Identification of Novel Herpes Simplex Virus Replicative Intermediates by Field Inversion Gel Electrophoresis: Implications for Viral DNA Amplification Strategies

Zhang, Xiaoliu; Efstathiou, Stacey; Simmons, Anthony

doi:10.1006/viro.1994.1375

Cited by 92 publications

(131 citation statements)

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“…It has previously been shown that during herpesvirus DNA packaging only the genomic terminus containing the pac2 signal is found associated with the high-molecular-weight concatemeric DNA (15,17,19,25,26,36), and this observation has formed the basis for a proposal that the pac2 sequence controls the initiation and directionality of DNA packaging (19). Our observations that mutations within pac2 have relatively greater effects during the first cycle of infection and that motifs in pac1 may be involved in the late stages of DNA encapsidation are compatible with this model.…”

Section: Discussionmentioning

confidence: 99%

Effects of Mutations within the Herpes Simplex Virus Type 1 DNA Encapsidation Signal on Packaging Efficiency

Hodge¹,

Stow²

2001

J Virol

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The cis-acting signals required for cleavage and encapsidation of the herpes simplex virus type 1 genome lie within the terminally redundant region or a sequence. The a sequence is flanked by short direct repeats (DR1) containing the site of cleavage, and quasi-unique regions, Uc and Ub, occupy positions adjacent to the genomic L and S termini, respectively, such that a novel fragment, Uc-DR1-Ub, is generated upon ligation of the genomic ends. The Uc-DR1-Ub fragment can function as a minimal packaging signal, and motifs have been identified within Uc and Ub that are conserved near the ends of other herpesvirus genomes (pac2 and pac1, respectively). We have introduced deletion and substitution mutations within the pac regions of the Uc-DR1-Ub fragment and assessed their effects on DNA packaging in an amplicon-based transient transfection assay. Within pac2, mutations affecting the T tract had the greatest inhibitory effect, but deletion of sequences on either side of this element also reduced packaging, suggesting that its position relative to other sequences within the Uc-DR1-Ub fragment is likely to be important. No single region essential for DNA packaging was detected within pac1. However, mutants lacking the G tracts on either side of the pac1 T-rich motif exhibited a reduced efficiency of serial propagation, and alteration of the sequences between DR1 and the pac1 T element also resulted in defective generation of Ub-containing terminal fragments. The data are consistent with a model in which initiation and termination of packaging are specified by sequences within Uc and Ub, respectively.Herpesviruses have linear double-stranded DNA genomes of 125 to 245 kbp that are circularized upon infection and replicate in an "endless" form, generating branched concatemeric structures. During the assembly of progeny particles, the concatemers are cleaved at specific sites corresponding to the genomic termini and, in a tightly coupled process, the viral DNA is packaged into preformed capsids (reviewed in references 13, 20, and 23).In the case of herpes simplex virus type 1 (HSV-1), the cis-acting sequences required for cleavage and packaging reside within the terminally redundant region or a sequence (21,29,32,34). The a sequence is 250 to 500 bp long and present as a single copy at the S terminus and one or more tandem copies at the L terminus. In addition, one or more copies are also present in inverted orientation at the junction between the L and S segments (23).Flanking the a sequences are direct repeats (DR1) of 17 to 20 bp, with single copies of DR1 separating tandem a sequences. The site of genomic cleavage occurs within the DR1 element such that a single copy is regenerated upon ligation of the two genomic ends (see Fig. 1). The central portion of the a sequence comprises multiple tandem reiterations of one or two other short sequence elements (11 to 24 bp) referred to as DR2 and DR4. The quasi-unique sequences, each of ca. 80 bp, which lie between DR1 and either side of the DR2 and DR4 repeats are termed the ...

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Section: Discussionmentioning

confidence: 99%

Effects of Mutations within the Herpes Simplex Virus Type 1 DNA Encapsidation Signal on Packaging Efficiency

Hodge¹,

Stow²

2001

J Virol

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“…To prepare intact HSV-1 DNA, HSV-1 infected cells and HSV-1 viral particles were embedded in low-melting point agarose and lysed with proteinase K in the agarose block (Zhang et al, 1994). The blocks were then treated with ATP-DNase (Toyobo Inc., Osaka, Japan), and the HSV-1 DNA was extracted with phenol after incubation at 65 °C for 10 min to melt the agarose.…”

Section: Shape Of Hsv-1 Dna After Infection Into Bhk-21 Cell Lines Anmentioning

confidence: 99%

“…BHK-21 cells infected with HSV-1 for 24 h were assumed to contain both circular and linear HSV-1 DNA, because HSV-1 DNA replicated by a rolling-circle mechanism would generate the circle plus tail molecule and unit-length linear HSV-I molecule (Fig. 6a) (Severini et aL, 1994;Zhang et al, 1994). Two SaII fragments of 18'0 and 20"2 kb derived from the L-S junction of circular HSV-1 DNA and possibly of concatemeric HSV-1 DNA, but not of unit-length linear HSV-1 DNA, would be resistant to ATP-DNase.…”

Section: Shape Of Hsv-1 Dna After Infection Into Bhk-21 Cell Lines Anmentioning

confidence: 99%

Replication of Herpes Simplex Virus Type 1 DNA is Inhibited in a Temperature-sensitive Mutant of BHK-21 Cells Lacking RCC1 (Regulator of Chromosome Condensation) and Virus DNA Remains Linear

Umene

Nishimoto

1996

Journal of General Virology

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“…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).…”

mentioning

confidence: 99%

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

Wang¹,

Nixon

McVoy³

2008

J Virol

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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...

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Identification of Novel Herpes Simplex Virus Replicative Intermediates by Field Inversion Gel Electrophoresis: Implications for Viral DNA Amplification Strategies

Cited by 92 publications

References 0 publications

Effects of Mutations within the Herpes Simplex Virus Type 1 DNA Encapsidation Signal on Packaging Efficiency

Effects of Mutations within the Herpes Simplex Virus Type 1 DNA Encapsidation Signal on Packaging Efficiency

Replication of Herpes Simplex Virus Type 1 DNA is Inhibited in a Temperature-sensitive Mutant of BHK-21 Cells Lacking RCC1 (Regulator of Chromosome Condensation) and Virus DNA Remains Linear

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

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