Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

University of California, Davis

Replicazione virale successo dipende da un ambiente favorevole cellulare; cos&igrave;, il virus deve essere sensibile allo stato delle loro cellule ospiti. Abbiamo esaminato l&#39;idea che un&#39;interazione tra fattori virali e cellulari normativi determina l&#39;interruttore da latenza herpesvirus associato al sarcoma di Kaposi (KSHV) replica litico. Il prodotto del gene immediato inizio K-Rta &egrave; la prima proteina virale espressa e un fattore essenziale nella riattivazione; di conseguenza, questa proteina virale &egrave; in una posizione chiave per servire come un sensore virale di fisiologia cellulare. Il nostro approccio mira a definire un fattore di trascrizione di host, cio&egrave;, sensore di host, che modula l&#39;attivit&agrave; dei K-Rta su promotori virali. A tal fine, abbiamo sviluppato un pannello di plasmidi reporter contenente tutte le 83 promotori virali putative per un&#39;indagine completa della risposta sia K-Rta e fattori di trascrizione cellulari. &Egrave; interessante notare che, membri della famiglia NF-kappaB hanno dimostrati di essere forti regolatori negativi di transattivazione K-Rta per tutti, ma due promotori virali (Ori-RNA e K12). Assunzione di K-Rta i promotori di ORF57 e K-bZIP, ma non il promotore K12, era notevolmente compromessa quando espressione di NF-kappaB &egrave; stata indotta. Molti promotori K-Rta-reattiva modulate da NF-kappaB contengono la sequenza del sito di legame di RBP-Jkappa, un coactivator principali che ancore K-Rta ai promotori di destinazione tramite motivi di consenso che si sovrappongono con quella di NF-kappaB. Gel shift analisi hanno dimostrato che la NF-kappaB inibisce il legame di RBP-Jkappa e forma un complesso con RBP-Jkappa. I nostri risultati supportano un modello in cui un equilibrio tra attivit&agrave; di NF-kappaB e K-Rta/RBP-Jkappa determina la riattivazione KSHV. Un&#39;importante caratteristica di questo modello &egrave; che l&#39;interazione tra RBP-Jkappa e NF-kappaB su promotori virali controlla l&#39;espressione di gene virale mediata da K-Rta.

NF-kappaB serve come un sensore cellulare di latenza associato al sarcoma di Kaposi herpesvirus e regola negativamente K-Rta di inimicarsi il coactivator RBP-Jkappa.

Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-kappaB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-kappaB expression was induced. Many K-Rta-responsive promoters modulated by NF-kappaB contain the sequence of the RBP-Jkappa binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-kappaB. Gel shift assays demonstrated that NF-kappaB inhibits the binding of RBP-Jkappa and forms a complex with RBP-Jkappa. Our results support a model in which a balance between K-Rta/RBP-Jkappa and NF-kappaB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jkappa and NF-kappaB on viral promoters controls viral gene expression mediated by K-Rta.

NF-kappaB serves as a cellular sensor of Kaposi's sarcoma-associated herpesvirus latency and negatively regulates K-Rta by antagonizing the RBP-Jkappa coactivator.

R&eacute;ussite de la r&eacute;plication virale est tributaire d&#39;un environnement cellulaire favorable ; ainsi, les virus doivent &ecirc;tre sensibles &agrave; l&#39;&eacute;tat de leurs cellules de l&#39;h&ocirc;te. Nous avons examin&eacute; l&#39;id&eacute;e qu&#39;une interaction entre des facteurs de r&eacute;gulation virus et cellulaires d&eacute;termine le passage de la latence des herp&egrave;svirus associ&eacute; au sarcome de Kaposi (KSHV) &agrave; r&eacute;plication lytique. Le produit de g&egrave;ne pr&eacute;coce imm&eacute;diat K-Rta est la premi&egrave;re prot&eacute;ine virale exprim&eacute;e et un facteur essentiel de r&eacute;activation ; en cons&eacute;quence, cette prot&eacute;ine virale est dans une position cl&eacute; pour servir un capteur viral de la physiologie cellulaire. Notre approche visant &agrave; d&eacute;finir un facteur de transcription h&ocirc;te, c&#39;est-&agrave;-dire, le capteur de h&ocirc;te, qui module l&#39;activit&eacute; de K-Rta sur promoteurs viraux. &Agrave; cette fin, nous avons d&eacute;velopp&eacute; un panel de plasmides journaliste contenant tous les promoteurs viraux putatifs 83 pour une &eacute;tude compl&egrave;te de la r&eacute;ponse &agrave; la fois K-Rta et les facteurs de transcription cellulaire. Fait int&eacute;ressant, les membres de la famille NF-kappaB se sont av&eacute;r&eacute;s &ecirc;tre des r&eacute;gulateurs n&eacute;gatifs forts de transactivation de K-Rta pour tous mais deux promoteurs viraux (Ori-RNA et K12). Recrutement de K-Rta pour les promoteurs des ORF57 et K-bZIP, mais pas le promoteur K12, a &eacute;t&eacute; consid&eacute;rablement affaiblie lorsque expression de NF-kappaB est induite. Nombreux promoteurs K-Rta-sensible, modul&eacute;s par le NF-kappaB contient la s&eacute;quence de l&#39;accepteur RBP-Jkappa, un co-activateur majeur qui ancre K-Rta aux promoteurs de cible via les motifs d&#39;un consensus qui recoupent celle de NF-kappaB. Gel Maj essais ont d&eacute;montr&eacute; que le NF-kappaB inhibe la liaison du RBP-Jkappa et forme un complexe avec la RBP-Jkappa. Nos r&eacute;sultats supportent un mod&egrave;le dans lequel un &eacute;quilibre entre K-Rta/RBP-Jkappa et activit&eacute;s de NF-kappaB d&eacute;termine r&eacute;activation KSHV. Une caract&eacute;ristique importante de ce mod&egrave;le est que l&#39;interaction entre la RBP-Jkappa et NF-kappaB sur promoteurs viraux contr&ocirc;le l&#39;expression de g&egrave;nes viraux m&eacute;di&eacute;e par K-Rta.

NF-kappaB sert un capteur cellulaire de latence des herp&egrave;svirus associ&eacute; au sarcome de Kaposi et r&eacute;gule n&eacute;gativement K-Rta de contrarier le co-activateur RBP-Jkappa.

The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.

Kaposi's sarcoma-associated herpesvirus K-Rta exhibits SUMO-targeting ubiquitin ligase (STUbL) like activity and is essential for viral reactivation.

The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb C(term)) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb C(term) phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4-E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

PRMT4-mediated arginine methylation negatively regulates retinoblastoma tumor suppressor protein and promotes E2F-1 dissociation.

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is a γ-herpesvirus linked to Kaposi's sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL or body-cavity B-lymphoma [BCBL]) and a subset of Multicentric Castleman's Disease. During lytic growth, pervasive viral transcription generating a variety of transcripts with uncertain protein-coding potential has been described on a genome-wide scale in β- and γ-herpesviruses. One class of such RNAs is called long non-coding RNAs (lncRNAs). KSHV encodes a viral lncRNA known as polyadenylated nuclear RNA (PAN RNA), a copious early gene product. PAN RNA has been implicated in KSHV gene expression, replication, and immune modulation. PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program. Latent KSHV episomes are coated with viral latency-associated nuclear antigen (LANA). LANA rapidly dissociates from episomes during reactivation. Here we review recent studies suggesting that PAN RNA may function as a viral lncRNA, including a role in the facilitation of LANA-episomal dissociation during lytic replication.

Long non-coding RNA and epigenetic gene regulation of KSHV.

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.

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

Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression while H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are "poised" to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function, thus we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication.Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta, and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate-early and early gene cluster regions of the KSHV genome, contributed to tethering Polycomb Repressive Complex 2 though physical interaction thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted balance of bivalent histone modifications towards more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.IMPORTANCE Post-translational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, an essential transcriptional factor for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts and maintains Polycomb Repressor Complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2 mediated local histone modification.

ZIC2 is Essential for Maintenance of Latency and is a Target of an Immediate-Early Protein during KSHV Lytic Reactivation.

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Currently, treatment options for patients with PEL are limited. Oncolytic viruses have been engineered as anticancer agents and have recently shown increased therapeutic promise. Similarly, lytic activation of endogenous viruses from latently infected tumor cells can also be applied as a cancer therapy. In theory, such a therapeutic strategy would induce oncolysis by viral replication, while simultaneously stimulating an immune response to viral lytic cycle antigens. We examined the combination of the FDA-approved drug ingenol-3-angelate (PEP005) with epigenetic drugs as a rational therapeutic approach for KSHV-mediated malignancies. JQ1, a bromodomain and extra terminal (BET) protein inhibitor, in combination with PEP005, not only robustly induced KSHV lytic replication, but also inhibited IL6 production from PEL cells. Using the dosages of these agents that were found to be effective in reactivating HIV (as a means to clear latent virus with highly active antiretroviral therapy), we were able to inhibit PEL growth in vitro and delay tumor growth in a PEL xenograft tumor model. KSHV reactivation was mediated by activation of the NF-κB pathway by PEP005, which led to increased occupancy of RNA polymerase II onto the KSHV genome. RNA-sequencing analysis further revealed cellular targets of PEP005, JQ1, and the synergistic effects of both. Thus, combination of PEP005 with a BET inhibitor may be considered as a rational therapeutic approach for the treatment of PEL. Mol Cancer Ther; 16(11); 2627-38. ©2017 AACR.

Oncolytic Reactivation of KSHV as a Therapeutic Approach for Primary Effusion Lymphoma.

The three-dimensional structure of chromatin organized by genomic loops facilitates RNA polymerase II access to distal promoters. The Kaposi's sarcoma-associated herpesvirus (KSHV) lytic transcriptional program is initiated by a single viral transactivator, K-Rta. Here we report the KSHV genomic structure and its relationship with K-Rta recruitment sites using Capture Hi-C analyses. High-resolution 3D viral genomic maps identify a number of direct physical, long-range, and dynamic genomic interactions. Mutant KSHV chromosomes harboring point mutations in the K-Rta responsive elements (RE) significantly attenuate not only the directly proximate downstream gene, but also distal gene expression in a domain-specific manner. Genomic loops increase in the presence of K-Rta, while abrogation of K-Rta binding impairs the formation of inducible genomic loops, decreases the expression of genes networked through the looping, and diminishes KSHV replication. Our study demonstrates that genomic architectural dynamics plays an essential role in herpesvirus gene expression.

Yoshihiro Izumiya

Department of Dermatology,
University of California Davis School of Medicine,
Department of Dermatology, University of California Davis School of Medicine

University of California, Davis

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Yoshihiro Izumiya

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Dermatology,University of California Davis School of Medicine,Department of Dermatology, University of California Davis School of Medicine

University of California, Davis

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Department of Dermatology,
University of California Davis School of Medicine,
Department of Dermatology, University of California Davis School of Medicine