Figure 1.
Identification of pUL79 interacting proteins.
(A) Schematic diagram for creating pADflagUL79, the recombinant HCMV BAC clone used to produce virus ADflagUL79. A cassette that contained a 3×FLAG tag followed by the FRT-bracketed GalK/kanamycin dual selection marker was amplified by PCR and recombined into the wildtype HCMV BAC clone (pADwt) at the 5′ terminus of the UL79 coding sequence. The selection marker was then removed by Flp/FRT recombination. The final clone, pADflagUL79, carried the UL79 coding sequence tagged at its 5′ terminus with 3×FLAG. (B) Single step viral growth analysis. HFF cells were infected with HCMV recombinant virus ADflagUL79 (derived from pADflagUL79) or ADwt (derived from pADwt) at an MOI of 3. Infected culture supernatants were collected at indicated days post infection and virus titers were determined by TCID50 assay. The mean virus titers were derived from two independent experiments and two technical replicates. Standard deviations are presented. The detection limit is indicated by the dashed line. (C) Viral protein expression profile. HFFs were infected as described in (B), and harvested at indicated times post infection. Accumulations of host and viral proteins were determined by immunoblot analysis. FLAG-tagged pUL79 was detected by an anti-FLAG antibody. Actin was used as a loading control. Representative results from three independent experiments are shown. (D) Polyacrylamide gel electrophoresis to resolve pUL79 protein complexes. HFFs were infected as described in (B), and at 72 hpi, cell lysates were prepared for immunoprecipitation using an anti-FLAG antibody. Immunoprecipitated proteins were resolved on a gradient polyacrylamide gel and silver stained. Protein bands containing RNAP II subunits identified by mass spectrometry are indicated. Molecular size markers (in kilodaltons) are shown.
Table 1.
pUL79 protein partners identified by mass spectrometry.
Figure 2.
pUL79 interacts with the RNAP II protein complex.
In (A–B), HFFs were infected as described in Fig. 1, and at 72 hpi cell lysates were immunoprecipitated using either an anti-FLAG antibody (A) or anti-Rpb1 antibody N-20 (B). Immunoprecipitated proteins and lysate inputs were analyzed by immunoblotting. To examine the efficiency of nuclease digestion, the immunoprecipitated samples were also analyzed on an ethidium bromide (EtBr)-stained agarose gel. In (C–D), nuclear lysates from HEK-293T cells transiently expressing HA-tagged pUL79 or empty vector control were prepared at 72 hours post transfection. Lysates were immunoprecipitated using either an anti-HA antibody (C) or anti-Rpb1 antibody 8WG16 (D). Immunoprecipitated proteins and lysate inputs were analyzed by immunoblotting. The clone names of antibodies used in immunoblot analysis are shown. Representative results from three independent experiments are presented.
Figure 3.
pUL79 does not alter protein accumulations of RNAP II.
HFFs were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shield-1 (Shld1). Cells were harvested at different times post infection and protein accumulation was analyzed by immunoblot analysis with antibodies recognizing various subunits and isoforms of RNAP II, cellular CTD kinases (cyclin T1, CDK9), or viral proteins (immediate-early protein IE1, early-late protein pUL44, late protein pp71). The protein accumulation of the ddFKBP tagged pUL79 was monitored by an antibody recognizing the FKBP-epitope. Representative results from three independent experiments are shown.
Figure 4.
pUL79 alters RNAP II occupancy at viral loci.
(A) Schematic representation of the HCMV genes and host GAPDH gene examined by chromatin immunoprecipitation assay (ChIP). Locations and sizes of primer-probe pairs used in ChIP-qPCR analysis are indicated. (B) HFF cells were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shld1. Cell extracts were prepared at 72 hpi and analyzed by ChIP assay using rabbit an anti-RNAP II antibody N-20. Normal rabbit IgG was included as a control for non-specific immunoprecipitation. Amounts of input and precipitated (output) DNAs were quantified by qPCR with primers specific for indicated viral loci or human GAPDH. The output-to-input DNA ratios were determined from four independent ChIP experiments with standard deviations calculated by Prism 6 software. Statistical analysis was performed using Student's t test (**, P<0.01; ***, P<0.005; ****, P<0.0001; NS, not significant). (C) HFF cells were infected with ADflagUL79 or ADwt at an MOI of 3. Cell extracts were prepared at 72 hpi and analyzed by ChIP assay using anti-FLAG antibody. Normal mouse IgG was included as a control. Amount of input and precipitated (output) DNAs were quantified by qPCR with primers used in (B). The output-to-input DNA ratios were determined from three independent ChIP experiments with standard deviations calculated by Prism 6 software. Statistical analysis was performed using Student's t test (*, P<0.05; **, P<0.01).
Figure 5.
pUL79 does not alter a particular phosphorylated form of the RNAP II CTD domain.
HFF cells were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shld1. Cell extracts were harvested at 72 hpi and analyzed by ChIP assays. Rabbit antibody to pSer2 CTD, rat antibody to pSer5-CTD, and mouse antibody to non-phosphorylated CTD (8WG16) were used in ChIP assays. Normal rabbit, rat, and mouse IgGs were included as controls for non-specific precipitation, respectively. Immunoprecipitated DNAs were analyzed as described in Fig. 4B and the output-to-input DNA ratios are presented in (A). In addition, the immunoprecipitated amount of each phosphor-isoform of RNAP II CTD relative to that of total RNAP II (immunoprecipitated with antibody N-20) was also calculated and presented in (B). Data from four independent experiments were collected with standard deviations calculated by Prism 6 software. Statistical analysis was performed using Student's t test (*, P<0.05; **, P<0.01; ***, P<0.005).
Figure 6.
pUL79 alters the rate of transcriptional elongation at viral loci.
(A) Schematic representation of the HCMV genes and host genes examined by the nuclear run-on (NRO) assay. Locations and sizes of primer-probe pairs used in subsequent RT-qPCR analysis are indicated. (B–D) HFFs were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shld1 (indicated by “+” or “−” sign, respectively). Nuclear extracts were prepared at 24 or 72 hpi and analyzed by NRO assays. Transcription elongation was allowed to resume for 30 minutes in the presence of biotin-labeled UTP, labeled RNA was isolated, and their amounts were determined by RT-qPCR. In addition, accumulations of total RNAs were also determined by RT-qPCR. The normalized amounts of viral run-on transcripts or total transcripts in the presence of Shld1 were set at 1 for the NRO assay or total transcript accumulation analysis, respectively. (B) Relative amounts of total and run-on transcripts of viral late genes UL99 and UL32. (C) Relative amounts of run-on and total transcripts of viral immediate-early and early genes. (D) Relative amounts of run-on and total transcripts of host genes. Data from three independent experiments were collected and standard deviations were calculated by Prism 6 software. Statistical analysis was performed using Student's t test (**, P<0.01; ***, P<0.005; NS, not significant).
Figure 7.
pUL79 alters the rate of transcriptional elongation at various regions of the UL48 gene.
(A) Schematic representation of the HCMV gene UL48. Primer-probe pairs used for analysis are indicated. (B–D) HFF cells were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shld1. Cell extracts were harvested at 72 hpi and analyzed by ChIP assays as described in Fig. 4 and Fig. 5. (B) represents the output-to-input DNA ratios of RNAP II ChIP. (C) represents the output-to-input DNA ratios of pSer5-CTD, pSer2-CTD, and non-phosphorylated CTD ChIPs. (D) represents the immunoprecipitated amount of each phosphor-isoform of RNAP II CTD relative to that of total RNAP II (immunoprecipitated with antibody N-20). Data from four independent experiments were collected with standard deviations calculated by Prism 6 software. (E–F) HFFs were infected with ADddUL79 at an MOI of 3 in the presence or absence of 1 µM Shld1 (indicated by “pUL79+” or “pUL79−”, respectively). Nuclear extracts were prepared at 72 hpi and analyzed by NRO assay as described in Fig. 6. Relative amounts of total transcripts and run-on are presented in (E) and (F), respectively. Statistical analysis was performed using Student's t test (*, P<0.05; **, P<0.01; ***, P<0.005).
Figure 8.
Potential role of pUL79 in RNAP II-mediated viral transcription.
During late times of viral infection where pUL79 is expressed, we propose two models where pUL79 may act as an elongation factor to facilitate viral transcription. (A) In the “promoter clearance” model, pUL87, pUL92, pUL95, and potentially other viral factors (shown as red dashed circles) form viral protein pre-initiation complexes (vPIC) to recruit RNAP II to viral promoters. Once transcription initiates, pUL79 interacts with the vPIC to release RNAP II from the vPIC for efficient elongation. In the absence of pUL79, RNAP II is unable to dissociate from vPIC and fails to recruit elongation factors for continued transcription. (B) In the “epigenetic reader” model, pUL79 acts as an epigenetic reader to recognize chromatin modification(s) to facilitate RNAP II elongation. During late times of infection, newly synthesized viral DNA is wrapped with specific histone modifications (shown as purple dashed ovals). pUL79 recognizes these modifications, and then dissociates viral DNA from chromatin binding, with or without other cellular/viral factors, to facilitate RNAP II elongation. In the absence of pUL79, RNAP II is unable to pass through the unopened chromatin, resulting in transcriptional stalling on viral loci.