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. 2019 Nov 13;93(23):e01052-19. doi: 10.1128/JVI.01052-19

FIG 5.

FIG 5

Increased BST2 transcription during infection restricts plaque size and is dependent on IKK signaling. (A) MRC5 cells were mock infected or infected with WT or UL26ΔN, UL26ΔC, or ΔUL26 mutant HCMV at an MOI of 3.0. The abundance of BST2 mRNA at 48 hpi was measured by real-time PCR and normalized to GAPDH. Values are means ± SEM (n = 6; *, P < 0.05; **, P < 0.01; ns, not significant). (B) MRC5 cells were mock infected or infected with WT, UL26(+/−), or ΔUL26 mutant HCMV. The abundance of BST2 mRNA at 48 hpi was measured by real-time PCR and normalized to GAPDH. Values are means ± SEM (n = 3; **, P < 0.01; ns, not significant). (C) HFF cells were lentivirally transduced with either a nontargeting control shRNA construct or a BST2-targeting shRNA construct. The abundance of BST2 transcript was measured by real-time PCR and normalized to GAPDH. Values are means ± SEM (n = 5; **, P < 0.01) (D) Nontargeting shRNA control and BST2 KD cells were infected with 40 PFU of either WT or ΔUL26 mutant HCMV. The relative efficiencies of plaque formation were quantified at 11 days postinfection (dpi). The averages are plotted after normalizing to the number of plaques for each virus counted on the infected shRNA controls. Values are means ± SEM (n = 8; ns, not significant). (E and F) shRNA control and BST2 KD cells were infected as in panel C with 40 PFU of either WT (E) or ΔUL26 mutant (F) HCMV. Individual plaque sizes were quantified using the ImageJ software, averaged, and normalized to the average plaque size for each virus in shRNA control cells. Values are means ± SEM (n = 40; *, P < 0.05; ns, not significant). (G and H) Nontargeting shRNA control and BST2 KD cells were infected with either mock infected or infected with WT or ΔUL26 mutant HCMV at an MOI of 3.0. Cellular RNA was harvested at 48 hpi, and IE1, pp28, and GAPDH mRNA levels were measured by quantitative PCR (qPCR). The ratio of IE1 mRNA to GAPDH mRNA (G) or pp28 mRNA to GAPDH mRNA (H) for each virus was normalized to the value measured in the shRNA control cell line and plotted as the means ± SEM (n = 4; **, P < 0.01; ns, not significant). (I) CRISPR control, IKKα(−/−), and IKKβ(−/−) BJ/hTert cells were either mock infected or infected with WT or ΔUL26 mutant HCMV at an MOI of 3.0. Cellular RNA was harvested at 48 hpi, and the abundance of the BST2 transcript was measured by real-time PCR and normalized to GAPDH. Values are plotted as the means ± SEM (n = 6; *, P < 0.05; **, P < 0.01; ns, not significant).