Growth of m74 knockout viruses. Multistep growth curves of BAC-derived parental MCMV and m74 knockout mutants are shown (upper panels). NIH 3T3 cells were infected at an MOI of 0.05, and supernatants were harvested and titrated daily. Titers of supernatant virus are expressed as TCID₅₀ per ml. Additionally, virus spread in MEF (lower panels) is depicted by staining of infected cells for MCMV ie1 protein 3 days postinfection. Shown are growth curves and spread patterns of w.t.-MCMV and vm74stop (A) and w.t.-FRT-MCMV and vΔm74 (B) representative for at least three independent experiments.

trans-complemented m74 knockout mutants share the growth phenotype of noncomplemented mutants. NIH 3T3 cells were infected at an MOI of 0.05, and supernatants were harvested after 5 days and titrated. Shown are means of three to four independent experiments. Titers of supernatant virus from knockout mutants (vm74stop and vΔm74) and trans-complemented mutants (vm74stop/m74trans and vΔm74/m74trans) were significantly lower (as indicated by asterisks) than titers of the respective parental viruses (w.t.-MCMV and w.t.-FRT-MCMV) (P < 0.014, Student's t test).

vΔm74 and w.t.-FRT-MCMV release comparable amounts of viral genomes. NIH 3T3 cells were infected with w.t.-FRT-MCMV and trans-complemented vΔm74 at an MOI of 1. (A) Initial infection of NIH 3T3 cells is shown by staining of infected cells for MCMV ie1 protein 16 h after infection. (B) At 16 h after infection, cells from three parallel cultures were harvested for real-time PCR analysis of total cellular DNA. (C) Input virus and supernatants from three parallel cultures harvested at different time points after infection were analyzed by real-time PCR for viral DNA. The TCID₅₀ ml⁻¹ ± standard deviations of the 30- and 48-h supernatants are indicated above the respective bars. Error bars depict the standard deviations of triplicates. n.d., not detectable.

m74 knockout renders MCMV entry into fibroblasts energy dependent and pH sensitive. (A) MEF were infected with the indicated BAC-derived viruses in the presence of energy depletion medium. Three hours after infection, cells were fixed and stained for ie1 expression. The percentage of ie1-positive cells after energy depletion is expressed relative to ie1-positive cells of mock-treated cell infections. Shown are means ± standard deviations of three independent experiments done in triplicate. As indicated by asterisks, inhibition of vm74stop and vΔm74 was significantly different from inhibition of wild-type parental or trans-complemented viruses (P < 0.015, Student's t test). (B and C) NIH 3T3 cells were infected with w.t.-FRT-MCMV (▴), vΔm74 (▵), and vΔm74/m74trans (⋄) in the presence of increasing concentrations of NH₄Cl and bafilomycin A1. The effect of inhibitors on virus entry was monitored as described for panel A. Shown are means ± standard deviations of three independent experiments done in triplicate. For 10 and 25 nM bafilomycin A1, two independent experiments were performed. NH₄Cl and bafilomycin A1 significantly inhibited entry of vΔm74 (P < 0.001, two-way ANOVA).

Schematic representation of the MCMV m74 knockout mutants. The m74 locus and, for pSM3fr-FRT, the m16-FRT-m17 locus are indicated. Cloning of the pm74stop and the pΔm74 mutants and cis complementation of the latter mutant by reinsertion of m74 at the m16-FRT-m17 locus are depicted. The names of the BACmids and the respective BAC-derived viruses are indicated on the right. Terminal repeats of the MCMV genome are indicated by open boxes.

Complementation of m74 knockout mutants. (A) cis complementation of vΔm74. Multistep growth curves of w.t.-FRT-MCMV and of the cis-complemented vΔm74-m74+/−HA. MEF-BL/6-1 cells were infected at an MOI of 0.05, and supernatants were harvested and titrated daily. (B and C) trans complementation of vm74stop (B) and vΔm74 (C). NIH 3T3-m74 cells were infected at an MOI of 0.05, and supernatants were harvested and titrated daily. Shown are growth curves representative for at least three independent experiments expressed either as PFU/ml (A) or as TCID₅₀/ml (B and C).

Electron microscopy studies on w.t.-FRT-MCMV- and vΔm74/m74trans-infected MEF. Panels A and C show pictures of conventional electron microscopy using epoxide resin sections stained with uranyl acetate and lead citrate of BL/6-1 cells. Panels B and D represent cryo-electron microscopy projections from frozen-hydrated sections of NIH 3T3 cells. Panels A and B show infections with w.t.-FRT-MCMV. Panels C and D show infections with vΔm74/m74trans. Enveloped cytoplasmic virus particles, partly in larger vesicles in the cytoplasm, are indicated by black and white arrows in panels A and B, respectively. A multicapsid particle in panel B is labeled with an asterisk. The wavy pattern in panel B depicts so-called crevasses, an inevitable cutting artifact in upper layers of vitreous sections thicker than 60 nm. Panels C and D show cytoplasmic accumulations of capsids (black arrows) surrounded by empty vesicles (white arrows). The darker left area of panel D marks the supporting carbon film beneath the section ribbon.

The m74 protein associates with gH. (A) 293 cells transfected with pCR3-m74HA or pCR3 as a control were harvested 24 h after transfection. MEF infected with vΔm74-m74HA or vΔm74-m74, as a control, were harvested 5 days after infection when the cytopathic effect was complete. Total cell lysates were analyzed by Western blotting using an anti-HA antibody to detect HA-tagged m74 protein. For treatment with Endo H, cells were treated either with buffer only or with Endo H as indicated. The arrows indicate the 70-, 55-, and 45-kDa m74 protein bands. (B) Immunoprecipitation of HA-tagged m74 and MCMV gH from extracts of vΔm74-m74HA-infected MEF lysed under nonreducing conditions. Proteins were precipitated with anti-HA or anti-gH antibodies or an IgG2a isotype control. Precipitated proteins were detected with an anti-HA antibody by Western blot analysis (WB). The total cell extract of infected cells is shown on the left. The high-molecular-weight complex is indicated by a black arrow. (C) The same experiment as that described for panel B but with lysis of cells under reducing conditions. The total cell extract is shown on the right. The 70- and 55-kDa m74 protein bands are indicated by black arrows. The heavy chain of the antibodies used for precipitation is detected by the secondary anti-rat antibody and is indicated by a white arrow. Two exposures (upper panel, 2 min; lower panel, 20 s) of the same blot are shown.

gO knockout renders HCMV entry into fibroblasts energy dependent and pH sensitive. (A) HFF were infected with vTB40-BAC4, vUL131Astop, and vΔgO in the presence of energy depletion medium as described for Fig. 8. Twenty-four hours after infection, cells were stained for HCMV ie1 protein. Shown are means ± standard deviations of three independent experiments done in triplicate. As indicated by the asterisk, inhibition of vΔgO was significantly different from inhibition of vTB40-BAC4 and vUL131Astop (P < 000.2, Student's t test). (B and C) HFF were infected with vTB40-BAC4 (▪), vUL131Astop (⋄), and vΔgO (□) in the presence of increasing concentrations of NH₄Cl (B) and bafilomycin A1 (C). Inhibitors were applied, and analysis was performed as described for Fig. 8. Shown are means ± standard deviations of three independent experiments done in triplicate. For 10 and 25 mM NH₄Cl and for 50 and 100 nM bafilomycin A1, two independent experiments were performed. NH₄Cl and bafilomycin A1 significantly inhibited entry of vΔgO (P < 0.001, two-way ANOVA).