The autonomous parvovirus LuIII, initially isolated from a human cell line, is unique among the mammalian parvoviruses because its genome has non-identical palindromic termini, yet, during replication it encapsidates both strands with equal frequency. With the exception of LuIII, parvoviruses of known nucleotide sequence that encapsidate equal amounts of both DNA strands, have identical 3' and 5' termini. The nucleotide sequence of the Lulll termini shares over 90% sequence identity with that of the rodent parvovirus Minute Virus of Mice (MVM) and an overall genome identity of over 80%. Unlike LuIII, MVM encapsidates 99% minus strand DNA. The minor nucleotide differences observed between the termini of LuIII and MVM very likely do not determine the encapsidation pattern observed for Lulll since similar differences are observed between the sequences of MVM and that of another rodent parvovirus, H-1 which also encapsidates 99% minus strand. Two regions differ significantly between LuIII and MVM. At map unit (m.u.) 92 LuIII has a single copy of a sequence present in tandem in the prototype strain of MVM (MVMp). Although this sequence has been suggested to be involved in replication it most likely is not determining the encapsidation pattern observed for LuIII because a lymphotrophic strain of MVM (MVMi) has a single copy of this sequence and it too encapsidates 99% minus strand. At m.u. 89 LuIII has a unique sequence, rich in A/T residues. Given the location and nature of this sequence and its absence from the genome of MVM this sequence very likely represent a major determinant in the encapsidation pattern observed for LuIII. Deciphering the sequences involved in the encapsidation of LuIII DNA will eventually lead to the characterization of the mechanism resulting in the encapsidation pattern observed for LuIII.
The aims of this research include construction of genomic clones of LuIII and MVM (for both MVMi and MVMp) containing a deletion and insert, respectively, of the A-T rich sequence and of minigenomes with and without the A/T rich sequence containing both termini or two copies of either the 3' or 5' terminus. The clones constructed will be transfected into NBE 324K cells and the nature of the progeny viral DNA analyzed. Gel retardation assays of LuIII infected and uninfected NBE 324K cell lysates will be done to identify DNA binding proteins of viral and cellular nature using radiolabelled LuIII termini, A/T rich sequence or a combination of termini including the A/T rich sequence. A cDNA library of HELA cells will also be screened for DNA binding proteins. The long term objective of this project is to elucidate the molecular and cellular changes which lead to the suppression of oncogenic transformation in the host as a result of parvoviral relication and describe a mechanism in which the infection of a parvovirus may be mimicked at a cellular level as to suppress transformation of the cell.
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