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Mutagenesis of murine cytomegalovirus using a Tn3-based transposon
1Program in Infectious Diseases and Immunity, School of Public Health, University of California, 140 Warren Hall, Berkeley, California, 94720, USA.
Abstract:
A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.
Insights
This study shows that M25 and M27 genes are not essential for murine cytomegalovirus (MCMV) replication. Transposon mutagenesis is a stable method for studying MCMV gene function in vitro and in vivo.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Murine cytomegalovirus (MCMV) is a significant pathogen in mice, with its gene functions often studied through genetic manipulation.
- Understanding MCMV gene essentiality is crucial for developing antiviral strategies and comprehending viral pathogenesis.
Purpose of the Study:
- To investigate the essentiality of specific MCMV genes (M25, M27, m155) for viral replication using transposon mutagenesis.
- To evaluate the stability of transposon insertions in the MCMV genome during in vitro and in vivo propagation.
- To assess the impact of transposon insertions on MCMV replication in cultured cells and animal models.
Main Methods:
- Generation of MCMV mutants using a transposon derived from Escherichia coli Tn3.
- Analysis of recombinant viruses with transposon insertions in M25, M27, and m155 open reading frames.
- Assessment of viral replication in NIH 3T3 cells and Balb/c mice, including viral titers in various organs.
Main Results:
- M25 and M27 genes were found not to be essential for MCMV replication in NIH 3T3 cells.
- Transposon insertions were stable during both in vitro and in vivo MCMV propagation.
- MCMV with a transposon insertion in M25 showed titers comparable to wild-type virus in multiple organs of infected mice, indicating M25 dispensability.
- The presence of the transposon sequence did not significantly impede viral replication in vivo.
Conclusions:
- The M25 and M27 genes are dispensable for MCMV replication in cultured cells.
- M25 is dispensable for MCMV growth in salivary glands, lungs, livers, spleens, and kidneys of Balb/c mice.
- The Tn3-based transposon system is a viable and stable mutagenesis approach for functional genomics studies of MCMV in both cell culture and animal models.