Sequence elements of the adeno-associated 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-associated 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 required for this effect a set of expression constructs was generated for Rep mutants with either N-terminal and/or C-terminal truncations, with small internal deletions, or with point mutations. In transient cotransfection assays these mutants were tested for the inhibition of HSV-induced DNA amplification and in parallel for DNA replication of a rep-defective AAV genome. Our data show that the C-terminal region of Rep where spliced and unspliced proteins differ is dispensable for both AAV DNA 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 inhibition of DNA amplification. Rep52 which starts at methionine 225 is neither sufficient, nor required for this effect. We further analyzed the region between amino acids 174 and 225: A stretch of 16 highly hydrophilic amino acids is dispensable for the inhibition of DNA amplification, 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 amplification, 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 replication 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.