Construction and characterization of murine cytomegaloviruses that containtransposon insertions at open reading frames m09 and M83

A transposon derived from Escherichia coli Tn3 was introduced into the geno me of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, in cluding two recombinant viruses that contained the transposon sequence with in open reading frames m09 and M83. Our studies provide the first direct ev idence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 se vere combined immunodeficient (SCID) mice indicated that the transposon ins ertion is stable during viral propagation both in vitro and in vivo. Moreov er, the virus that contained the insertion mutation in m09 exhibited a tite r similar to that of the wild-type virus in the salivary glands, lungs, liv ers, spleens, and kidneys of both the BALB/c and SCID mice and was as virul ent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence o f the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the inser tion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCM V genes in both immunocompetent and immunodeficient animals.