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

Abstract

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (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 pac1 and pac2 were essential for cleavage whereas others were not. Withinpac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Withinpac2, 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.

Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome

“…4; Supplementary Video 3). Within the 66-bp Ub sequence, a stretch consisting of pac1 T element flanked by two short G tracts (GGGGGG and GGGGGGGG from pac1 proximal and distal GC elements of Ub , respectively) forms the major conserved motif at the termini of herpesvirus genomes, constituting the minimal sequence necessary for proper concatemeric cleavage 17,18 . Whereas both flanking G tracts are sequence critical, the T element tolerates substitutions, though not deletions, suggesting it may function as a regulatory spacer element 15 .…”

“…Exceptionally, the projection of tentacle helices from the portal clip toward the terminase docking site/portal cap and α4/α5’s coiled-coil arrangement (widely implicated in propagating conformation changes 28 ) are evocative of a signaling pathway. In light of evidence of sequence- and headful-sensing regulatory effects in genome packaging 17,18,29,30 , that α4 contacts the DNA-interacting region of portal clip and α5 interacts with the probe-like, capsid penetrating N-anchor of Tri1 provide tantalizing structural clues as to possible mechanistic bases of these modes of genome packaging regulation.…”

Putative Functional Domains of Human Cytomegalovirus pUL56 Involved in Dimerization and Benzimidazole D-Ribonucleoside Activity

“…The authors showed that when murine CMV is engineered to carry an additional copy of the joined terminal sequence a (which occurs naturally within the pac1 and pac2 motifs) in inverted orientation, the viral genome uses these sequences to support inversion as well as cleavage/packaging of progeny genomes. By contrast, when the same fragment is inserted in direct orientation, only packaging occurs [42]. Thus, two roles are suggested for this sequence: one in the recombination process involving genome inversion and a second in the cleavage process employed during DNA packaging.…”

Bifunctional Protein Conferring Enhanced Green Fluorescence and Puromycin Resistance

“…The culture supernatant was again used to infect a fresh flask of cells, and the selection procedure was repeated. Recombinant viruses were cloned from this culture supernatant by 96-well limiting dilution as previously described (19). The recombinant virus was considered pure when all virus-infected wells exhibited green fluorescence upon repeated 96-well limiting dilution.…”