Murine cytomegalovirus with a transposon insertional mutation at open reading frame M35 is defective in growth in vivo.

We had previously constructed a pool of murine cytomegalovirus (MCMV) mutants that contained a Tn3-based transposon sequence randomly inserted in the viral genome. In the study reported here, one of the mutants, RvM35, which contains the transposon insertion at open reading frame M35, was characterized both in vitro in tissue cultures and in immunocompetent Balb/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M35 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M35 region, the viral mutant was attenuated in growth in both the intraperitoneally infected Balb/c and SCID mice. At 21 days postinfection, the titers of the mutant in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice were lower than the titers of the wild-type Smith strain and the rescued virus by 50,000-, 100-, 10-, 100-, and 50-fold, respectively. Moreover, the growth of RvM35 is severely attenuated in the salivary glands. The virulence of the mutant virus also appears to be attenuated, because no death was observed in SCID mice infected with RvM35 until 35 days postinfection, while all the animals infected with the wild-type and rescued viruses died 27 days postinfection. Our results suggest that M35 is important for MCMV virulence in killing SCID mice and is required for optimal viral growth in vivo, including in the salivary glands.

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166.

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 30000-, 10000-, 1000-, 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.

Genetic analyses of gene function and pathogenesis of murine cytomegalovirus by transposon-mediated mutagenesis.

Murine cytomegalovirus (MCMV) has a linear genome of 230 kb and encodes more than 170 genes, many of which have not been extensively studied for their functions in pathogenesis in vivo. A Tn3-based transposon was constructed and used to generate MCMV mutants by disrupting viral gene targets. The functions of the mutated genes were investigated by studying the viral mutants in cultured cells and in immunocompetent Balb/c and immunodeficient SCID mice. A pool of MCMV mutants that contained the transposon sequence randomly inserted at the viral genome was generated. Studies of several mutants (e.g. a viral mutant with the transposon inserted at open reading frame m09) in cultured cells and in mice indicate that the presence of the transposon sequence per se in the viral genome does not significantly affect viral growth in vitro and in vivo. Moreover, the genome structures of the viral mutants, including the transposon insertion regions, were stable during replication in cultured cells and in animals. Several viral mutants (e.g. a viral mutant with the transposon at M27) that are attenuated in growth and virulence in animals were identified. These results suggest that the genes mutated in these viral mutants may be important for viral virulence and pathogenesis. The Tn3-based system may be a useful tool for the systematic construction of CMV mutants and for studies of CMV gene functions in viral replication in vitro and in pathogenesis in vivo.