Definition of the Minimal cis -Acting Sequences Necessary for Genome Maturation of the Herpesvirus Murine Cytomegalovirus

Abstract

Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large (ϳ180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of transacting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.Herpesviruses have large (130-to 235-kb) linear doublestranded DNA genomes that replicate via concatemeric intermediates consisting of head-to-tail-linked genomes (1,2,14,17,18,25,27,32). The concatemers are packaged into preformed capsids and cleaved at precise locations to release unit-length genomes within the capsids (3). The cis-acting sequences that are recognized by the cleavage/packaging machinery have not been fully defined, but two motifs denoted pac1 and pac2 were initially identified by their strong conservation near the ends of herpesvirus genomes (7). Typically, pac1 motifs consist of 3-to 7-bp A-or T-rich regions flanked on each side by short poly(C) tracts (7, 29), whereas pac2 motifs consist of 5-to 10-bp A-rich regions (7). In some viruses a consensus CGCGGCG motif is located just distal of pac2 (relative to the terminus) (7).In order to mutagenically define the cis cleavage/packaging sequences of a herpesvirus, we developed an assay based on cleavage at a second or "ectopic" cleavage site that was inserted into the murine cytomegalovirus (MCMV) genome. In 1984, Marks and Spector (15) showed that the two ends of the MCMV genome (designated X and C, respectively) ( Fig. 1) fuse shortly after infection to form "junctions." Upon concatemer synthesis, such junctions presumably serve as natural cleavage/packaging sites. We predicted tha...

Analysis of Herpes Simplex Virus Type 1 DNA Packaging Signal Mutations in the Context of the Viral Genome

“…This corresponds very closely with the minimal essential region for pac1 function identified in MCMV (32). The results obtained with the pac1 substitution mutants are in good agreement with previous studies using HSV-1 amplicons and ectopic-site MCMV recombinants (10,15,32). These confirm that important sequences are present within the proximal and distal GC-rich regions but that the sequence of the T element is not crucial.…”

“…The results reported here confirm that the pac2 region is essential for the initiation of the cleavage-packaging process and that while the sequence of the T element is critical, extensive nucleotide substitutions within the consensus, unconserved, and GC elements are tolerated. It has recently been reported that in MCMV a second essential packaging element, which is GC rich, is present within pac2 located further from the cleavage site than the T element (32). It remains possible that a corresponding signal is present in HSV-1 within the region designated "rest of Uc" and is supported by our previous observation that in the amplicon assay, deletion but not substitution of the intervening unconserved region caused a significant impairment in DNA packaging (10).…”

“…In the case of pac2, the T-rich element is most highly conserved with a consensus CGCGGCG motif also frequently being present (32). Detailed studies, employing primarily HSV-1 and murine cytomegalovirus (MCMV), have highlighted the roles of the major conserved motifs and suggested the following general mechanism by which concatemers are cleaved and packaged (1,10,13,15,16,23,25,29,32). Within Uc the most critical sequence is the pac2 T element, which is essential for cleavage to initiate DNA packaging.…”

“…In the first, amplicons (i.e., bacterial plasmids containing a viral DNA replication origin and packaging signal) are transfected into mammalian cells and their ability to be replicated and packaged is assessed following the provision of viral helper functions, either by superinfection with virus particles or by cotransfection of virion DNA (7,20,24,27,29,35). The second assay introduces an additional copy of the packaging signal under test at an ectopic site within the viral genome and determines whether it functions as a site for the cleavage of concatemeric DNA and the generation of novel terminal fragments of virion DNA (5,15,18,23,29,32). Both these approaches, however, suffer from the disadvantage that recombination occurs between the test packaging signal and the wild-type (wt) signal present either in the helper virus or in its normal location within an ectopic-site recombinant (5,8,15,23,32).…”

“…The second assay introduces an additional copy of the packaging signal under test at an ectopic site within the viral genome and determines whether it functions as a site for the cleavage of concatemeric DNA and the generation of novel terminal fragments of virion DNA (5,15,18,23,29,32). Both these approaches, however, suffer from the disadvantage that recombination occurs between the test packaging signal and the wild-type (wt) signal present either in the helper virus or in its normal location within an ectopic-site recombinant (5,8,15,23,32). Additionally, concatemers generated following replication of amplicons have a significantly different structure from standard herpesviral genomes in that multiple copies of the packaging signal are present, spaced at regular intervals corresponding to the size of the input plasmid.…”