 to 8 days after transfection. Expression of capsid proteins from transfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [35S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus.

Analysis of the termini of the DNA of bovine parvovirus: demonstration of sequence inversion at the left terminus and its implication for the replication model

“…Transfection with genomic clones of BPV containing defined conformations at the ends resulted in progeny virion DNAs with distributions identical to that found in a normal infection. The same ratio of plus to minus strands (27), as well as the same ratios of flip to flop forms at the ends of reannealed virion DNAs (Fig. 4), were observed.…”

“…This suggested that, at the left end, one conformation could generate the other and that the flip form always accumulated preferentially in the viral DNA, regardless of the conformation present in the clone used for transfection. Similar analyses were performed on a different infectious genomic clone, pGCSma2O (flip at both ends [27]), which is deleted by 3 bases at the left end and 35 bases at the right end. Both ends were repaired to wild-type length and again the ratio of flip to flop at each terminus was indistinguishable from that for wild-type virion DNA (data not shown).…”

“…Fate of flip and flop forms of transfecting genomic clones. Full-length genomic clones pVT501 (flop at left end) and pVT502 (flip at left end) have the same right end (in the flip form) and differ only in the orientation at the left end (27). When the left ends of the progeny DNA of amplified transfections initiated with either pVT501 or pVT502 were analyzed by end labeling and SmaI digestion, both flip and flop conformations were observed and the ratio of the two forms was identical to that found in a normal infection (Fig.…”

“…Clone construction. The construction and characterization of BPV infectious genomic clpnes pVT501, pVT502, and pGCSma20 have been described previQusly (27). Left and right terminal fragments of BPV were cloned into pUC8, and terminal SmaI fragments were cloned 'into M13mpl8 by standard procedures (18).…”

Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein

“…Previous results have indicated that the cis sequences required for parvovirus DNA replication are contained in or near the terminal inverted repeat sequences (29). Defective interfering particles that can be serially passaged have been described that are as small as 500 base pairs with intact terminal palindromes (8).…”