FIG. 6.

Recombinant viruses with point mutations in either the LxCxE RB binding motif or the kinase motif are impaired in their abilities to stabilize and phosphorylate RB. HFF cells were infected (at an MOI of 2 PFU/cell) with the wt virus HB5 or with recombinant viruses containing the point mutations in pUL97, as shown. Cell lysates were harvested at 24 h following infection, separated on polyacrylamide gels, and transferred to nitrocellulose membranes. Shown are immunoblots, with the antibodies to the proteins indicated to the left of the figure. The accumulation of RB occurred in cells infected with HB5, but was reduced in cells infected with the K355M mutant and the C151G mutant. The phosphorylation of RB on serine 780 was determined with specific antisera. Cells infected with HB5 contained increased levels of RB phosphorylated on serine 780. Cells infected with viruses containing point mutations that abrogated UL97 kinase activity (K355M) or disrupted the LxCxE motif (C151G) exhibited reduced levels of RB phosphorylated on serine 780. The expression of IE1 confirmed that cells were infected, and tubulin (tub) was included as a loading control.