A dominant selectable marker for the construction of recombinant poxviruses

doi:10.1016/0378-1119(88)90424-6

Gene

Volume 65, Issue 1, 15 May 1988, Pages 123-128

https://doi.org/10.1016/0378-1119(88)90424-6 Get rights and content

Abstract

Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

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Cited by (113)

A generic protocol for the affinity-purification of native macromolecular complexes from poxvirus-infected cells
2022, STAR Protocols
Citation Excerpt :
In order to isolate the recombinant viruses, 3 to 4 rounds of plaque purification are performed in the presence of mycophenolic acid (MPA), xanthine (XT) and hypoxanthine (HXT). MPA blocks the synthesis of purine nucleotides, but viruses expressing gpt can initiate an alternative pathway for purine synthesis using XT and HXT as precursors (Boyle and Coupar, 1988). Recombinant viruses expressing gpt are able to form plaques under MPA selection (Lorenzo at al., 2004).
The functional and structural characterization of macromolecular complexes requires protocols for their native isolation. Here, we describe a protocol for this task based on the recombinant poxvirus Vaccinia expressing tagged proteins of interest in infected cells. Tagged proteins and their interactors can then be isolated via affinity chromatography. The procedure is illustrated for the Vaccinia virus encoded multi-subunit RNA polymerase. Our protocol also allows the expression and isolation of heterologous proteins and hence is suitable for a broader application.
For complete details on the use and execution of this profile, please refer to Grimm et al. (2019).
Capripoxvirus vectors for vaccine development
2020, Gene Reports
Citation Excerpt :
The selection of recombinant viruses can be carried out using antibiotics that co-integrate into the viral genome simultaneously with the target genes. It has been shown that neomycin (Franke et al., 1985), just like mycophenolic acid (Falkner and Moss, 1988; Boyle and Coupar, 1988), blocks vaccinia virus replication in normal cell lines. Expression of the neomycin resistance gene or xanthine–guanine phosphoribosyl transferase (gpt) allowed only recombinant viruses to form plaques in a selective medium.
The Capripoxvirus genus of the Poxviridae family comprises sheep poxvirus, goat poxvirus, and lumpy skin disease virus, which are the causative agents of economically important diseases of ruminants. Live attenuated capripoxvirus vaccines have been used for many years in veterinary practice, and have demonstrated their safety, reliability, and ability to provide long-term protection. A limited host range and non-pathogenicity to humans are the main advantages for the use of capripoxviruses in the development of vector vaccines to induce effective immune protection. Here, we review the approaches used to generate recombinant poxviruses and discuss the experience of developing capripoxvirus vector vaccines.
Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology
2017, Molecular Therapy
Citation Excerpt :
VACV-CHIK was constructed by homologous recombination to replace the A39R open reading frame (ORF) of VACV with a poxvirus expression cassette expressing the CHIKV 26S-structural polyprotein (GenBank: AM258992), under the control of VACV promoters,58 together with a poxvirus expression cassette for the expression of the E. coli guanine phosphoribosyltransferease (Ecogpt). Homologous recombination and positive selection of recombinant viruses containing the co-insertion of the Ecogpt expression cassette was undertaken as described (protocol 6),59–61 in which the homologous recombination arms flanking the CHIKV-26S and Ecogpt expression cassettes were designed to be homologous with the sequences flanking the A39R ORF. After successful replacement of the A39R ORF with CHIKV-26S and Ecogpt expression cassettes, the Ecogpt expression cassette was removed by a second homologous recombination and counterselection against Ecogpt expression as described (protocol 7),59,62 whereby the homologous recombination arms were designed to be homologous to the sequences flanking the Ecogpt expression cassette within the recombinant VACV-CHIK genome.
Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction.
Vaccinia Virus Recombinants: Expression Vectors and Potential Vaccines
2012, Animal Cell Biotechnology
Recombinant capripoxviruses expressing proteins of bluetongue virus: Evaluation of immune responses and protection in small ruminants
2007, Vaccine
The development of recombinant capripoxviruses for protective immunization of ruminants against bluetongue virus (BTV) infection is described. Sheep (n = 11) and goats (n = 4) were immunized with BTV recombinant capripoxviruses (BTV-Cpox) individually expressing four different genes encoding two capsid proteins (VP2 and VP7) and two non-structural proteins (NS1, NS3) of BTV serotype 2 (BTV-2). Seroconversion was observed against NS3, VP7 and VP2 in both species and a lymphoproliferation specific to BTV antigens was also demonstrated in goats. Finally, partial protection of sheep challenged 3 weeks after BTV-Cpox administration with a virulent strain of BTV-2, was observed.
Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine
2005, Virology
The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3′-azido-2′,3′-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

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A dominant selectable marker for the construction of recombinant poxviruses

Abstract

Gene

Vaccinia virus recombinants expressing the SA11 rotavirus VP7 glycoprotein gene induce serotype-specific neutralizing antibodies

J. Virol.

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants

Mol. Cell. Biol.

Infection of human and simian tissue cultures with Rous sarcoma virus

Expression of rabies virus glycoprotein from a recombinant vaccinia virus

Nature