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. 1998 Jan;72(1):48-56.
doi: 10.1128/JVI.72.1.48-56.1998.

Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome

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Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome

M A McVoy et al. J Virol. 1998 Jan.

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. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, 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.

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Figures

FIG. 1
FIG. 1
Structures of recombinant viruses containing ectopic cleavage sites. Shown is a representation of the wild-type MCMV genome and HindIII restriction map, with X- and C-terminal sequences shown as hatched boxes. The expanded regions show the HindIII site between L and J in wild-type virus, the lacZ insertion (black box) at this HindIII site in recombinant virus RM461, and the ectopic cleavage site inserted at the same location in recombinant virus RM4072. The ectopic site consists of a fusion of C- and X-terminal sequences (hatched boxes labeled X and C) adjacent to a gpt expression cassette (open box). The point at which cleavage is predicted to occur (where C and X sequences are fused) is indicated. The XhoI site marked with an asterisk was engineered into this region (see Materials and Methods). The thick lines indicate the DNA sequences contained in the indicated hybridization probes. (B) BamHI and XhoI fragments generated from recombinant viruses containing ectopic cleavage sites are predicted based on the locations of BamHI and XhoI sites and the predicted point of cleavage. The fragment sizes in kilobases are indicated.
FIG. 2
FIG. 2
Sequences of ectopic cleavage sites and mutations. The ectopic cleavage site with flanking HindIII L and J sequences is shown as in Fig. 1 except that the orientation has been reversed. The predicted point of cleavage, restriction enzyme sites used in plasmid construction, and locations of oligonucleotide primers (horizontal arrows) used for PCR, mutagenesis, and sequencing are indicated. The expanded region shows the wild-type sequences (25) for cleavage sites with one or two copies of the 30-bp repeat (italics) found in plasmids used to make RM4072 and RM4074. The cleavage point predicted for each site is indicated with an arrow, and the predicted components of pac1 and pac2 are boxed. Mutations introduced into the ectopic cleavage sites of the indicated recombinant viruses are shown immediately above (RMA39) or below (RM4077, RM4122, RM4098, RM4092, RM4091, and RM4097) the wild-type sequences. Bases differing from wild type are in lowercase, and deleted bases are indicated by dots. Restriction sites used for mutagenesis or created by mutagenesis are underlined. The sequences of the ectopic cleavage sites of viruses RM4091Δ and RM4077ins which contain spontaneous deletions and an insertion are depicted at the bottom. Boxes enclose predicted components of pac1 and pac2 that have wild-type sequences (solid borders) or contain mutations (dashed borders).
FIG. 3
FIG. 3
Cleavage at the ectopic cleavage site of RM4072. Autoradiograms show cell-associated (-c) and virion (-v) DNAs from the parental virus RM461 and from RM4072 digested with BamHI or XhoI and hybridized with 32P-labeled probes following electrophoresis and transfer to nylon. (A and B) Results of hybridization with a BamHI/XhoI fragment from pON432 which detected fragments from ectopic junctions and termini; (C) results of hybridization with pON4048 DNA to detect fragments from both ectopic and natural cleavage sites and termini. The positions of molecular size markers are shown on the left of panel A, and the locations and sizes in kilobases (in parentheses) of BamHI and XhoI ectopic junction (JE), natural junction (JN), ectopic terminal (TE), natural terminal (TN), and comigrating ectopic and natural terminal (TE/N) fragments are indicated. Note that in panel B, an irrelevant 2.0-kb XhoI fragment adjacent to the ectopic cleavage site also hybridizes to the probe.
FIG. 4
FIG. 4
Cleavage at mutated ectopic cleavage sites. Autoradiograms of virion DNAs from recombinant viruses digested with the indicated restriction enzymes and hybridized with 32P-labeled pMA34 DNA to detect ectopic junction and terminal fragments following electrophoresis and transfer to nylon. For each virus, the fold reduction in cleavage is estimated from densitometric quantitation of the 2.5- and 3.5-kb fragments in the lane above (for details, see Results). The positions of molecular size markers are shown on the left, and arrows indicate the 3.5-kb XhoI ectopic junction fragments (JE) and 2.5-kb XhoI ectopic terminal (TE) fragments. Analysis of RMA39 was carried out in an experiment separate from the other viruses.
FIG. 5
FIG. 5
Cleavage at ectopic cleavage sites containing spontaneous mutations. The autoradiogram shows virion DNAs from recombinant viruses digested with the indicated restriction enzymes and hybridized with 32P-labeled pMA34 DNA to detect ectopic junction and terminal fragments following electrophoresis and transfer to nylon. The positions of molecular size markers are shown on the left, and arrows indicate the 3.5-kb XhoI ectopic junction fragments (JE) and 2.5-kb XhoI ectopic terminal (TE) fragments.
FIG. 6
FIG. 6
Distribution of 30-bp repeats at ectopic cleavage sites of recombinant viruses. (A) Representation of the ectopic cleavage site as in previous figures, showing the positions of primers MOL24 and CNEW, the predicted PCR product, and SmaI restriction digest products. Ectopic cleavage sites with one 30-bp repeat are predicted to result in 146-bp SmaI fragments, and those with two 30-bp repeats are predicted to result in 176-bp SmaI fragments. (B) Ethidium bromide-stained polyacrylamide gel showing SmaI-digested PCR products amplified from plasmids pON4072 and pON4074 or from virion DNAs of recombinant viruses, using MOL24 and CNEW. The final lane contains the PCR product from RM4097 virion DNA without SmaI digestion. Below the lanes are indicated the number of 30-bp repeats in plasmids pON4072 and pON4074 or in the plasmids used to construct each recombinant virus, whether the ectopic cleavage site is cleaved (+), not cleaved (−), or inefficiently cleaved (±), and the estimated ratio of double- to single-repeat-containing fragments. The sizes of HaeIII fragments of φX174 replicative DNA (markers) are indicated on the left, and arrows on the right indicate the 146- and 176-bp SmaI fragments.
FIG. 7
FIG. 7
Conserved sequences at herpesvirus termini. Alignments of the terminal sequences of HSV-1 (33), varicella-zoster virus (VZV) (13), Epstein-Barr virus (EBV) (65), HCMV (30), human herpesvirus (HHV-6) (55), human herpesvirus rat (HHV-7) (42), cytomegalovirus (RCMV) (59), GPCMV (28), and MCMV (25). Genomic termini are indicated, with the exception of the HCMV pac2-containing terminus, which has an additional 97 bp between the left end of the sequence and the terminus. The conserved components of pac1 and pac2 are shown in boldface and set off by spaces. Bases within the CGCGGCG motifs of varicella-zoster virus and Epstein-Barr virus that differ from consensus are in lowercase. GPCMV has two pac2-containing termini called M and O. An alternative terminus found in some MCMV genomes is indicated (25).

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