FIG. 1.

In vitro and in vivo expression of the MCMV homologs of HCMV DNA polymerase (M54), primase (M70), and helicase (M105). (A) The MCMV ORFs, cloned into pcDNA3.1/V5-His-TOPO, were expressed using the T7 promoter by coupled in vitro transcription/translation reactions (TNT T7 Quick) with [³⁵S]methionine. A portion of each reaction mixture was subjected to reducing SDS-PAGE on a 7% polyacrylamide gel, and the labeled proteins were detected by autoradiography. The numbers and lines on the left indicate the positions and relative molecular masses (in kDa) of the proteins in the marker (not shown). Vect indicates the TNT reaction performed with the DNA vector alone. The predicted molecular masses for the encoded proteins are M54, 128.8 kDa; M70 (untagged), 109.6 kDa; and M105, 111.4 kDa. (B) After the M70 clone was mutated to yield the V5- and six-His-tagged M70*, plasmids were transiently transfected into COS-7 cells and the whole cells were lysed and solubilized in reducing SDS-PAGE sample buffer 48 h posttransfection. The lysate proteins were resolved by SDS-PAGE as described above and electroblotted to a nitrocellulose membrane that was subsequently probed with a mouse anti-V5 monoclonal antibody. The numbers on the left are as described for panel A, and Vect indicates the lysate from cells transfected with empty plasmid vector. The predicted molecular mass for the M70* protein is 114.7 kDa.