The autonomous parvovirus LuIII 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 LuIII termini shares over 90% sequence identify with that of the rodent parvovirus Minute Virus of Mice (MVM) and an overall genome and an overall genome identify 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 encapsulation pattern observed for LuIII since similar differences are observed between the sequences of MVM and that of another rodent parvovirus, H-1 which also 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 represents a major determinant in the encapsidation pattern observed for LuIII. Preliminary data suggest that this A/T sequence is acting as an autonomously replicating sequence (ARS), function which could explain the synthesis of plus strand from the 5' terminus of the LuIII genome. Parvovirus are known to interfere with, and in some cases suppress the formation of tumors. Understanding parvovirus replication is important in the elucidation of the mechanisms used by these viruses to suppress oncogenic transformation in the host cell. Most importantly is the potential use of parvoviral genomes as vectors in gene therapy as evidence from the extensive experimentation involving heterologous gene constructs for many human diseases. Recently the construction of chimeric LuIII molecules in which the coding sequences of LuIII molecules in which the coding sequences of LuIII have been replaced with those of a reporter gene in hope of using these in gene transfer applications, when transient expression is desired were reported.
The aims of this research include identification of important sequences in the replication and particularly the encapsulation of LuIII by constructing mutated clones of LuIII and MVM Gel retardation assays of LuIII infected and uninfected NBE 324 I cell lysates and putative regulatory LuIII sequences will be done to identify DNA binding proteins of viral and cellular nature. The possibility of the A/T rich sequence acting as an autonomously replicating sequence will be addressed by transforming a clone which has the A/T sequence inserted next to a Ura-3 gene cloned into pUC19 in yeast cells and assays for URA prototropy. Due to inconclusive findings the possible integration of LuIII into the genome of the host cell will also be studied. The long term objective of this project is to elucidate the encapsidation mechanism of parvovirus LuIII. This in turn will start to unveil the molecular and cellular changes that occur during an infection which lead to the suppression of oncogenic transformation in the host as a result of parvoviral replication. Ultimately, infection of a parvovirus may be mimicked at a cellular level as to suppress transformation of the cell. By elucidating the natural mechanisms used by parvoviruses to replicate in their host the possible strategies used in gene therapy can be designed to simulate to the extent possible, normal pathways of the cell. By elucidating the natural mechanisms used by parvoviruses to replicate in their host the possible strategies used in gene therapy can be designed to stimulate to the extent possible, normal pathways of infection, therewith minimizing the changes of failure in the strategy employed.
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