STRUCTURE AND FUNCTION OF THE ADV GENOME

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The purpose of this project is to study various aspects of genome structure and function of the Aleutian mink disease parvovirus (ADV). In the past year we have initiated studies on full-length chimeric clones of ADV. The strategy has been to replace segments of an infectious molecular clone of the nonpathogenic ADV-G strain with corresponding fragments of molecular clones from pathogenic ADVs derived directly from infected animal organs. Two of the 3 constructs assayed to date failed to produce infectious ADV after transfection and serial passage in cell culture; these were clones chimeric for either the 15-88 or the 43-65 map unit segments. A clone chimeric for the 65-73 map unit segment was able to replicate similar to the parent ADV-G. These results suggested that at least one determinant for permissive replication in cell culture maps to the 43-65 map unit portion of the genome, which contains the amino terminus of the virion proteins, the carboxy terminus of the non-structural proteins and sequences associated with one of the splices. We have also begun to study the expression of ADV proteins in a vaccinia based eukaryotic expression system. A segment representing the spliced R3 mRNA that codes for both ADV structural proteins has been recombined into an infectious vaccinia virus. This recombinant vaccinia (VV-lL1) directed synthesis of both VP1 (p85) and VP2 (p75). The viral antigen was localized to the nucleus. Furthermore, lysates of VV-IL1 infected cells could be used as an ADV test antigen in counterimmunoelectrophoresis. Finally, parvovirus particles were observed by electron microscopy. Thus, all signals necessary for production and nuclear transport of both VPl/VP2 as well as those required for particle assembly were present.