HCMV preparation and infection

HCMV AD169 strain (ATCC VR538) was prepared as previously described [71,72], and viral stocks used in the experiments contained ~2 x 10⁷ PFU/ml. Standard plaque assays were used to titrate viral stocks, as well as in different experiments to determine viral titers in the supernatants harvested from infected cells [71,72]. UVB-inactivated AD169 was prepared using a double pulse of UV-B light (1.2 J/cm²).

HCMV TR was kindly provided by Prof. Jay A. Nelson (Oregon Health and Science University, USA) and reconstitution was performed as previously described [72]. HCMV VR1814 was previously described [71,72]. Merlin strain was kindly provided by Drs. Klaus Hamprecht and Gerhard Jahn (University of Tubingen, Germany). HCMV clinical isolates were obtained from urines of infants affected by congenital HCMV infection, as previously described [73] (Ethical Approval n. 007816 obtained by the Research Ethics Committee of the University Hospital of Turin “A.O.U. Città della Salute e della Scienza Torino–A.O. Ordine Mauriziano–A.S.L. TO1”).

Cells were infected at 80–90% confluence at the indicated MOI in their respective culture medium, without FCS. After 3 h (AD169 and TR strains) or 5 h (VR-1814 strain) at 37°C, virus inoculum was replaced with fresh culture medium (day 0). Mock-infected control cells were exposed for the same amount of time to an equal volume of medium. At various days post-infection (dpi), cells were harvested and analyzed.

When the pUL97 HCMV kinase inhibitors Maribavir (MBV) or Gö6976 (Gö; Merck Calbiochem), or the Cdk inhibitors CGP74514A (CGP; Merck Millipore), RO-3306, KO-3861 and BMS-265246 (Sellekchem) were used, HFFs were treated, infected and cultured as described in figure legends. As controls, cells were not treated or treated with DMSO.