Cells, viruses, and reagents

Primary human foreskin fibroblasts (HFFs) were grown in DMEM containing 10% newborn calf serum. Cells were used between passage 5 and 15 in all experiments. Replication defective lentiviruses expressing eIF4AIII-specific shRNAs (TRCN 0000061855 or 0000061856) were generated as described previously (Lenarcic et al., 2014). HFFs were transduced with lentivirus overnight in the presence of polybrene (4 µg/ml). The next day the media was replaced with serum free DMEM, and the cells were used for experiments at 72 h after transduction. Efficient eIF4AIII depletion was routinely assessed by quantitative real-time PCR (qRT-PCR) or Western blot.

A variant of the HCMV AD169 strain containing a green fluorescent protein expression cassette driven by the SV40 promoter (ADGFP; Wang et al., 2004) was used as the wild type virus strain in all studies. Unless otherwise noted, all infections were performed at a multiplicity of infection (MOI) of three in a minimal volume of serum free media. Infected cells were harvested by scraping and stored at −80 °C until analyzed. Quantification of cell-free virus by the TCID50 method was performed as described previously (Moorman and Shenk, 2010).

DMDA-pateamine A (PatA) (Kuznetsov et al., 2009), a synthetic analogue of pateamine A, was generously provided by Dr. Daniel Romo (Texas A&M University).