Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

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.

The canonical Wnt/ß-catenin signaling pathway stimulates herpes simplex virus 1 productive infection.

The ability of herpes simplex virus 1 (HSV-1) to replicate efficiently in differentiated cells is regulated by cellular factors that stimulate viral gene expression, cell survival, and viral morphogenesis. Activation of the canonical Wnt signaling pathway generally increases ß-catenin protein levels, cell survival, and growth in dividing cells suggesting this important signaling pathway regulates productive infection. In this study, we demonstrated that a ß-catenin specific small molecule inhibitor (iCRT14) reduced HSV-1 titers approximately 10-fold in primary human lung fibroblasts and Vero cells. Furthermore, ß-catenin dependent transcription was increased at late times after infection and as expected iCRT14 reduced ß-catenin dependent transcription. Although HSV-1 infection increased ß-catenin steady state protein levels approximately 4-fold in Vero cells, there was only a nominal increase in human lung fibroblasts. We hypothesized that VP16 regulates ß-catenin dependent transcription because VP16 is a viral regulatory protein expressed at late times after infection. In the absence of other viral proteins, VP16 increased ß-catenin dependent transcription and ß-catenin steady state protein levels. Collectively, these studies suggested the cellular transcription factor ß-catenin stimulates productive infection, in part because VP16 enhances ß-catenin dependent transcription.

Multiple Posttranscriptional Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease.

The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with the aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from cotransfected plasmids were also retained in the same nuclei where vhs mRNA was located, while poly(A) binding protein (PABP) was relocalized to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Coexpression of VP16 and VP22 rescued the cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous green fluorescent protein transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and autoinduced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV-1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs but that must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted posttranscriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and coexpressed mRNAs for nuclear retention, an activity that is relieved by coexpression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors coordinate gene expression at the time that they are needed. These findings are broadly relevant to both virus and cellular gene expression.

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.

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles.

Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the UL37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion.IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while others vary substantially. Interestingly, this correlates with the VP16 trans-activating viral protein and indirectly with VP22, a second virion component whose modulation profoundly alters virion composition. This reaffirms that not only the presence but also the amount of specific tegument proteins is an important determinant of viral fitness.

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids.

UNLABELLED: Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs (1766)WD(1767) and (1862)WE(1863) are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17(+))Lox-pUL36-WD/AA-WE/AA and HSV-1(17(+))Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE: Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread.

Humoral cross reactivity between α-synuclein and herpes simplex-1 epitope in Parkinson's disease, a triggering role in the disease?

Environmental factors are implicated in the development of Parkinson's disease (PD). We have investigated on the role of molecular mimicry between HSV1 and α-synuclein that could foster the progression of PD. The antibody response against homologous peptides in PD patients and healthy controls was evaluated, showing that these antibodies are highly prevalent among PD patients to healthy controls. The competitive assay demonstrated cross-reactivity between HSV1 and human α-synuclein peptides. The results may suggest the hypothesis of the involvement of HSV1 in stimulating the immune cells against the neurons of the substantia nigra as a consequence of the cross reactivity.

CD40 ligand exhibits a direct antiviral effect on Herpes Simplex Virus type-1 infection via a PI3K-dependent, autophagy-independent mechanism.

The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. Herpes Simplex Virus type-1 (HSV-1) is a neurotropic virus capable of manipulating host responses and exploiting host proteins to establish productive infection. Herein we have examined the impact of CD40L-mediated CD40 activation on HSV-1 replication in U2OS cells stably expressing the CD40 receptor. Treatment of these cells with CD40L significantly reduced the HSV-1 progeny virus compared to non-treated cells. The activation of CD40 signaling did not affect the binding of HSV-1 virions on the cell surface but rather delayed the translocation of VP16 to the nucleus, affecting all stages of viral life cycle. Using pharmacological inhibitors and RNAi we show that inhibition of PI3 kinase but not autophagy reverses the effects of CD40L on HSV-1 replication. Collectively, these data demonstrate that CD40 activation exerts a direct inhibitory effect on HSV-1, initiating from the very early stages of the infection by exploiting PI3 kinase-dependent but autophagy-independent mechanisms.

Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression.

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis.

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

Bovine herpesvirus 1 regulatory proteins bICP0 and VP16 are readily detected in trigeminal ganglionic neurons expressing the glucocorticoid receptor during the early stages of reactivation from latency.

Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viral trans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons.

Noninvasive visualization of microRNA-16 in the chemoresistance of gastric cancer using a dual reporter gene imaging system.

MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of (131)I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo (99m)Tc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.

Activation domains for controlling plant gene expression using designed transcription factors.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.

A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.

C-terminal trans-activation sub-region of VP16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells.

The HSV-1 tegument protein VP16 contains a trans-activation domain (TAD) that is required for induction of immediate early (IE) genes during lytic infection and induced reactivation from latency. Here we report the differential contributions of the two sub-regions of the TAD in neuronal and non-neuronal cells during activation of IE gene expression, virus replication, and reactivation from quiescently infected (QIF)-PC12 cells. Our studies show that VP16- and chemical (hexamethylenebisacetamide)-induced IE gene activation is attenuated in neuronal cells. Irrespective of neuronal or non-neuronal cell backgrounds, IE gene activation demonstrated a greater requirement for the N-terminal sub-region of VP16 TAD (VP16N) than the C-terminal sub-region (VP16C). In surprising contrast to these findings, a recombinant virus (RP4) containing the VP16N deletion was capable of modest forskolin-induced reactivation whereas a recombinant (RP3) containing a deletion of VP16C was incapable of stress-induced reactivation from QIF-PC12 cells. These unique process-dependent functions of the VP16 TAD sub-regions may be important during particular stages of the virus life cycle (lytic, entrance, and maintenance of a quiescent state and reactivation) when viral DNA would be expected to be differentially modified.

Apoptotic DNA fragmentation may be a cooperative activity between caspase-activated deoxyribonuclease and the poly(ADP-ribose) polymerase-regulated DNAS1L3, an endoplasmic reticulum-localized endonuclease that translocates to the nucleus during apoptosis.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.

Effects of endogenous proteins and microRNA target sequence in a positive feedback system.

A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins.

Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics.

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.