Mutagenesis of murine cytomegalovirus using a Tn3-based transposon

A transposon derived from Escherichia coil Tn3 was introduced into the geno me of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the t ransposon within the M25, M27, and m155 open reading frames. Our studies pr ovide the first direct evidence to suggest that M25 and M27 are not essenti al for Viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during Vi ral propagation both in vitro and in vivo. Moreover the virus that containe d the insertion mutation in M25 exhibited a titer similar to that of the wi ld-type virus in the salivary glands, lungs, livers, spleens, and kidneys o f the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these org ans and the presence of the transposon sequence in the viral genome does no t significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes i n both tissue culture and in animals. (C) 2000 Academic Press.