Models of the biological consequences of herpesvirus infection--cytocidal infection, persistent infection, biological and/or oncogenic transformation--were developed using the three equine herpesviruses. EHV-1 (abortigenic and systemic infections), ECMV (a cytomegalovirus) and EHV-3 (venereal disease) share DNA sequences, differ in overall organization. All have oncogenic potential, and EHV-1, ECMV, and EHV-3 oncogenically transformed mouse embryo and hamster embryo (HE) cells, persistently infected HE cells, and EHV biochemically transformed cells have been established. Overall goals are to: i) elucidate the genomic structure of the three herpesviruses and of EHV-1 DI particles by restriction enzyme mapping techniques, ii) quantitate and map DNA sequences shared among the DNAs, iii) identify DNA sequences associated with oncogenic transformation and persistency, their arrangement within the cell, and gene products, and iv) ascertain whether these viral genes are related and colinearly arranged and how DNA organization relates to gene function. EHV DNA fragments cloned in the pBR322 or pACYC plasmid-E. coli systems and radiolabeled by nick translation will be used as probes in liquid and blot hybridization to quantitate and map type-shared sequences. Transformed and tumor cell DNAs restricted with a variety of enzymes will be analyzed by hybridization to our library of cloned EHV fragments to reveal the identity of the viral genes. Conjoint sequences from these cells will be cloned in LambdaCh4A phage (as accomplished recently) for fine mapping and eventually for sequencing to elucidate the nature, number, and organization of integration sites. DNA sequences so identified will be transfered (transfection and protoplast fusion) into cells and assayed for biological activities. Expression of these viral sequences will be monitored by mapping viral transcripts by identification of DNA fragments released from S1 degested RNA-DNA hybrids. Viral polypeptides will be identified by immunofluorescence, gel analysis of immunoprecipated viral polypeptides, and in vitro translation of transcripts. Lastly, EHV DI particles with the capacity to mediate persistent infection in cell culture and/or animals will be mapped to determine whether specific sequences are associated with persistency.
Showing the most recent 10 out of 99 publications
