ABSTRACT
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.