Herpesvirus genetics has come of age

doi:10.1016/S0966-842X(02)02394-6

Trends in Microbiology

Volume 10, Issue 7, 1 July 2002, Pages 318-324

https://doi.org/10.1016/S0966-842X(02)02394-6 Get rights and content

Abstract

The genetic analysis of the large and complex herpesviruses has been a constant challenge to herpesvirologists. Elegant methods have been developed to produce mutants in infected cells that rely on the cellular recombination machinery. Bacterial artificial chromosomes (BACs), single copy F-factor-based plasmid vectors of intermediate insert capacity, have now enabled the cloning of complete herpesvirus genomes. Infectious virus genomes can be shuttled between Escherichia coli and eukaryotic cells. Herpesvirus BAC DNA engineering in E. coli by homologous recombination requires neither restriction sites nor cloning steps and allows the introduction of a wide variety of DNA modifications. Such E. coli-based technology has provided a safe, fast and effective approach to the systematic mining of the information stored in herpesvirus genomes as a result of their intimate co-evolution with their specific hosts for millions of years. Use of this technique could lead to new developments in clinical virology and basic virology research, and increase the usage of viral genomes as investigative tools and vectors.

Section snippets

Forward or classical herpesvirus genetics

The main objects of interest in viral genetic analysis are mutant alleles, where changes in the genetic material result in phenotypic alterations. The mutation rate in DNA viruses such as herpesviruses is low and thus the experimental generation of viral mutants is instrumental in genetic studies. Originally, random mutations were introduced and mutants sensitive or resistant to particular physical, chemical or biological conditions were isolated. The characterization of conditional alleles,

Bacterial artificial chromosomes (BACs)

The needs of functional genomics stimulated the development of cloning vehicles suitable for the maintenance of large DNA inserts. Yeast artificial chromosomes (YACs) were the first cloning vectors for large genomic DNA fragments. Unfortunately, YACs cause difficulties such as frequent spontaneous rearrangements and insert instability, and there can be substantial contamination of purified YACs with yeast DNA 17., 18.. For this reason, alternative, E. coli-based cloning vectors for large DNA

Conclusions and perspective

The recent increased interest in functional genomics has led to the development of new genetic engineering techniques to generate single-copy BAC plasmids containing large genomic inserts. The properties of BACs fit perfectly with the needs of herpesvirus genomics. Any mutation can be introduced into any viral gene once a herpesvirus BAC has been constructed. There is increased safety, as the viral genome is maintained as a BAC most of the time. All mutagenesis steps can be controlled and

Acknowledgements

The work was supported by the Deutsche Forschungsgemeinschaft through SFB 455, SFB 567, and SPP New vaccination strategies.

References (67)

P.A. Schaffer
Genetics of herpes simplex virus
J. Invest. Dermatol.
(1984)
E.S. Mocarski
Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions
Cell
(1980)
L.E. Post et al.
A generalized technique for deletion of specific genes in large genomes: alpha gene 22 of herpes simplex virus 1 is not essential for growth
Cell
(1981)
W.C. Manning et al.
Insertional mutagenesis of the murine cytomegalovirus genome: one prominent alpha gene (ie2) is dispensable for growth
Virology
(1988)
C. Cunningham et al.
A cosmid-based system for constructing mutants of herpes simplex virus type 1
Virology
(1993)
R.B. Register et al.
A facile system for construction of HSV-1 variants: site-directed mutation of the UL26 protease gene in HSV-1
J. Virol. Methods
(1996)
L.C. Schalkwyk
Techniques in mammalian genome mapping
Curr. Opin. Biotechnol.
(1995)
A. McGregor et al.
Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli
Mol. Genet. Metab.
(2001)
Y. Zhang
A new logic for DNA engineering using recombination in Escherichia coli
Nat. Genet.
(1998)
A. Bubeck
The glycoprotein gp48 of murine cytomegalovirus. Proteasome-dependent cytosolic dislocation and degradation
J. Biol. Chem.
(2002)

J. Rudolph et al.

Equine herpesvirus type 1 devoid of gM and gp2 is severely impaired in virus egress but not direct cell-to-cell spread

Virology

(2002)

P.P. Cherepanov et al.

Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic–resistance determinant

Gene

(1995)

P.A. Schaffer

Temperature-sensitive mutants of herpesviruses

Curr. Top. Microbiol. Immunol.

(1975)

J.R. Smiley

Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus

Nature

(1980)

R.R. Spaete et al.

Insertion and deletion mutagenesis of the human cytomegalovirus genome

Proc. Natl. Acad. Sci. U. S. A.

(1987)

M. van Zijl

Regeneration of herpesviruses from molecularly cloned subgenomic fragments

J. Virol.

(1988)

J.I. Cohen et al.

Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro

Proc. Natl. Acad. Sci. U. S. A.

(1993)

B. Tomkinson

Epstein–Barr virus recombinants from overlapping cosmid fragments

J. Virol.

(1993)

G. Kemble

Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids

J. Virol.

(1996)

T. Cihlar

Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments

J. Virol.

(1998)

M.E. Ehsani

Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones

J. Virol.

(2000)

N.A. DeLuca et al.

Activation of immediate-early, early, and late promoters by temperature- sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4

Mol. Cell Biol.

(1985)

M. Ramsay

Yeast artificial chromosome cloning

Mol. Biotechnol.

(1994)

H. Shizuya

Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector

Proc. Natl. Acad. Sci. U. S. A.

(1992)

P.A. Ioannou

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments

Nat. Genet.

(1994)

U.J. Kim

Stable propagation of cosmid sized human DNA inserts in an F factor based vector

Nucleic Acids Res.

(1992)

Q. Tao et al.

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors

Nucleic Acids Res.

(1998)

M. Messerle

Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome

Proc. Natl. Acad. Sci. U. S. A.

(1997)

T.A. Stavropoulos et al.

An enhanced packaging system for helper-dependent herpes simplex virus vectors

J. Virol.

(1998)

H.J. Delecluse

Propagation and recovery of intact, infectious Epstein–Barr virus from prokaryotic to human cells

Proc. Natl. Acad. Sci. U. S. A.

(1998)

Y. Saeki

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors

Hum. Gene Ther.

(1998)

E.M. Borst

Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants

J. Virol.

(1999)

G.A. Smith et al.

Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus

J. Virol.

(1999)

Cited by (92)

Alphaherpesvirus-vectored vaccines against animal diseases: Current progress
2020, Journal of Integrative Agriculture
Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity, are multivalent and multieffective, and have high safety ratings. Animal alphaherpesviruses have a large genome, contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein. Furthermore, animal alphaherpesviruses have a wide host spectrum, can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen, thus making them ideal carriers for recombinant virus-vectored vaccines. With the development of gene-editing technology, recombinant viruses capable of expressing foreign genes can be constructed by various methods. Currently, studies on recombinant virus-vectored vaccines constructed based on animal alphaherpesviruses have involved poultry, pigs, cattle, sheep, and companion animals. Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases. This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines, aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.
Utilization of herpesviridae as recombinant viral vectors in vaccine development against animal pathogens
2019, Virus Research
Throughout the past few decades, numerous viral species have been generated as vaccine vectors. Every viral vector has its own distinct characteristics. For example, the family herpesviridae encompasses several viruses that have medical and veterinary importance. Attenuated herpesviruses are developed as vectors to convey heterologous immunogens targeting several serious and crucial pathogens. Some of these vectors have already been licensed for use in the veterinary field. One of their prominent features is their capability to accommodate large amount of foreign DNA, and to stimulate both cell-mediated and humoral immune responses. A better understanding of vector-host interaction builds up a robust foundation for the future development of herpesviruses-based vectors. At the time, many molecular tools are applied to enable the generation of herpesvirus-based recombinant vaccine vectors such as BAC technology, homologous and two-step en passant mutagenesis, codon optimization, and the CRISPR/Cas9 system. This review article highlights the most important techniques applied in constructing recombinant herpesviruses vectors, advantages and disadvantages of each recombinant herpesvirus vector, and the most recent research regarding their use to control major animal diseases.
Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination
2018, Journal of Virological Methods
Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli. Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, methods have been developed that express Cre from the virus genome and enable auto-excision of the BAC plasmid. We report a versatile and rapid in vitro method based on purified Cre enzyme to carry out the same process in a test tube and does not require cell line generation or cloning into the virus genome. This method greatly increases the repertoire of methods available to modify the genome prior to reconstitution of virus infectivity in a mammalian host.
Use of a current varicella vaccine as a live polyvalent vaccine vector
2016, Vaccine
Varicella-zoster virus (VZV) is the causative agent of varicella and zoster. The varicella vaccine was developed to control VZV infection in children. The currently available Oka vaccine strain is the only live varicella vaccine approved by the World Health Organization. We previously cloned the complete genome of the Oka vaccine strain into a bacterial artificial chromosome vector and then successfully reconstituted the virus. We then used this system to generate a recombinant Oka vaccine virus expressing mumps virus gene(s). The new recombinant vaccine may be an effective polyvalent live vaccine that provides protection against both varicella and mumps viruses. In this review, we discussed about possibility of polyvalent live vaccine(s) using varicella vaccine based on our recent studies.
A precise excision of the complete Epstein-Barr virus genome in a plasmid based on a bacterial artificial chromosome
2011, Journal of Virological Methods
The Maxi-EBV is a bacterial artificial chromosome (BAC)-based plasmid that contains the complete Epstein-Barr virus (EBV) genome of 172 kb. This plasmid also carries an additional cassette of 11.5 kb in size for the expression of a mini F factor, selection markers and GFP. In the intracellular study of EBV infection based on the Maxi-EBV system, a parallel control that only contains this 11.5 kb vector is desirable but unavailable. In order to construct the vector in this approach, a clean deletion of the complete EBV genome from the Maxi-EBV was performed. This was achieved by homologous recombination using the bacteriophage λ Red system. Initially, an FRT-flanked kanamycin-resistance (kan) fragment of 1.4 kb with 61 bp homologies on the ends was introduced into the Maxi-EBV plasmid, replacing the 172-kb EBV genome. The kan gene was then removed by Flp/FRT excision. The results of identification demonstrated that the mutation was generated precisely. The results highlight the feasibility for a genome as large as 172 kb to be replaced by a greatly shorter fragment and for a much smaller vector backbone to be retrieved. Cell lines derived from the transfection of the vector will subsequently be appropriate controls in the related study.
Equine herpesvirus 4: Recent advances using BAC technology
2011, Veterinary Microbiology
The equine herpesviruses are major infectious pathogens that threaten equine health. Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease, known as rhinopneumonitis, among horses worldwide. EHV-4 genome manipulation with subsequent understanding of the viral gene functions has always been difficult due to the limited number of susceptible cell lines and the absence of small-animal models of the infection. Efficient generation of mutants of EHV-4 would significantly contribute to the rapid and accurate characterization of the viral genes. This problem has been solved recently by the cloning of the genome of EHV-4 as a stable and infectious bacterial artificial chromosome (BAC) without any deletions of the viral genes. Very low copy BAC vectors are the mainstay of present genomic research because of the high stability of inserted clones and the possibility of mutating any gene target in a relatively short time. Manipulation of EHV-4 genome is now feasible using the power of BAC technology, and should aid greatly in assessing the role of viral genes in the virus–host interaction.

View all citing articles on Scopus

View full text

Trends in Microbiology

ReviewHerpesvirus genetics has come of age

Abstract

Section snippets

Forward or classical herpesvirus genetics

Bacterial artificial chromosomes (BACs)

Conclusions and perspective

Acknowledgements

J. Invest. Dermatol.

Cell

Cell

Virology

Virology

J. Virol. Methods

Curr. Opin. Biotechnol.

Mol. Genet. Metab.

Nat. Genet.

J. Biol. Chem.

Virology

Gene

Temperature-sensitive mutants of herpesviruses

Curr. Top. Microbiol. Immunol.

Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus

Nature

Insertion and deletion mutagenesis of the human cytomegalovirus genome

Proc. Natl. Acad. Sci. U. S. A.

Regeneration of herpesviruses from molecularly cloned subgenomic fragments

J. Virol.

Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro

Proc. Natl. Acad. Sci. U. S. A.

Epstein–Barr virus recombinants from overlapping cosmid fragments

J. Virol.

Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids

J. Virol.

Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments

J. Virol.

Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones

J. Virol.

Activation of immediate-early, early, and late promoters by temperature- sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4

Mol. Cell Biol.

Yeast artificial chromosome cloning

Mol. Biotechnol.

Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector

Proc. Natl. Acad. Sci. U. S. A.

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments

Nat. Genet.

Stable propagation of cosmid sized human DNA inserts in an F factor based vector

Nucleic Acids Res.

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors

Nucleic Acids Res.

Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome

Proc. Natl. Acad. Sci. U. S. A.

An enhanced packaging system for helper-dependent herpes simplex virus vectors

J. Virol.

Propagation and recovery of intact, infectious Epstein–Barr virus from prokaryotic to human cells

Proc. Natl. Acad. Sci. U. S. A.

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors

Hum. Gene Ther.

Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants

J. Virol.

Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus

J. Virol.

Review
Herpesvirus genetics has come of age