Fig 2.

DZnep treatment primes nonpermissive THP1 cells for HCMV AD169 lytic mRNA expression. (A) Schematic diagram of the treatment and infection protocol. Cells were infected with AD169 at an MOI of 0.02. d, day. (B) Quantification of viral copy numbers in infected treated cells. Total DNA was prepared at 2 hpi, and relative viral copy numbers were quantified by qPCR. The relative genome copy number is reported as the mean fold change from the copy number in DMSO-treated cells for three biological replicates. Error bars, standard errors of the means. (C) Gene expression analysis in HCMV AD169-infected THP1 cells pretreated with DMSO, DZnep, or PMA. The expression levels of the major immediate early genes IE1/2, the early gene UL54, and the late gene UL99 were measured by qRT-PCR at the indicated times postinfection and are represented as the mean fold changes from the expression levels in DMSO-treated cells. Data are averages for three biological replicates, and error bars reflect the standard errors of the means of the normalized expression levels. Asterisks indicate P values of <0.05 (*), <0.01 (**), <0.001 (***), or <0.0001 (****) by two-way ANOVA followed by Dunnett's multiple-comparison posttest. (D) THP1 cells were infected with TB40E at an MOI of 0.2 as outlined in panel A. At 24 hpi, the expression of IE, E, and L loci, as indicated, in HCMV TB40E-infected THP1 cells pretreated with DMSO or DZnep was analyzed. Data are averages for three biological replicates, and error bars reflect the standard errors of the means of the normalized expression levels. Asterisks indicate P values of <0.05 (*), <0.01 (**), or <0.001 (***) by paired, two-tailed t tests. (E) Western blot analysis of AD169 IE1 protein accumulation in DZnep-treated THP1 cells. THP1 cells were treated as described in the legend to Fig. 1 and were infected with AD169 at an MOI of 1.0. Whole-cell lysates were prepared at 24 hpi, and IE1 protein expression was detected by Western blotting. HP1 served as a loading control.