Figure 1.
Total RNAP II levels decrease during WT HSV-1 KOS infection.
HeLa cells were either mock infected or infected with WT HSV-1 KOS or with an ICP27 null mutant, 27-GFP in which the ICP27 coding sequence was replaced with GFP. Cells lysates were prepared at 1, 3, 5, 7 and 9 h after infection and separated on a 5-15% linear gradient SDS-polyacrylamide gel. Western blots were probed with polyclonal antibody N20, which detects all forms of RNAP II The cellular protein YY1 served as the loading control.
Figure 2.
RNAP II CTD phosphoserine-2 levels decreased with increasing amounts of DRB.
HeLa cells were mock infected or infected with WT HSV-1 KOS at an MOI of 10. DRB stocks were prepared in Dimethyl formamide. At 1, 3 and 5 h after infection, Dimethyl formamide alone (D) or increasing amounts of DRB from 0 to 100µµM were added to cell monolayers. At 8 h after infection, whole cell lysates were prepared and separated on 5-15% linear gradient SDS-polyacrylamide gels. Western blots were probed with polyclonal antibody N20, which detects all forms of RNAP II; monoclonal antibody H5, which detects phosphoserine-2, and monoclonal antibody H14, which detects phosphoserine-5. For HSV-1 proteins, antibody P1011 was used to detect IE protein ICP4, P1119 for ICP27, P1123 for gB and P1103 for gD. Lamin A/C served as a sample loading control.
Figure 3.
Addition of FVP reduced RNAP II CTD phosphoserine-2 levels and levels of HSV-1 IE proteins ICP4 and ICP27 and late proteins gB and gD were also reduced.
A) HeLa cells were mock infected or infected with WT HSV-1 KOS at MOI of 10. . Flavopiridol (FVP) stocks were prepared in DMSO. At 1 h after infection, DMSO alone or increasing amounts of FVP from 0 to 450 nM were added to cell monolayers. At 8 h after infection, whole cell lysates were prepared and fractionated on 5-15% gradient SDS-polyacrylamide gels. Western blots were probed with N20, H5 and H14 as indicated. Lamin A/C served as the loading control. B) DMSO alone was added at 0 h after infection or FVP (450 nM) was added at 1, 3 or 5 h after infection as indicated. Whole cell lysates were prepared at 8 h. Western blots were probed with anti-RNAP II antibody N20 or phosphoserine-2 antibody H5 or phosphoserine-5 antibody H14. HSV-1 protein ICP4 was detected using antibody P1101; ICP27 was detected with antibody P1119; gB was detected with antibody P1103 and gD was detected with antibody 1123. Lamin A/C served as a loading control.
Figure 4.
DRB and FVP altered the staining pattern of H5 antibody.
A) RSF cells were either mock infected or infected with WT HSV-1 KOS at an MOI of 10. Cells were either left untreated (no drug) or were treated with 100 µM DRB or 450 nM FVP as indicated. Cells were fixed at 8 h and stained with RNAP II antibody ARNA3, which recognizes all forms of RNAP II or phosphoserine-2 antibody H5. For the H5 no drug panels, cells from mock and KOS samples have been shown at higher magnification to show the nuclear staining patterns and are identified by white arrows. B-C) To account for cell toxicity, HeLa cells were either mock infected, or infected with HSV-1 KOS at an MOI of 1 for up to 16 hours as indicated. Infected cells were either untreated or treated with 100 µM DRB or 450 nM FVP. Cells were collected at 4 h intervals, stained with Trypan Blue vital dye, and counted in triplicate using a hemocytometer to estimate B) percent cell death and C) total cell counts.
Figure 5.
DRB and FVP reduced RNA synthesis in mock and HSV-1-infected cells.
RSF cells were mock infected or infected with WT HSV-1 KOS at MOI of 10. At 1 h after infection, cells were treated with DRB (100 µM) or FVP (450 nM). A) Bromouridine (BrU) was added to the media (4 mM) at 7.5 h to label nascent RNA. Cells were fixed at 8 h after infection and stained with anti-BrU antibody (green). Mock infected cells were stained with DAPI to mark nuclei. KOS-infected cells were stained with DAPI and with anti-ICP4 antibody as a marker of infection. B) Cells were fixed at 8 h after infection and in situ hybridization with a biotinylated oligo-dT probe was performed to detect poly(A+) RNA. Mock infected cells were stained with DAPI and KOS infected cells were stained with DAPI and anti-ICP4 antibody as an infection marker. All images were captured on a Zeiss Axiovert 200M microscope at 100X magnification.
Figure 6.
The inhibitory effects of cdk9 inhibiters DRB and FVP on RNA synthesis during HSV-1 infection could be reversed after removing the drugs.
RSF cells were infected with HSV-1 KOS at an MOI of 10. A, D) Infected cells were left untreated. B, E) Infected cells were treated with 100 µM DRB or 450 nM FVP at 3 h after infection. C,F) Infected cells were treated with 100 µM DRB or 450 nM FVP as indicated for the first 4 h of infection after which, cells were washed and drug-free medium was added. A-C). BrU was added to the media at 7.5 h after infection to label nascent RNA. Cells were fixed at 8 h and stained with anti-BrU and anti-ICP4 antibodies and with DAPI. D-F). Cells were fixed at 8 h after infection and in situ hybridization was performed with a biotinylated oligo dT-probe, which was subsequently detected by FITC-conjugated streptavidin. ICP4 staining served as an infection marker and DAPI staining marked nuclei. All images were captured on a Zeiss Axiovert 200M microscope at 100X magnification.
Figure 7.
DRB and FVP hindered HSV-1 transcription-replication compartment formation but pre-replicative compartments were apparent after drug removal.
RSF cells were infected with WT KOS at an MOI of 10. A,D) Infected cells were untreated and were fixed at 4 h and 8 h as indicated. B,E) Cells were treated with 100 µM DRB or 450 nM FVP as indicated at the beginning of infection and were fixed at 8 h. C,F) DRB or FVP were added at the start of infection and were removed at 4 h and cells were incubated in drug-free medium for an additional 4 h. Infected cells were fixed at 8 h. A,B,C). Cells were stained with anti-ICP4 antibody to monitor viral transcription-replication compartment formation and DAPI to mark nuclei. D,E,F). Infected cells were stained with anti-ICP27 antibody to monitor ICP27 sub-cellular localization and DAPI to mark nuclei. Images were captured on a Zeiss Axiovert 200M microscope at 100X magnification.
Figure 8.
A global reduction in HSV-1 mRNA expression was seen in the presence of DRB and FVP.
HeLa cells were infected with HSV-1 KOS at an MOI of 10 and were either left untreated (no drug) or were treated with 100 µM DRB or 450 nM FVP starting at 3 h after infection. Total RNA was isolated at 8 h. Poly(A+) RNA was selected and reverse transcribed and the cDNA from each fraction was hybridized to HSV-1-specific microarray chips and quantified by using Array Vision software. The graph shown displays the averages of three independent experiments in which each transcript was represented three times on the array. The y axis represents the intensity of the light scattering signal and the x axis represents individual HSV-1 transcripts. Red bars represent immediate-early transcripts; early transcripts are in blue; late transcripts are in green, and LAT transcripts are in violet.
Figure 9.
Viral replication was greatly reduced in the presence of DRB and FVP but viral replication resumed when the drugs were removed after 4 h.
HeLa cells were infected with HSV-1 KOS at MOI of 1. A) Infected cells were either untreated (no drugs) or DRB (100 µM) or FVP (450 nM) were added at the time of infection for the duration of infection, or DRB and FVP were present for the first 4 h and then were removed and cells were incubated in drug-free medium for the remainder of the time, as indicated. Samples were harvested at 0, 4, 8, 12 and 16 h after infection and virus titers were determined by plaque assays. B) HeLa cells were infected with KOS at an MOI of 1 and were untreated (no drugs) or treated with DRB or FVP starting at 0 h or 3 h as indicated and the drugs were present for the duration of the experiment. Samples were harvested at 0, 4, 8, 12 and 16 h as described in panel A. The experiments were performed in triplicate and error bars are shown.
Figure 10.
Expression of a dominant-negative kinase-dead cdk9 mutant or the cdk9 negative-regulator HEXIM1 inhibited nascent RNA synthesis.
A) HeLa cells were either transfected with empty vector or with 2 µg of DN-cdk9-HA DNA, which expresses an HA-tagged kinase-dead cdk9 dominant negative mutant. After 24 h, whole cell lysates were prepared and fractionated by SDS-PAGE. Western blots were probed with anti-cdk9 antibody (left panel) or anti-HA antibody (right panel). B) HeLa cells transfected with DN-cdk9-HA were fixed 24 h after transfection and stained with anti-HA antibody to visualize DN-cdk9-HA and DAPI to mark nuclei. C) HeLa cells were transfected with empty vector or with plasmid DNA expressing FLAG-tagged HEXIM1. Whole cell lysates were prepared as in panel A and Western blots were probed with anti-HEXIM1 antibody (left) and anti-FLAG (right). D) Indirect immunofluorescence was performed on HeLa cells transfected with HEXIM1-FLAG. Anti-FLAG antibody was used to visualize HEXIM1-FLAG and DAPI staining marked the nuclei. E) HeLa cells were transfected with DN-cdk9-HA for 24 h and were subsequently mock infected or infected with HSV-1 KOS. Bromouridine (BrU) was added at 7.5 h after infection for 30 min, at which time cells were fixed and stained with anti-BrU antibody to visualize newly transcribed RNA and anti-HA antibody to visualize DN-cdk9-HA. DAPI staining in the upper mock panels marked nuclei and staining with anti-ICP4 antibody in the lower KOS panels was used as a marker for infection. White arrows point to cells expressing DN-cdk9-HA in the BrU and merged panels. F) HeLa cells transfected with HEXIM1-FLAG for 24 h were mock infected or infected with HSV-1 KOS and BrU was added at 7.5 h after infection for 30 min. Cells were fixed at 8 h and stained with anti-BrU to detect newly transcribed RNA and anti-FLAG antibody to detect HEXIM1-FLAG. DAPI was used to mark nuclei in the upper mock panels and anti-ICP4 antibody was used in the lower KOS panels as a marker for infection. White arrows point to cells expressing HEXIM1-FLAG. All images were captured on a Zeiss Axiovert 200M microscope under 100X magnification.