Mutagenesis of a Herpes Simplex Virus Origin of DNA Replication and its Effect on Viral Interference

Abstract

Supercoiled plasmid molecules containing cloned copies of a DNA fragment which includes a functional herpes simplex virus type 1 origin of DNA replication were cleaved preferentially at 2 positions within the viral insert by nuclease S1. Plasmids with molecular linker insertions at these sites were constructed, and analysis of 2 representative plasmids demonstrated the presence of palindromic DNA sequences at the preferred cleavage positions. One of these palindromic sequences occurred within a 90 bp [base-pair] region in which the cis-acting sequences essential for viral origin function had previously been located. Insertion of a linker at this position abolished origin activity, demonstrating an essential role for sequences within the palindrome in the initiation of DNA synthesis. In transfection assays, plasmids containing a functional viral origin of DNA replication markedly interfered with the infectivity of non-defective viral DNA even in the absence of viral encapsidation signals. Inactivation of the origin greatly reduced this effect on DNA infectivity, suggesting that viral interference may be mediated by a mechanism involving the replicative machinery.