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. 2017 Oct 29;8(57):96536-96552.
doi: 10.18632/oncotarget.22106. eCollection 2017 Nov 14.

Human cytomegalovirus and Herpes Simplex type I virus can engage RNA polymerase I for transcription of immediate early genes

Ourania N Kostopoulou  1 , Vanessa Wilhelmi  1 , Sina Raiss  1 , Sharan Ananthaseshan  1 , Mikael S Lindström  2 , Jiri Bartek  2 , Cecilia Söderberg-Naucler  1
Affiliations

Human cytomegalovirus and Herpes Simplex type I virus can engage RNA polymerase I for transcription of immediate early genes

Ourania N Kostopoulou et al. Oncotarget. .

Abstract

Human cytomegalovirus (HCMV) utilizes RNA polymerase II to transcribe viral genes and produce viral mRNAs. It can specifically target the nucleolus to facilitate viral transcription and translation. As RNA polymerase I (Pol I)-mediated transcription is active in the nucleolus, we investigated the role of Pol I, along with relative contributions of the human Pol II and Pol III, to early phases of viral transcription in HCMV infected cells, compared with Herpes Simplex Virus-1 (HSV-1) and Murine cytomegalovirus (MCMV). Inhibition of Pol I with siRNA or the Pol I inhibitors CX-5461 or Actinomycin D (5nM) resulted in significantly decreased IE and pp65 mRNA and protein levels in human fibroblasts at early times post infection. This initially delayed replication was compensated for later during the replication process, at which stage it didn't significantly affect virus production. Pol I inhibition also reduced HSV-1 ICP0 and gB transcripts, suggesting that some herpesviruses engage Pol I for their early transcription. In contrast, inhibition of Pol I failed to affect MCMV transcription. Collectively, our results contribute to better understanding of the functional interplay between RNA Pol I-mediated nucleolar events and the Herpes viruses, particularly HCMV whose pathogenic impact ranges from congenital malformations and potentially deadly infections among immunosuppressed patients, up to HCMV's emerging oncomodulatory role in human tumors.

Keywords: Herpes Simplex type 1 virus; RNA Polymerase I; cytomegalovirus; nucleolus; replication.

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Conflict of interest statement

CONFLICTS OF INTEREST There is no conflict of interest.

Figures

Figure 1
Figure 1. Effect of Pol I inhibition by CX-5461 on the HCMV immediate early (IE) and pp65 proteins in HCMV infected MRC5 cells
A. Representative images of immunofluorescent staining (IF) for HCMV IE (green on the left column) and pp65 (green on the right column) proteins of pre-treated HCMV infected MRC5 cells. The cells were treated for 1.5h with 0.1μΜ, 1μΜ CX-5461, 5nM ActD or 1mM GCV and then HCMV (VR1814) infected for 48h. Actinomycin D (ActD) was used as control drug for Pol I inhibition and Ganciclovir (GCV) as antiviral control drug. UBF was stained as control for nucleolus. Nuclei were stained with DAPI (blue). B. Quantification of the percent of the IE and pp65 positive untreated and treated HCMV infected cells by IF (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). C. Protein levels of HCMV IE (IE1:IE72, IE2:IE86) and pp65 were detected by WB analysis in untreated and pre-treated HCMV infected cells 72hpi. D. Quantification of the density of IE (IE72, IE86) and pp65 bands was performed using Image Studio Lite programme. Data were presented as percent of IE72, IE86 and pp65 levels after normalization to β-actin.
Figure 2
Figure 2
A. Effect of Pol I inhibition by CX-5461 on the HCMV IE (IE1:UL123, IE2:UL122), pp65 (UL83) and gB (UL55) transcripts in MRC5 HCMV infected cells. Cells were pre-treated for 1.5h with 1μΜ CX-5461 or 5nM ActD and then infected with HCMV (VR1814) for 6h, 24h and 48h. ActD was the control drug for Pol I activity. 47S was used as control for Pol I activity. Beta 2-microglobulin (B2M) was the endogenous control. Bars represent mean±SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001) B. Cells were stained for 5-FUrd incorporation using anti-BrdU antibody in order to confirm the inhibitory effect of CX-5461 on rRNA production. Nucleus was stained with DAPI and Fibrillarin was used as nucleolar marker. Representative immunofluorescence images are shown. Fibrillarin (red), BrdU (green), DAPI (blue).
Figure 3
Figure 3. Inhibition of Pol I by siRNA affects 47S and IE (transcripts and proteins) in HCMV infected MRC5 cells
A. Relative expression of IE transcripts in HCMV infected cells transfected with negative control or specific siRNA targeting Pol I for 6h, 24h, 48h determined by qPCR. B2M was used as endogenous control. B. Representative images of transfected (with negative control and siRNA targeting Pol I) and HCMV infected cells for 24h and 72h. Quantification of IE positive HCMV infected cells. C. Protein levels of IE (IE72 and IE86) in transfected, HCMV infected cells were determined by WB analysis and D) quantified using Image Studio Lite programme. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001
Figure 4
Figure 4. Percent of relative expression of IE and 47S transcripts in non treated treated, non synchronized and synchronized HCMV infected MRC5 cells 6h, 24h, 48hpi
*p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001
Figure 5
Figure 5. Effect of Pol I inhibition by CX-5461 on the HCMV IE and pp65 proteins and transcripts in untreated and pre-treated HCMV infected HUVEC cells
Protein analysis was performed by A. IF staining (IE in first column and pp65 in second column: green and Dapi: blue) and B. by WB analysis. C. Percent of relative expression of IE and pp65 transcripts in pre-treated HCMV infected cells. 47S was the control for CX-5461 activity. The percent of each graph is the average percentage of three independent experiments. Bars represent mean±SD, *p < 0.05. NI: Non-infected, NT: Non-treated
Figure 6
Figure 6. Production of infectious HCMV is not affected by Pol I inhibition at late time post infection
The picture depicts percent of IE positive cells after transferring supernatants from treated HCMV infected cells to MRC5.
Figure 7
Figure 7. Effect of the Pol I inhibition by CX-5461 on HSV-1 infected MRC5 cells
A. Percent of relative expression of immediate early (ICP0) and gB transcripts of HSV-1 infected (6h, 24h, 48h) cells pre-treated for 1.5h with 1μΜ CX-5461 or 1mM GCV. 47S transcript was used as control of the Pol I inhibitory effect of CX-5461. B. C. Infected cell protein 0 (ICP0) levels in pre-treated HSV-1 infected cells, were detected by WB (quantitative analysis of three independent experiments) and D. IF staining (IE: green, Dapi: blue).
Figure 8
Figure 8. Effect of CX-5461 on MCMV infected 3T3 cells
A. Relative expression (%) of IE1 and IE3 MCMV transcripts 6h, 24hpi. Bars represent mean±SD (n = 3). B. Representative images of immunofluorescent staining for MCMV IE1 (green) in untreated and pre-treated with 0.1 μM or 1μM CX-5461 or 1mM GCV MCMV infected 3T3 cells 48hpi. Nucleus was stained with DAPI (blue) UBF and as nucleolar marker was used (red). C. Protein levels of MCMV IE1 were determined also by WB at 48hpi. Vinculin was used as loading control.
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