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Cited by 186Herpesvirus Infections and TreatmentsDNA Repair MechanismsCytomegalovirus and herpesvirus research

Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions

Mocarski, Edward S., Post, Leonard E., Roizman, Bernard·1980·Cell
DOI 10.1016/0092-8674(80)90172-5
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Abstract

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

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  • Prior methods for HSV1 genome editing require amplification and purification to obtain viral genomic DNA, then co-transfection with homologous DNA sequences into cells followed by two-step counter-selection and multiple plaque isolation or insertion into a BAC system and several in vitro molecular biological procedures.

    “The genomic DNA is then co-transfected with homologous DNA sequences into cells followed by two-step counter-selection and multiple plaque isolation [30] or by inserting the viral genome into a BAC system and performing several in vitro molecular biological procedures [31].”

    From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [30]

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Authors
Mocarski, E. S., Post, L. E., & Roizman, B.
Affiliations
University of Chicago (US)
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
1980-11
Volume
22
Issue
1
Pages
243-255
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/0092-8674(80)90172-5
OpenAlex
W2027690720
Cited by
186
References
16
Citations by year
2026: 2 · 2023: 1 · 2021: 1 · 2020: 1 · 2017: 3 · 2016: 2 · 2014: 4 · 2013: 2 · 2012: 1
FWCI
9.70
Type
journal article
Language
en
Open access
closed
Licence
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Epidemiology › Herpesvirus Infections and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Health Sciences › Medicine › Epidemiology › Cytomegalovirus and herpesvirus research
Keywords
herpes simplex virus, viral genome, genome inversion, homologous recombination, site-specific recombination, inverted repeats, thymidine kinase gene, molecular engineering
MeSH terms
Base Sequence, Cloning, Molecular, DNA, Viral, Genes, Viral, Genetic Engineering, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Thymidine Kinase, Simplexvirus

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