Sequences within the Herpesvirus-Conserved pac 1 and pac 2 Motifs Are Required for Cleavage and Packaging of the Murine Cytomegalovirus Genome

Abstract

ABSTRACT The DNA sequence motifs pac 1 [an A-rich region flanked by poly(C) runs] and pac 2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac 1 and pac 2 were essential for cleavage whereas others were not. Within pac 1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac 2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.