Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory.
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 fluorescencein situRNA 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.IMPORTANCEB 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.
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.
Christopher Phillip
CP
Chen
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.
Yuanzhi
Y
Lyu
Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, California, USA.
Frank
F
Chuang
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.
Kazushi
K
Nakano
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.
Chie
C
Izumiya
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.|College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, People's Republic of China.
Di
D
Jin
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA.
Mel
M
Campbell
Department of Dermatology, University of California Davis School of Medicine, Sacramento, California, USA yizumiya@ucdavis.edu.|Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Sacramento, California, USA.|University of California Davis Comprehensive Cancer Center, Sacramento, California, USA.
Yoshihiro
Y
Izumiya
D000956:Antigens, Viral / Q000235:genetics; D002467:Cell Nucleus / Q000821:virology; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000235:genetics; D006801:Humans; D021621:Imaging, Three-Dimensional; D017874:Immediate-Early Proteins / Q000235:genetics*; D007438:Introns; D012319:RNA Polymerase II / Q000378:metabolism*; D012514:Sarcoma, Kaposi / Q000821:virology*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics; D017735:Virus Latency / Q000235:genetics; D014779:Virus Replication
D000956:Antigens, Viral; D017874:Immediate-Early Proteins; D014764:Viral Proteins; D012319:RNA Polymerase II
KSHV; Kaposi's sarcoma-associated herpesvirus; RNA polymerase II; reactivation; regulation of gene expression; transcription; transcriptional factory