Mutagenesis of Murine Cytomegalovirus Using a Tn3-Based Transposon

Zhan, Xiaoyan; Lee, Manfred; Abenes, Gerardo; Reis, Ilse Von; Kittinunvorakoon, Chonticha; Ross‐Macdonald, Petra; Snyder, M; Liu, Fenyong

doi:10.1006/viro.1999.0089

Cited by 36 publications

(86 citation statements)

References 39 publications

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“…A 2.1-kb mRNA transcript was detected ( Fig. 2A), similar to that previously observed for MCMV-infected NIH 3T3 cells (1,49). To examine m155 protein expression, an influenza hemagglutinin (HA) epitope tag was inserted at the C-terminal end of the m155 ORF in the bacterial artificial chromosome (BAC) genome of the MCMV-GFP virus as previously described (28).…”

supporting

confidence: 71%

The Mouse Cytomegalovirus Glycoprotein m155 Inhibits CD40 Expression and Restricts CD4 T Cell Responses

Loewendorf

Steinbrueck

Peter

et al. 2011

J Virol

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Healthy individuals infected with human cytomegalovirus (HCMV) are largely asymptomatic, but HCMV infection can result in high morbidity and death in immunosuppressed patients (12,38,48). HCMV establishes lifelong persistence, which is thought to require manipulation of the host's immune system by the coordinated function of many viral immunomodulatory genes (34).As adaptive immune responses develop during infection, interactions occur between mature antigen-presenting cells (APC) and their cognate T cells. The quality and quantity of the resulting T cell responses are regulated in large part by the affinity of peptide/major histocompatibility complex-T cell receptor (MHC-TCR) interactions and commensurate cosignals mediated by members of the tumor necrosis factor receptor (TNFR) and CD28 families. CD40 is a costimulatory TNFR superfamily member that is upregulated upon APC maturation and plays a role in promoting both CD4 and CD8 T cell responses (reviewed in references 14 and 29). The infection of dendritic cells with mouse CMV (MCMV) or HCMV restricts antigen presentation, inhibits costimulatory molecule expression, and enhances the expression of negative cosignaling ligands, resulting in reduced priming, expansion, and survival of antigen-specific T cells that encounter these APC (3,7,8,16,17,19,22,35). Consequently, it is quite possible that these mechanisms contribute to the immune suppression that is observed for cases of clinical HCMV disease (10,15,44).MCMV infection results in reduced cell surface expression of CD40 in dendritic cells (DC) (3,8). Two cell lines were utilized to investigate this effect; the RAW264.7 monocytic macrophage cell line and the DC2.4 bone marrow-derived DC line (kindly provided by K. Rock) (41). Throughout the study, fluorescence-activated cell sorter (FACS) and immunofluorescence experiments with MCMV that expresses green fluorescent protein (MCMV-GFP) (33) were performed using multiplicities of infection (MOI) of 0.4 and centrifugal enhancement, resulting in approximately 30% infected RAW264.7 and 20% infected DC2.4 cells, respectively. Immunoblot and RNA analysis of infected cells used MOI of 1.2 and centrifugal enhancement. Microscopy of infected and unin-

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supporting

confidence: 71%

The Mouse Cytomegalovirus Glycoprotein m155 Inhibits CD40 Expression and Restricts CD4 T Cell Responses

Loewendorf

Steinbrueck

Peter

et al. 2011

J Virol

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“…The transposon (Tn3gpt), which is derived from the Tn3 transposon from E. coli (5,34,37), contains the expression cassette encoding guanine phosphoribosyltransferase (gpt) (which contains the gpt coding sequence driven by a promoter and a transcription termination signal) and an additional transcription termination site (Fig. 1A) (48). Construction of an MCMV genomic subclone pool and the procedure of transposon-based shuttle mutagenesis to generate a pool of MCMV DNA fragments (MCMV-Tn3gpt) containing a Tn3gpt insertion were performed as described by Zhan et al (48).…”

Section: Animalsmentioning

confidence: 99%

“…1A) (48). Construction of an MCMV genomic subclone pool and the procedure of transposon-based shuttle mutagenesis to generate a pool of MCMV DNA fragments (MCMV-Tn3gpt) containing a Tn3gpt insertion were performed as described by Zhan et al (48). To generate a pool of MCMV mutants that contained the transposon sequence, full-length MCMV genomic DNA and plasmid DNA containing MCMV-Tn3gpt fragments were cotransfected into NIH 3T3 cells by using a calcium phosphate precipitation protocol (Gibco-BRL).…”

Section: Animalsmentioning

confidence: 99%

“…Viral DNA was purified from NIH 3T3 cells infected with the viruses as described previously (44,48). Briefly, cells that exhibited 100% cytopathic effect were washed with phosphate-buffered saline and then lysed with a solution that contained sodium dodecyl sulfate and VOL.…”

Section: Animalsmentioning

confidence: 99%

“…We have previously generated a pool of MCMV mutants by using a Tn3 transposon-mediated shuttle mutagenesis system (48,49). In this approach, the transposon is randomly inserted into the MCMV genomic DNA fragments in a plasmid library in Escherichia coli.…”

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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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Identification of mutations in a temperature‐sensitive mutant (tsm5) of murine cytomegalovirus using complementary genome sequencing

Timoshenko

Al-Ali

Martin

et al. 2009

Journal of Medical Virology

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Identification of mutations in mutants derived chemically is a difficult and relatively random process. NimbleGen Comparative Genome Sequencing (CGS) was assessed as an inexpensive, rapid method of identifying mutations in the temperature-sensitive mutant tsm5 of the K181 (Birmingham) variant of murine cytomegalovirus (MCMV). This genome resequencing approach requires an established genome sequence as a reference. Comparison of tsm5 and the K181 (Birmingham) variant with the published K181 (Perth) MCMV genomic sequence revealed a total of 10 synonymous and 15 non-synonymous SNPs in tsm5 and 14 of the latter were confirmed by sequencing. Thus, while CGS cannot be relied upon to identify correctly all mutations it was helpful for identifying a large number of mutations for further investigation that could contribute to the ts phenotype of tsm5.

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Mutagenesis of Murine Cytomegalovirus Using a Tn3-Based Transposon

Cited by 36 publications

References 39 publications

The Mouse Cytomegalovirus Glycoprotein m155 Inhibits CD40 Expression and Restricts CD4 T Cell Responses

The Mouse Cytomegalovirus Glycoprotein m155 Inhibits CD40 Expression and Restricts CD4 T Cell Responses

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

Identification of mutations in a temperature‐sensitive mutant (tsm5) of murine cytomegalovirus using complementary genome sequencing

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