Slug, a Stress-Induced Transcription Factor, Stimulates Herpes Simplex Virus 1 Replication and Transactivates a cis-Regulatory Module within the VP16 Promoter.

Stress-mediated activation of the glucocorticoid receptor (GR) and specific stress-induced transcription factors stimulate herpes simplex virus 1 (HSV-1) productive infection, explant-induced reactivation, and immediate early (IE) promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Several published studies concluded the virion tegument protein VP16, ICP0, and/or ICP4 drives early steps of reactivation from latency. Notably, VP16 protein expression was induced in trigeminal ganglionic neurons of Swiss Webster or C57BL/6J mice during early stages of stress-induced reactivation. If VP16 mediates reactivation, we hypothesized stress-induced cellular transcription factors would stimulate its expression. To address this hypothesis, we tested whether stress-induced transcription factors transactivate a VP16 cis-regulatory module (CRM) located upstream of the VP16 TATA box (-249 to -30). Initial studies revealed the VP16 CRM cis-activated a minimal promoter more efficiently in mouse neuroblastoma cells (Neuro-2A) than mouse fibroblasts (NIH-3T3). GR and Slug, a stress-induced transcription factor that binds enhancer boxes (E-boxes), were the only stress-induced transcription factors examined that transactivated the VP16 CRM construct. GR- and Slug-mediated transactivation was reduced to basal levels when the E-box, two 1/2 GR response elements (GREs), or NF-κB binding site was mutated. Previous studies revealed GR and Slug cooperatively transactivated the ICP4 CRM, but not ICP0 or ICP27. Silencing of Slug expression in Neuro-2A cells significantly reduced viral replication, indicating Slug-mediated transactivation of ICP4 and VP16 CRM activity correlates with enhanced viral replication and reactivation from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency. Viral regulatory proteins are not abundantly expressed during latency, indicating cellular transcription factors mediate early stages of reactivation. Notably, the glucocorticoid receptor (GR) and certain stress-induced transcription factors transactivate cis-regulatory modules (CRMs) essential for expression of infected cell protein 0 (ICP0) and ICP4, key viral transcriptional regulatory proteins linked to triggering reactivation from latency. Virion protein 16 (VP16) specifically transactivates IE promoter and was also reported to mediate early stages of reactivation from latency. GR and Slug, a stress-induced enhancer box (E-box) binding protein, transactivate a minimal promoter downstream of VP16 CRM, and these transcription factors occupy VP16 CRM sequences in transfected cells. Notably, Slug stimulates viral replication in mouse neuroblastoma cells suggesting Slug, by virtue of transactivating VP16 and ICP4 CRM sequences, can trigger reactivation in certain neurons.

Pparg signaling controls bladder cancer subtype and immune exclusion.

Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.

Alphaherpesvirus Latency and Reactivation with a Focus on Herpes Simplex Virus.

We are at an interesting time in the understanding of alpha herpesvirus latency and reactivation and their implications to human disease. Conceptual advances have come from both animal and neuronal culture models. This review focuses on the concept that the tegument protein and viral transactivator VP16 plays a major role in the transition from latency to the lytic cycle. During acute infection, regulation of VP16 transactivation balances spread in the nervous system, establishment of latent infections and virulence. Reactivation is dependent on this transactivator to drive entry into the lytic cycle. In vivo de novo expression of VP16 protein is mediated by sequences conferring pre-immediate early transcription embedded in the normally leaky late promoter. In vitro, alternate mechanisms regulating VP16 expression in the context of latency have come from the SCG neuron culture model and include the concepts that (i) generalized transcriptional derepression of the viral genome and sequestration of VP16 in the cytoplasm for ~48 hours (Phase I) precedes and is required for VP16-dependent reactivation (Phase II); and (ii) a histone methyl/phospho switch during Phase I is required for Phase II reactivation. The challenge to the field is reconciling these data into a unified model of virus reactivation. The task of compiling this review was uncomfortably humbling, as if cataloging the stars in the universe. While not completely dark, our night sky is missing a multitude of studies which are among the many points of light contributing to our field. This article is a focused review in which we discuss from the vantage point of our expertise, just a handful of concepts that have or are emerging. A lookback at some of the pioneering work that grounds our field is also included.

Typical gene expression profile of pseudorabies virus reactivation from latency in swine trigeminal ganglion.

Pseudorabies virus (PRV) establishes a lifelong latent infection in swine trigeminal ganglion (TG) following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Muscle injection combined with intravenous deliver of the synthetic corticosteroid dexamethasone (DEX) consistently induces reactivation from latency in pigs. In this study, PRV-free piglets were infected with PRV. Viral shedding in nasal and ocular swabs demonstrated that PRV infection entered the latent period. The anti-PRV antibody was detected by enzyme-linked immunosorbent assay and the serum neutralization test, which suggested that the PRV could establish latent infection in the presence of humoral immunity. Immunohistochemistry and viral genome detection of TG neurons suggested that PRV was reactivated from latency. Viral gene expressions of IE180, EP0, VP16, and LLT-intron were readily detected at 3-h post-DEX treatment, but gB, a γ1 gene, was not detectable. The differentially expressed phosphorylated proteins of TG neurons were analyzed by ITRAQ coupled with LC-MS/MS, and p-EIF2S2 differentially expression was confirmed by western blot assay. Taken together, our study provides the evidence that typical gene expression in PRV reactivation from latency in TG is disordered compared with known lytic infection in epithelial cells.

Heat-Triggered Remote Control of CRISPR-dCas9 for Tunable Transcriptional Modulation.

CRISPR-associated proteins (Cas) are enabling powerful new approaches to control mammalian cell functions, yet the lack of spatially defined, noninvasive modalities limits their use as biological tools. Here, we integrate thermal gene switches with dCas9 complexes to confer remote control of gene activation and suppression with short pulses of heat. Using a thermal switch constructed from the heat shock protein A6 (HSPA6) locus, we show that a single heat pulse 3-5 °C above basal temperature is sufficient to trigger expression of dCas9 complexes. We demonstrate that dCas9 fused to the transcriptional activator VP64 is functional after heat activation, and, depending on the number of heat pulses, drives transcription of endogenous genes GzmB and CCL21 to levels equivalent to that achieved by a constitutive viral promoter. Across a range of input temperatures, we find that downstream protein expression of GzmB closely correlates with transcript levels (R2 = 0.99). Using dCas9 fused with the transcriptional suppressor KRAB, we show that longitudinal suppression of the reporter d2GFP depends on key thermal input parameters including pulse magnitude, number of pulses, and dose fractionation. In living mice, we extend our study using photothermal heating to spatially target implanted cells to suppress d2GFP in vivo. Our study establishes a noninvasive and targeted approach to harness Cas-based proteins for modulation of gene expression to complement current methods for remote control of cell function.

Heat-shock protein 90α is involved in maintaining the stability of VP16 and VP16-mediated transactivation of α genes from herpes simplex virus-1.

BACKGROUND: Numerous host cellular factors are exploited by viruses to facilitate infection. Our previous studies and those of others have shown heat-shock protein 90 (Hsp90), a cellular molecular chaperone, is involved in herpes simplex virus (HSV)-1 infection. However, the function of the dominant Hsp90 isoform and the relationship between Hsp90 and HSV-1 α genes remain unclear. METHODS AND RESULTS: Hsp90α knockdown or inhibition significantly inhibited the promoter activity of HSV-1 α genes and downregulated virion protein 16(VP16) expression from virus and plasmids. The Hsp90α knockdown-induced suppression of α genes promoter activity and downregulation of α genes was reversed by VP16 overexpression, indicating that Hsp90α is involved in VP16-mediated transcription of HSV-1 α genes. Co-immunoprecipitation experiments indicated that VP16 interacted with Hsp90α through the conserved core domain within VP16. Based on using autophagy inhibitors and the presence of Hsp90 inhibitors in ATG7-/- (autophagy-deficient) cells, Hsp90 inhibition-induced degradation of VP16 is dependent on macroautophagy-mediated degradation but not chaperone-mediated autophagy (CMA) pathway. In vivo studies demonstrated that treatment with gels containing Hsp90 inhibitor effectively reduced the level of VP16 and α genes, which may contribute to the amelioration of the skin lesions in an HSV-1 infection mediated zosteriform model. CONCLUSION: Our study provides new insights into the mechanisms by which Hsp90α facilitates the transactivation of HSV-1 α genes and viral infection, and highlights the importance of developing selective inhibitors targeting the interaction between Hsp90α and VP16 to reduce toxicity, a major challenge in the clinical use of Hsp90 inhibitors.

Antiviral activity of the mineralocorticoid receptor NR3C2 against Herpes simplex virus Type 1 (HSV-1) infection.

Analysis of a genome-scale RNA interference screen of host factors affecting herpes simplex virus type 1 (HSV-1) revealed that the mineralocorticoid receptor (MR) inhibits HSV-1 replication. As a ligand-activated transcription factor the MR regulates sodium transport and blood pressure in the kidney in response to aldosterone, but roles have recently been elucidated for the MR in other cellular processes. Here, we show that the MR and other members of the mineralocorticoid signalling pathway including HSP90 and FKBP4, possess anti-viral activity against HSV-1 independent of their effect on sodium transport, as shown by sodium channel inhibitors. Expression of the MR is upregulated upon infection in an interferon (IFN) and viral transcriptional activator VP16-dependent fashion. Furthermore, the MR and VP16, together with the cellular co-activator Oct-1, transactivate the hormone response element (HRE) present in the MR promoter and those of its transcriptional targets. As the MR induces IFN expression, our data suggests the MR is involved in a positive feedback loop that controls HSV-1 infection.

Real-time observation of light-controlled transcription in living cells.

Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.

Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors.

Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.

OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts.

SOX2 and OCT4, in conjunction with KLF4 and cMYC, are sufficient to reprogram human fibroblasts to induced pluripotent stem cells (iPSCs), but it is unclear if they function as transcriptional activators or as repressors. We now show that, like OCT4, SOX2 functions as a transcriptional activator. We substituted SOX2-VP16 (a strong activator) for wild-type (WT) SOX2, and we saw an increase in the efficiency and rate of reprogramming, whereas the SOX2-HP1 fusion (a strong repressor) eliminated reprogramming. We report that, at an early stage of reprogramming, virtually all DNA-bound OCT4, SOX2, and SOX2-VP16 were embedded in putative enhancers, about half of which were created de novo. Those associated with SOX2-VP16 were, on average, stronger than those bearing WT SOX2. Many newly created putative enhancers were transient, and many transcription factor locations on DNA changed as reprogramming progressed. These results are consistent with the idea that, during reprogramming, there is an intermediate state that is distinct from both parental cells and iPSCs.

Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms.

Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.

De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia.

The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.

Circadian Oscillations of NADH Redox State Using a Heterologous Metabolic Sensor in Mammalian Cells.

It is known that there are mechanistic links between circadian clocks and metabolic cycles. Reduced nicotinamide adenine dinucleotide (NADH) is a key metabolic cofactor in all living cells; however, it is not known whether levels of NADH oscillate or not. Here we employed REX, a bacterial NADH-binding protein, fused to the VP16 activator to convert intracellular endogenous redox balance into transcriptional readouts by a reporter gene in mammalian cells. EMSA results show that the DNA binding activity of both T- and S-REX::VP16 fusions is decreased with a reduced-to-oxidized cofactor ratio increase. Transient and stabilized cell lines bearing the REX::VP16 and the REX binding operator (ROP) exhibit two circadian luminescence cycles. Consistent with these results, NADH oscillations are observed in host cells, indicating REX can act as a NADH sensor to report intracellular dynamic redox homeostasis in mammalian cells in real time. NADH oscillations provide another metabolic signal for coupling the circadian clock and cellular metabolic states.

TRIC: Capturing the direct cellular targets of promoter-bound transcriptional activators.

Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p-benzoyl-L-phenylalanine (Bpa), a genetically incorporated photo-crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast.

Hyperactivated Stat3 boosts axon regeneration in the CNS.

Axonal regeneration after spinal cord injury (SCI) is intrinsically and extrinsically inhibited by multiple factors. One major factor contributing to intrinsic regeneration failure is the inability of mature neurons in the central nervous system (CNS) to activate regeneration-associated transcription factors (TFs) post-injury. A prior study identified TFs overexpressed in neurons of the peripheral nervous system (PNS) compared to the CNS; some of these could be involved in the ability of PNS neurons to regenerate. Of these, signal transducer and activator of transcription 3 (STAT3), as well its downstream regeneration-associated targets, showed a significant upregulation in PNS neurons relative to CNS neurons, and a constitutively active variant of Stat3 (Stat3CA) promoted neurite growth when expressed in cerebellar neurons (Lerch et al., 2012; Smith et al., 2011). To further enhance STAT3's neurite outgrowth enhancing activity, Stat3CA was fused with a viral activation domain (VP16). VP16 hyperactivates TFs by recruiting transcriptional co-factors to the DNA binding domain (Hirai et al., 2010). Overexpression of this VP16-Stat3CA chimera in primary cortical neurons led to a significant increase of neurite outgrowth as well as Stat3 transcriptional activity in vitro. Furthermore, in vivo transduction of retinal ganglion cells (RGCs) with AAV constructs expressing VP16-Stat3CA resulted in regeneration of optic nerve axons after injury, to a greater degree than for those expressing Stat3CA alone. These findings confirm and extend the concept that overexpression of hyperactivated transcription factors identified as functioning in PNS regeneration can promote axon regeneration in the CNS.

Identification of the Nuclear Localization Signal Region of Duck Enteritis Virus UL14 and Its Interaction with VP16.

OBJECT: Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae viruses. VP16 and pUL14 are both predicted to be tegument proteins of DEV. METHODS: An indirect immunofluorescence assay (IFA) was performed for preliminary analysis of the colocalization of pUL14 and VP16, which detected their subcellular localization in duck embryo fibroblasts (DEFs) during virus replication. The coexpression of pUL14 and VP16 was detected in transfected DEFs. A bimolecular fluorescence complementation (BiFC) assay was used to confirm a direct interaction between pUL14 and VP16. To better characterize the nuclear localization domain of pUL14, we designed a series of deletion mutants and transfected them with VP16. RESULTS: Our IFA findings indicated that pUL14 binds to VP16 in the cytoplasm and that pUL14 leads VP16 import into the nucleus during DEV replication. The BiFC assay revealed the presence of pUL14 and VP16 complexes. Furthermore, 1-98 amino acid (aa) at the N-terminus of pUL14 played a role in the nuclear localization signal (NLS) region and promoted translocation of VP16 into the nucleus to complete the virus life cycle. CONCLUSIONS: Our findings indicated that pUL14 could transport VP16 into the nucleus and that the N-terminal 1-98 aa may contain the NLS domain of pUL14.

[Research Advances in VP16 of the Herpes Virus].

Viral protein(VP)16is an important tegument protein of the herpes virus. It is involved in early transcription activation of viral genes as well as virion assembly and release in host cells.VP16 of some herpes viruses have deubiquitinating protease activity and can help the virus counteract the host immune response. In this review, we explain the function and complex interactions between VP16 and other proteins based of the structural characteristics of VP16.This summary provides a reference for further research of maturation of the herpes virus as well as interactions between VP16 and other proteins.

Dysregulated serum response factor triggers formation of hepatocellular carcinoma.

UNLABELLED: The ubiquitously expressed transcriptional regulator serum response factor (SRF) is controlled by both Ras/MAPK (mitogen-activated protein kinase) and Rho/actin signaling pathways, which are frequently activated in hepatocellular carcinoma (HCC). We generated SRF-VP16iHep mice, which conditionally express constitutively active SRF-VP16 in hepatocytes, thereby controlling subsets of both Ras/MAPK- and Rho/actin-stimulated target genes. All SRF-VP16iHep mice develop hyperproliferative liver nodules that progresses to lethal HCC. Some murine (m)HCCs acquire Ctnnb1 mutations equivalent to those in human (h)HCC. The resulting transcript signatures mirror those of a distinct subgroup of hHCCs, with shared activation of oncofetal genes including Igf2, correlating with CpG hypomethylation at the imprinted Igf2/H19 locus. CONCLUSION: SRF-VP16iHep mHCC reveal convergent Ras/MAPK and Rho/actin signaling as a highly oncogenic driver mechanism for hepatocarcinogenesis. This suggests simultaneous inhibition of Ras/MAPK and Rho/actin signaling as a treatment strategy in hHCC therapy.

VP16 fusion induces the multiple-knockout phenotype of redundant transcriptional repressors partly by Med25-independent mechanisms in Arabidopsis.

Biological functions of only some plant transcriptional repressors are known owing to the lack of knockout lines or unclear phenotypes because of redundancy. Here we show that strong viral activation domain VP16 fusion to the transcriptional repressor FLOWERING LOCUS C reversed its function and caused a stronger phenotype than that of the multiple-knockout line of redundant genes, suggesting the potential of this technique to identify transcription factor function that cannot be detected in a single-knockout line. Loss-of-function of transcriptional coactivator Mediator25 did not affect VP16 activity despite their in vivo interaction, suggesting the existence of other key mechanism(s) in plants.

Antagonistic control of a dual-input mammalian gene switch by food additives.

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.