The protective effects of Ninjin'yoeito against liver steatosis/fibrosis in a non-alcoholic steatohepatitis model mouse.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin'yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-ß was observed in CDAHFD-fed mice. Masson's trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson's trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH.

Serial Cultivation of an MSC-Like Cell Line with Enzyme-Free Passaging Using a Microporous Titanium Scaffold.

In vitro studies on adherent cells require a process of passage to dissociate the cells from the culture substrate using enzymes or other chemical agents to maintain cellular activity. However, these proteolytic enzymes have a negative influence on the viability and phenotype of cells. The mesenchymal stem cell (MSC)-like cell line, C3H10T1/2, adhered, migrated, and proliferated to the same extent on newly designed microporous titanium (Ti) membrane and conventional culture dish, and spontaneous transfer to another substrate without enzymatic or chemical dissociation was achieved. The present study pierced a 10 µm-thick pure Ti sheet with 25 µm square holes at 75 µm intervals to create a dense porous structure with biomimetic topography. The pathway of machined holes allowed the cells to access both sides of the membrane frequently. In a culture with Ti membranes stacked above- and below-seeded cells, cell migration between the neighboring membranes was confirmed using the through-holes of the membrane and contact between the membranes as migration routes. Furthermore, the cells on each membrane migrated onto the conventional culture vessel. Therefore, a cell culture system with enzyme-free passaging was developed.

Conventional and Kampo Medicine Treatment for Mild-to-moderate COVID-19: A Multicenter, Retrospective, Observational Study by the Integrative Management in Japan for Epidemic Disease (IMJEDI Study-observation).

Objective Patients in whom coronavirus disease 2019 (COVID-19) was suspected or confirmed between January 1, 2020, and October 31, 2021, were enrolled from Japanese hospitals in this multicenter, retrospective, observational study. Methods Data on the treatment administered (including conventional and Kampo medicine) and changes in common cold-like symptoms (such as fever, cough, sputum, dyspnea, fatigue, and diarrhea) were collected from their medical records. The primary outcome was the number of days without a fever (with a body temperature <37°C). The secondary outcomes were symptomatic relief and the worsening of illness, defined as the presence of a condition requiring oxygen inhalation. The outcomes of patients treated with and without Kampo medicine were compared. Patients We enrolled 962 patients, among whom 528 received conventional and Kampo treatment (Kampo group) and 434 received conventional treatment (non-Kampo group). Results Overall, after adjusting for the staging of COVID-19 and risk factors, there were no significant between-group differences in the symptoms or number of days being afebrile. After performing propensity score matching and restricting the included cases to those with confirmed COVID-19 who did not receive steroid administration and initiated treatment within 4 days from the onset, the risk of illness worsening was significantly lower in the Kampo group than in the non-Kampo group (odds ratio=0.113, 95% confidence interval: 0.014-0.928, p=0.0424). Conclusion Early Kampo treatment may suppress illness worsening risk in COVID-19 cases without steroid use. Further randomized controlled studies are needed to confirm the clinical benefit of Kampo medicine for COVID-19.

Impact of Ninjin'yoeito on frailty and short life in klotho-hypomorphic (kl/kl) mice.

With the recent aging of society, the prevention of frailty has become an important issue because people desire both a long and healthy lifespan. Klotho-hypomorphic (kl/kl) mice are known to show phenotypes of premature aging. Ninjin'yoeito (NYT) is a traditional Japanese Kampo medicine used to treat patients with vulnerable constitution, fatigue or physical exhaustion caused by aging and illness. Recent studies have reported the potential efficacy of NYT against frailty. We therefore evaluated the effect of NYT on the gait function, activity, the histopathological status of organs and survival using kl/kl mice as a model of aging-related frailty. Two sets of 28-day-old male kl/kl mice were assigned to the vehicle (non-treated; NT), 3% or 5% NYT dietary groups. One set of groups (NT, n = 18; 3% NYT, n = 11; 5% NYT, n = 11) was subjected to the analysis of free walking, rotarod, and spontaneous activity tests at approximately 58 days old. Thereafter, we measured triceps surae muscles weight and myofiber cross-sectional area (CSA), and quantified its telomere content. In addition, we evaluated bone strength and performed histopathological examinations of organs. Survival was measured in the second set of groups (NT, 3% NYT and 5% NYT group, n = 8 each). In the walking test, several indicators such as gait velocity were improved in the NYT 3% group. Similar results were obtained for the latency to fall in the rotarod test and spontaneous motor activity. Triceps muscle mass, CSA and its telomere content were significantly improved in the NYT 3% group. Bone density, pulmonary alveolus destruction and testicular atrophy were also significantly improved in the NYT 3% group. Survival rate and body weight were both significantly improved in the NYT3% group compared with those in the NT group. Continuous administration of NYT from the early stage of aging improved not only gait performance, but also the survival in the aging-related frailty model. This effect may be associated with the improvements in aging-related organ changes such as muscle atrophy. Intervention with NYT against the progression of frailty may contribute to a longer, healthier life span among the elderly individuals.

Evaluation of Preosteoblast MC3T3-E1 Cells Cultured on a Microporous Titanium Membrane Fabricated Using a Precise Mechanical Punching Process.

The surface topography of Titanium (Ti) combined toughness and biocompatibility affects the attachment and migration of cells. Limited information of morphological characteristics, formed by precise machining in micron order, is currently available on the Ti that could promote osteoconduction. In the present study, a pure Ti membrane was pierced with precise 25 µm square holes at 75 µm intervals and appear burrs at the edge of aperture. We defined the surface without burrs as the "Head side" and that with burrs as the "Tail side". The effects of the machining microtopography on the proliferation and differentiation of the preosteoblasts (MC3T3-E1 cells) were investigated. The cells were more likely to migrate to, and accumulate in, the aperture of holes on the head side, but grew uniformly regardless of holes on the tail side. The topography on the both surfaces increased osteopontin gene expression levels. Osteocalcin expression levels were higher on the head side than one on the blank scaffold and tail side (p < 0.05). The osteocalcin protein expression levels were higher on the tail side than on the head side after 21 days of cultivation, and were comparable to the proportion of the calcified area (p < 0.05). These results demonstrate the capacity of a novel microporous Ti membrane fabricated using a precise mechanical punching process to promote cell proliferation and activity.

Two cases of pseudo-achalasia with lung cancer: Case report and short literature review.

Pseudo-achalasia with lung cancer is a rare complication. We present 2 cases of pseudo-achalasia with lung cancer and summarize previous reports. The previous reports suggested that lung cancer can be complicated with pseudo-achalasia caused by paraneoplastic neurological syndromes rather than direct invasion of the tumor cells to the lower esophageal sphincter, irrespective of the histology of the lung cancer; this can strongly influence the performance status. Treatment for pseudo-achalasia improves not only the symptoms, but also the performance status. Therefore, pseudo-achalasia should be considered when lung cancer patients present with dysphagia without other known causes.

Serum extracellular vesicular miR-21-5p is a predictor of the prognosis in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis. Although the median survival is 3 years, the clinical course varies to a large extent among IPF patients. To date, there has been no definitive prognostic marker. Extracellular vesicles (EVs) are known to hold nucleic acid, including microRNAs, and to regulate gene expression in the recipient cells. Moreover, EVs have been shown to express distinct surface proteins or enveloped microRNAs depending on the parent cell or pathological condition. We aimed to identify serum EV microRNAs that would be prognostic for IPF. METHODS: To determine target microRNAs in IPF, we measured serum EV microRNA expression profiles using microRNA PCR arrays in a bleomycin mouse model and validated the microRNAs in additional mice using RT-PCR. Secondly, we enrolled 41 IPF patients and conducted a 30-month prospective cohort study. Expression of serum EV miR-21-5p was normalized by dividing by the EV amount. The relative amount of EVs was measured using the ExoScreen method. We calculated the correlations between baseline serum EV miR-21-5p expression and other clinical variables. Furthermore, we determined if serum EV miR-21-5p can predict mortality during 30 months using the Cox hazard model. According to the median level, we divided the IPF patients into two groups. Then we compared the survival rate during 30 months between the two groups using the Kaplan-Meier method. RESULTS: Serum EV miR-21-5p was elevated in both the acute inflammatory phase (day 7) and the chronic fibrotic phase (day 28) in the mouse model. In the clinical setting, serum EV miR-21-5p was significantly higher in IPF patients than in healthy control subjects. The baseline serum EV miR-21-5p was correlated with the rate of decline in vital capacity over 6 months. Furthermore, serum EV miR-21-5p was independently associated with mortality during the following 30 months, even after adjustment for other variables. In the survival analysis, IPF patients whose baseline serum EV miR-21-5p was high had a significantly poorer prognosis over 30 months. CONCLUSIONS: Our results suggest that serum EV miR-21-5p has potential as a prognostic biomarker for IPF.

Synthesis of new tricyclic thiolactams as potent antitumor agent for pancreatic cancer.

We synthesized the novel tricyclic thiolactams 2a-d, 3d-k, having a benzyl or substituted benzyl substituent on the nitrogen of indole subunit, and their preferential cytotoxicity under both nutrient-deprived medium (NDM) and Dulbecco's modified Eagle's medium (DMEM) was evaluated against a human pancreatic cancer cell line PANC-1. Among the tested compounds, the 4'-hydroxy derivative 3d showed the most potent cytotoxicity in NDM (PC50 1.68µM) although the moderate preferential cytotoxicity (PC50 1.68µM in NDM vs PC50 20µM in DMEM). The 3'-hydroxy derivative 3e exhibited the most preferential cytotoxicity (PC50 1.96µM in NDM vs less than 50% inhibition at 30µM in DMEM). The benzyl 2a and halogenated benzyl derivatives 2b,c showed no cytotoxicity in NDM. In addition, the indole (10, PC50 173.7µM), lactone (11, PC50 131.7µM), and lactam (12, PC50 44.8µM) derivatives showed week or moderate cytotoxicity in NDM. These results indicated that the hydroxy group on the benzyl substituent and tricyclic thiolactam ring were essential for the cytotoxicity in NDM against PANC-1 cell line. Moreover, 3'-hydroxy derivative 3e compound exhibited antitumor activity against the pancreatic ductal adenocarcinoma (PDAC) xenograft model in vivo.

Regulation of cancer stem cell activities by tumor-associated macrophages.

Recent studies revealed that tumor-associated macrophages play a decisive role in the regulation of tumor progression by manipulating tumor oncogenesis, angiogenesis and immune functions within tumor microenvironments. However, the role of cancer stem cells in the tumorigenic activities of tumor-associated macrophages during the course of transformation and treatment remains largely unknown. Recent studies have clarified the functional aspects of tumor-associated macrophages in the regulation of the tumorigenic activities and anticancer drug responsiveness of cancer stem cells through complex networks formed by distinct sets of cytokines, chemokines and growth factors. In this article we discuss recent advances and future perspectives regarding the molecular interplay between cancer stem cells and tumor-associated macrophages and provide future perspective about the therapeutic implication against treatment-resistant variants of cancer.

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1.

The mechanisms by which tumor microenvironments modulate nucleic acid-mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

MFG-E8 regulates the immunogenic potential of dendritic cells primed with necrotic cell-mediated inflammatory signals.

Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity.

The Diagnostic Value of the Interstitial Biomarkers KL-6 and SP-D for the Degree of Fibrosis in Combined Pulmonary Fibrosis and Emphysema.

The combined pulmonary fibrosis and emphysema (CPFE) was reported first in 1990, but it has been comparatively underestimated until recently. Although the diagnostic findings of both emphysematous and fibrotic regions are detectable by high-resolution computed tomography (HRCT) of the chest, the degree of progressive fibrosis, which increases with emphysematous lesions, is difficult to evaluate. In this study, we hypothesized that the biomarkers for pulmonary fibrosis, surfactant protein D (SP-D), and KL-6 would serve as good indicators of fibrotic lesions in CPFE. We recruited 46 patients who had been diagnosed in our hospital with both emphysema and fibrosis by their CT scan image from April 2003 to March 2008. The correlation among their pulmonary function tests, composite physiologic index (CPI), and the serum levels of SP-D and KL-6 was evaluated. We found a correlation between KL-6 and %VC, %TLC, or CPI and between SP-D and %VC or CPI. Interestingly, the combined product of KL-6 and SP-D (KL-6xSP-D) was found to highly correlate with %VC and %TLC or CPI. These results show that both KL-6 and SP-D, and especially the product of SP-D and KL-6, are good indicators of the presence of fibrotic lesions in the lungs of CPFE patients.

Increased prevalence of high anti-Cladosporium antibody titers in interstitial lung diseases.

Interstitial lung diseases (ILDs) represent a large group of different diseases, with a large part comprising idiopathic interstitial pneumonias. Differentiating hypersensitivity pneumonitis (HP), especially its chronic form and other ILDs, is difficult because of similarities in radiological manifestation and clinical course, and the difficulty of identifying causative antigens. We recently experienced a patient with Cladosporium-induced chronic HP that developed in a household environment, but the cause had been misdiagnosed as idiopathic interstitial pneumonia for several years. This case highlighted the need for measures differentiating HP from idiopathic interstitial pneumonia. In this study, we examined fungal exposure in ILDs using an antibody titer in serum to identify possible fungus-related HP. We measured the antibody titer to Cladosporium spp. in 34 patients with various ILDs, 17 patients with bronchial asthma, and 21 control subjects using an immunofluorescence assay. ILDs included HP (5 patients), idiopathic interstitial pneumonias (21 patients), and ILDs with collagen vascular diseases (8 patients). Results showed a significantly higher tendency for high anti-Cladosporium antibody titers in ILD groups (12 patients out of 34 patients), compared to patients with bronchial asthma (0/17) or control subjects (0/21). This increase in antibody titers was observed not only in patients with HP, but also in those with idiopathic interstitial pneumonias and those exhibiting collagen vascular diseases with ILDs. This report highlights the pathogenic role of fungal antigens in various ILDs. In conclusion, fungi commonly observed in our living environment such as Cladosporium could be involved in the development of ILDs.

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis.

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-ß (TGF-ß), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-ß decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-ß.

ATM-mediated DNA damage signals mediate immune escape through integrin-αvß3-dependent mechanisms.

Although the tumor microenvironment plays a critical role in tumor progression and metastasis, the relationship between chemotherapy resistance and modulation of the tumor microenvironment remains unclear. Here, we report a novel mechanism showing how constitutive DNA damage signals in therapy-resistant tumor cells suppress antitumor immunity in an integrin-αvß3-dependent manner. Integrin-αvß3 was upregulated on various therapy-resistant tumor cells through chronic activation of ATM/Chk2-and NFκB-mediated pathways. Inhibiting tumor-specific expression of integrin-αvß3 improved therapeutic responses to anticancer drugs by stimulating endogenous host immune systems. Mechanistic investigations revealed that tumor-specific integrin-αvß3 expression targeted dendritic cells, facilitating their ability to phagocytose viable therapy-resistant tumor cells and thereby impaired their ability to cross-prime antigen-specific T lymphocytes. Together, our results clarify the detrimental effects of constitutive DNA damage signals to chemosensitivity and antitumor immunity. Furthermore, these findings suggest that integrin-αvß3 targeting may benefit patients' refractory to current anticancer regimens by defeating DNA damage signaling-induced immune escape.

Tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells.

Recent evidence has unveiled the critical role of tumor cells with stem cell activities in tumorigenicity and drug resistance, but how tumor microenvironments regulate cancer stem/initiating cells (CSCs) remains unknown. We clarified the role of tumor-associated macrophages (TAMs) and their downstream factor milk-fat globule-epidermal growth factor-VIII (MFG-E8) in the regulation of CSC activities. Bone marrow chimeric systems and adoptive cell transfers elucidated the importance of MFG-E8 from TAMs in conferring to CSCs with the ability to promote tumorigenicity and anticancer drug resistance. MFG-E8 mainly activates signal transducer and activator of transcription-3 (Stat3) and Sonic Hedgehog pathways in CSCs and further amplifies their anticancer drug resistance in cooperation with IL-6. Thus, the pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.

A Normal Range of KL-6/MUC1 Independent of Elevated SP-D Indicates a Better Prognosis in the Patients with Honeycombing on High-Resolution Computed Tomography.

Both SP-D and KL-6/MUC1 are established biomarkers of the interstitial pneumonias, including idiopathic pulmonary fibrosis (IPF), but the causes and clinical outcomes based on their independent effects are not known. Eleven asymptomatic patients, detected with honeycombing on high-resolution computed tomography (HRCT), were compared with 17 other IPF outpatients having slight respiratory symptoms and honeycombing as well. Although SP-D was increased in both groups, KL-6 was significantly higher in the symptomatic IPF group. When the patients (n = 11) having both biomarkers elevated were compared with the other patients (n = 6) with only SP-D elevated, the distribution of fibrotic lesions with honeycombing on HRCT was larger and the survival time was shorter in the patients having both biomarkers elevated. Immunohistochemical analysis also differentiated these biomarkers in the lung. These results suggest both a cause and the prognostic value of dissociation of these biomarkers.

The human cytomegalovirus UL76 gene regulates the level of expression of the UL77 gene.

BACKGROUND: Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5' mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth. CONCLUSIONS/SIGNIFICANCE: While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.

Transcriptional repression by sumoylation of Epstein-Barr virus BZLF1 protein correlates with association of histone deacetylase.

The transition from latent to lytic phases of the Epstein-Barr virus life cycle is triggered by expression of a viral transactivator, BZLF1, that then induces expression of the viral immediate-early and early genes. The BZLF1 protein is post-translationally modified by a small ubiquitin-related modifier-1 (SUMO-1). Here we found that BZLF1 is conjugated at lysine 12 not only by SUMO-1 but also by SUMO-2 and 3. The K12R mutant of BZLF1, which no longer becomes sumoylated, exhibits stronger transactivation than the wild-type BZLF1 in a reporter assay system as well as in the context of virus genome with nucleosomal structures. Furthermore, exogenous supply of a SUMO-specific protease, SENP, caused de-sumoylation of BZLF1 and enhanced BZLF1-mediated transactivation. Immunoprecipitation experiments proved that histone deacetylase 3 preferentially associated with the sumoylated form of BZLF1. Levels of the sumoylated BZLF1 increased as lytic replication progressed. Based on these observations, we conclude that sumoylation of BZLF1 regulates its transcriptional activity through histone modification during Epstein-Barr virus productive replication.

Transient increases in p53-responsible gene expression at early stages of Epstein-Barr virus productive replication.

Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.