Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter

Abstract

On the basis of a previous finding that the 7.8-kb HindIII-O fragment of the human cytomegalovirus strain Towne genome is nonessential for viral replication, we constructed a vector, pKM, that directs introduction of foreign genes by homologous recombination precisely replacing the O fragment. Using this vector, we constructed Towne-strain-derived recombinant virus in which a chimeric lacZ gene fused to the simian virus 40 promoter and a poly(A) signal were inserted in place of the O fragment. Two types of recombinants were obtained which carried the chimeric gene in opposite directions, beta-Galactosidase (beta Gal) was produced throughout the infection cycle in human embryonic lung cells infected with these recombinants, and the rate of its synthesis in the early stages of infection was comparable to that of synthesis of a 65-kDa viral glycoprotein, one of the abundantly produced viral proteins. The chimeric lacZ gene introduced was stable and no lacZ- revertants have been observed so far.