FIGURE 2.

Eltanexor treatment does not affect viral entry or nuclear trafficking. (A) Eltanexor add-on and removal assays showed viral replications was suppressed after 24 hpi. Culture supernatants collected from add-on or removal assays and HCMV titers were determined by a TCID₅₀ assay. Data represent mean ± SEM, n = 3. Statistical analyses were performed between indicated hpi and 0 hpi, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Nuclear localization of viral tegument protein pp65 is not affected by Eltanexor treatment. HFFs were infected with HCMV at a MOI = 1 and treated with Eltanexor (0.4 μM) or DMSO. At 6 hpi, cells were fixed and immunostained with mouse antibodies against pp65, followed by Cy3-conjugated goat anti-mouse antibody (red). Nuclei were stained with DAPI (blue). Percentages of cells with pp65 nuclear localization were calculated based on counts from 5 random fields with at least three hundred cells. The experiments were repeated 3 times. Data represent mean ± SEM, n = 3. NS stands for not significant. (C) Entry of the viral genome is not affected by Eltanexor treatment. HFFs were infected with HCMV at a MOI = 1 and treated with Eltanexor at different concentrations or DMSO for 2 h. Viral genomic DNA was isolated and quantified by qPCR with primers for UL122. Fold changes of viral genome in HCMV infected cells treated with 0 μM of Eltanexor were normalized to 100%. Statistical analyses were performed between different concentrations of Eltanexor and vehicle (0 mM). Data represent mean ± SEM, n = 3. (D) Nuclear trafficking of viral genomic DNA is not affected by Eltanexor treatment. HFFs were infected with HCMV at a MOI = 1 and then treated with Eltanexor (0.4 μM) or DMSO. Viral DNA isolated from total cells or nuclei at 2, 4, and 6 hpi were quantified by qPCR with primers for UL122. Fold changes of viral genome in HCMV infected cells treated with DMSO at 2 hpi were normalized to 100%. Data represent mean ± SEM, n = 3. Statistical analyses were performed between Eltanexor and DMSO treatments. Total cell lysates, cytoplasm and nuclear components were isolated with commercial cytoplasm-nuclear isolation kits and purity was confirmed by immunoblot against the nuclear marker Lamin B and the cytoplasmic marker β-actin. The experiments were repeated three times.