RNA sequence reads for the presence of a selection of exon-exon junctions within HCMV RNA transcripts (). Treatment with XMD7-1 resulted in a general decrease in the number of reads representing junctions, including those encoding IE1 or IE2 (Fig. 3A) at 24, 48 and 72 h.p.i. (Table S7), which likely parallels the reduction in the total number of HCMV sequence reads (Fig. 3C). To ensure that this analysis was not biased by the method of RNA sample preparation, we performed a similar analysis of the same total RNA samples from which poly(A) RNA had been selected. Similar results were obtained in all respects (data not shown). Therefore, the reduction in IE2 proteins in cells treated with XMD7-1 was associated with reduction in RNA transcription across the entire HCMV genome and not with reduction of specific exon-exon junctions required for production of IE2 mRNAs. 3.6 Examination of CDK inhibition by XMD7 compounds. Production of IE2 proteins, but not IE1, is compromised when HCMV infected cells are treated with the broad-spectrum cyclin-dependent kinase (CDK) inhibitor roscovitine (), suggesting that CDK inhibition and cell cycle arrest are related to inhibition of IE2 production. We found that XMD7 compounds inhibited CDK7 (Figs. 2A-2C, indicated with crosses), but not other CDK proteins (Tables S4-S6), and that treatment of uninfected cells with XMD7 compounds had no effect of cell proliferation as determined using MTT assays (data not shown). Therefore, CDK inhibition upon treatment of infected cells with XMD7 compounds was unlikely to be related to the anti-HCMV activity of XMD7 compounds.
RNA sequence reads for the presence of a selection of exon-exon junctions within HCMV RNA transcripts (). Treatment with XMD7-1 resulted in a general decrease in the number of reads representing junctions, including those encoding IE1 or IE2 (Fig. 3A) at 24, 48 and 72 h.p.i. (Table S7), which likely parallels the reduction in the total number of HCMV sequence reads (Fig. 3C). To ensure that this analysis was not biased by the method of RNA sample preparation, we performed a similar analysis of the same total RNA samples from which poly(A) RNA had been selected. Similar results were obtained in all respects (data not shown). Therefore, the reduction in IE2 proteins in cells treated with XMD7-1 was associated with reduction in RNA transcription across the entire HCMV genome and not with reduction of specific exon-exon junctions required for production of IE2 mRNAs. 3.6 Examination of CDK inhibition by XMD7 compounds. Production of IE2 proteins, but not IE1, is compromised when HCMV infected cells are treated with the broad-spectrum cyclin-dependent kinase (CDK) inhibitor roscovitine (), suggesting that CDK inhibition and cell cycle arrest are related to inhibition of IE2 production. We found that XMD7 compounds inhibited CDK7 (Figs. 2A-2C, indicated with crosses), but not other CDK proteins (Tables S4-S6), and that treatment of uninfected cells with XMD7 compounds had no effect of cell proliferation as determined using MTT assays (data not shown). Therefore, CDK inhibition upon treatment of infected cells with XMD7 compounds was unlikely to be related to the anti-HCMV activity of XMD7 compounds.
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