Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene

Greaves, Richard F.; Brown, Janice; Vieira, Jeffrey; Mocarski, Edward S.

doi:10.1099/0022-1317-76-9-2151

Cited by 60 publications

(72 citation statements)

References 38 publications

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“…On the basis of design, r⌬MSVgpt enabled selective mutagenesis of the MIE regulatory region by homologous recombination in conjunction with the elimination of gpt. Such construction afforded dominant selection of resultant recombinant viruses in HGPRT-deficient fibroblasts exposed to 6-thioguanine (14). Replacement of gpt with the gfp ORF further assisted in the selection of growth-defective recombinant viruses.…”

Section: Construction Of An Hcmv Without the Mie Distal Enhancer Regionmentioning

confidence: 99%

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The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Meier

Pruessner

2000

J Virol

117

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The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type-and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediateearly (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.Human cytomegalovirus (HCMV) replicates in many cell types, including endothelial, smooth muscle, fibroblast, hepatocyte, neuronal, glial, and macrophage cells (reviewed in reference 45). It replicates poorly or negligibly in lymphocytes, neutrophils, and certain embryonal cells (11,12,25,44,45), and it resides latently in monocytes and their precursors (16,21,22,33,34,42,46,52,53,57). The mechanisms that govern HCMV replication or latency are poorly understood. Products of the HCMV major immediate-early (MIE) genes (e.g., IE1 p72 and IE2 p86) are required for viral replication (15,18,36,38,47). They are not expressed in latently or certain nonpermissively infected cell types (21,22,25,33,37,42,53). Hence, the regulation of their expression may be a pivotal step in controlling viral replication.The MIE regulatory region controls transcription of its genes through interplay of both positive and negative cis-acting elements. The enhancer component of the MIE regulatory region contains many of these cis-acting elements. The enhancer's boundaries are inexact but are often considered to span base positions -65 to -550 with respect to the ϩ1 start site of MIE RNAs (32). A variety of cellular and viral proteins interact with the enhancer's cis-acting elements to regulate activity of MIE promoter segments when assayed in transfec...

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Section: Construction Of An Hcmv Without the Mie Distal Enhancer Regionmentioning

confidence: 99%

“…This virus was used for homologous recombination with the plasmids listed below. The resultant recombinant viruses lack gpt, which allows for their selection in HGPRT-deficient fibroblasts and 6-thioguanine (50 g/ml) as described by Greaves et al (14). Viral DNA was prepared for recombination as described previously (31).…”

Section: Cells and Virusmentioning

confidence: 99%

The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Meier

Pruessner

2000

J Virol

117

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“…Construction of the rescued virus was carried out by using a procedure similar to that used for generating the viral mutant. A DNA fragment that contained the m166 coding region was cotransfected with the full-length Rvm166 genomic DNA into mouse cells and the recombinant viruses were selected in STO cells in the presence of 6-thioguanine, which selects against gpt expression (14,28). The rescued virus, designated as Rqm166, which did not express the gpt protein and no longer contained the transposon, was isolated after multiple rounds of selection and plaque purification.…”

Section: Isolation Of a MCMV Mutant Containing The Transposon Insertimentioning

confidence: 99%

“…The full-length intact Rvm166 genomic DNA and the DNA sequence that contained the coding sequence of m166 were subsequently cotransfected into mouse cells by using a calcium phosphate precipitation protocol (Gibco-BRL). The recombinant virus was selected in STO cells in the presence of 25 g/ml of 6-thioguanine (Sigma) and purified by six rounds of amplification and plaque purification, according to the protocol described previously (14). For each cotransfection, several viral plaques were picked and expanded.…”

Section: Animalsmentioning

confidence: 99%

“…To generate a pool of MCMV mutants, a library of MCMV genomic fragments containing randomly inserted Tn3gpt sequence were first constructed as described previously and then cotransfected with the full-length genomic DNA of the wild-type virus (Smith strain) into mouse NIH 3T3 cells where homologous recombination took place. The cells that harbored the progeny viruses expressing the gpt gene were selected for growth in the presence of mycophenolic acid and xanthine (14,28,44). After multiple rounds of selection and plaque purification, individual recombinant viruses were isolated and the location of the inserted transposon was determined by directly sequencing the genomic DNA of the recombinants.…”

Section: Isolation Of a MCMV Mutant Containing The Transposon Insertimentioning

confidence: 99%

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In Vitro and In Vivo Characterization of a Murine Cytomegalovirus with a Mutation at Open Reading Frame m166

Zhu

Chen

Hai

et al. 2003

J Virol

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We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30,000-, 10,000-, 1,000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.

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Cloning of herpesviral genomes as bacterial artificial chromosomes

Adler¹,

Messerle

Koszinowski

2003

Reviews in Medical Virology

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Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs). Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as a plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in E. coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example.

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Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene

Cited by 60 publications

References 38 publications

The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

In Vitro and In Vivo Characterization of a Murine Cytomegalovirus with a Mutation at Open Reading Frame m166

Cloning of herpesviral genomes as bacterial artificial chromosomes

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