SEQUENCE ELEMENTS OF THE ADENOASSOCIATED VIRUS REP GENE REQUIRED FOR SUPPRESSION OF HERPES-SIMPLEX-VIRUS-INDUCED DNA AMPLIFICATION

Herpes simplex virus (HSV) has been shown to induce DNA amplification in the host cell genome, which can be suppressed by the adeno-associat ed virus type 2 (AAV-2) rep gene (Heilbronn et al., 1990, J. Virol. 64 , 3012-3018), In an attempt to define domains of Rep which are require d for this effect a set of expression constructs was generated for Rep mutants with either N-terminal and/or C-terminal truncations, with sm all internal deletions, or with point mutations. In transient cotransf ection assays these mutants were tested for the inhibition of HSV-indu ced DNA amplification and in parallel for DNA replication of a rep-def ective AAV genome. Our data show that the C-terminal region of Rep whe re spliced and unspliced proteins differ is dispensable for both AAV D NA replication and inhibition of HSV-induced DNA amplification. The N- terminus of Rep is required for AAV DNA replication, whereas the first 174 amino acids can be deleted without loss of function for the inhib ition of DNA amplification. Rep52 which starts at methionine 225 is ne ither sufficient, nor required for this effect. We further analyzed th e region between amino acids 174 and 225: A stretch of 16 highly hydro philic amino acids is dispensable for the inhibition of DNA amplificat ion, but it is required for AAV DNA replication. Deletion of two short motifs spanning putative protein kinase C phosphorylation sites each strongly reduce both AAV DNA replication and inhibition of DNA amplifi cation, whereas a single amino acid substitution of one of these sites abolished AAV DNA replication with no effect on the inhibition of DNA amplification. Our data show that most, but not all, of the sequence elements within the N-terminus of Rep78 required for AAV DNA replicati on coincide with those required for the inhibition of HSV-induced DNA amplification. A replication-negative version of Rep78 comprising the internal 60% of the protein still carry the entire inhibitory function for HSV-induced DNA amplification. (C) 1995 Academic Press, Inc.