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. 2017 May 12;91(11):e02491-16.
doi: 10.1128/JVI.02491-16. Print 2017 Jun 1.

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

Christopher Phillip Chen  1 , Yuanzhi Lyu  1 , Frank Chuang  2 , Kazushi Nakano  1 , Chie Izumiya  1 , Di Jin  1   3 , Mel Campbell  1 , Yoshihiro Izumiya  4   2   5
Affiliations

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

Christopher Phillip Chen et al. J Virol. .

Abstract

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

Keywords: KSHV; Kaposi's sarcoma-associated herpesvirus; RNA polymerase II; reactivation; regulation of gene expression; transcription; transcriptional factory.

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Figures

FIG 1
FIG 1
Establishment of immune-FISH approach. (A) Design of RNA-FISH probes. Fluorescence-labeled DNA oligonucleotides were designed to hybridize to the intron region of the K-Rta transcript. Premature and mature mRNA encoding K-Rta are illustrated. ORFs located around ORF50 (K-Rta) are also depicted. (B) Visualization of active episomes in situ. Unsynchronized BCBL-1 cells were treated with TPA (20 ng/ml) for 24 h and stained by immune-FISH. LANA was stained with specific antibody and K-Rta transcripts were visualized by fluorescent labeled oligonucleotides. A phase-contrast image is also shown. (C) Multicolor fluorescence image of BCBL-1 cells viewed in 3D perspective. Reactivation was induced by TPA (20 nM) for 24 h. White arrows indicate reactivating cells. Green, LANA; red, K-Rta RNA.
FIG 2
FIG 2
Hijacking cellular RNA Pol II. Cellular RNA Pol II (green) colocalizes with K-Rta RNA (red). The indicated PEL cells were stimulated by a mixture of TPA (20 nM) with sodium butyrate (1 mM) for 4 h and stained at 24 h after the end of stimulation. DNA was counterstained with DAPI. Areas defined by rectangles in the merge + DAPI images are enlarged at the far right.
FIG 3
FIG 3
The transcriptional factory predominantly colocalizes with the RNA Pol II p-S5 form. Immune-FISH was performed with phospho-specific RNA Pol II antibodies. KSHV reactivation was induced in TREx-K-Rta BCBL-1 by combination of Dox and TPA for 4 h, and slides were prepared after 24 h after the end of the stimulation. KSHV transcribing sites were marked by RNA-FISH with K-Rta intron probes.
FIG 4
FIG 4
Degradation of RNA Pol II by the proteasome in KSHV-reactivating cells. (A) KSHV reactivation in synchronized TREx-K-Rta BCBL-1 after treatment with a combination of TPA and doxycycline. The cell cycle was synchronized with a double thymidine block, and KSHV reactivation was induced by a combination of Dox (1 μg/ml) and TPA (20 ng/ml). The indicated molecules were stained with immune-FISH at 28 h postinduction. (B) Immunoblotting. TREx-K-Rta BCBL-1 cells were reactivated by TPA and Dox for 4 h after cell cycle synchronization. Cell lysates were prepared at different time points after reactivation, and 50-μg portions of total cell lysates were subjected to immunoblotting. The indicated proteins were probed with specific antibodies. No Dox, no KSHV reactivated cells; Dox & TPA, reactivated by TPA and Dox for 4 h. (C) Proteasome-mediated degradation of RNA Pol II. (a) TREx-K-Rta BCBL-1 cells were reactivated by TPA and Dox for 4 h after cell cycle synchronization. At 28 h after stimulation, MG132 (final concentration, 10 μM) was added to the culture media and remained another 20 h. The indicated proteins were probed with specific antibodies. (b) Bortezomib (final concentration, 16 nM) was added to the culture media after stimulation of KSHV reactivation for 4 h and treatment lasted another 44 h. At 48 h poststimulation, the cells were harvested, and cell lysates were prepared. The indicated proteins were probed with specific antibody.
FIG 5
FIG 5
Biased shifting of RNA Pol II onto viral genomes during reactivation. (A and B) Cellular (A) and viral (B) gene expression. TREx-K-Rta BCBL-1 cells were reacted by TPA and Dox for 4 h after cell cycle synchronization. The indicated cellular or viral gene expression was measured by qRT-PCR. rRNA 18S, which is transcribed by RNA Pol I, was used for normalization. The values of gene-normalized expression at the indicated time points are shown in the bar graph. Blue, No Dox; red, Dox and TPA. (C) Chromatin immunoprecipitation. RNA Pol II occupancies at cellular or viral promoters were determined before and during reactivation (i.e., at 28 h postreactivation). ChIP assays were performed with the indicated genomic regions with specific primers. The relative enrichment over input DNA is shown.
FIG 6
FIG 6
(A) RNA-FISH showing the assembly of LANA dots with RNA Pol II in BCBL-1. RNA Pol II (red immunofluorescence), LANA (green immunofluorescence), and K-Rta RNA (light blue, RNA-FISH) were visualized. BCBL-1 was stimulated with TPA and sodium butyrate for 4 h. Cells were fixed after 24 h after the end of stimulation. Merge and enlarged merge images overlaid with K-Rta RNA staining are shown. (B) 3D view of KSHV transcriptional factory. Z-stack images were taken, and 3D images without or with DAPI staining were constructed. Green, LANA; red, RNA Pol II; orange, K-Rta transcripts; blue, DAPI.
FIG 7
FIG 7
DNA replication and transcriptional factory formation. (A) Colocalization between single-stranded viral DNA and RNA Pol II. The indicated molecules were probed with either antibody or fluorescent oligonucleotide probes. Merged images: green, replicating KSHV genomes; red, RNA Pol II; blue, DAPI. (B) Transcriptional factory formation in presence or absence of thymidine. TREx-K-Rta BCBL-1 cells were reactivated in the presence or absence of thymidine. The aggregation of RNA Pol II is shown. (C) Pictures of five randomly selected fields were taken, and cells with complete colocalization of all three factors (RNA Pol II, LANA, and K-Rta RNA similar to Fig. 6) were counted as transcriptional factories. The numerical results are summarized in panel C.
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