The Effects of Endosomal Toll-like Receptor Inhibitors in an EBV DNA-Exacerbated Inflammatory Bowel Disease Mouse Model.

Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.

[Effect of HBV DNA load on the safety and prognosis of systematic therapy in advanced hepatocellular carcinoma].

Objective: To study the effect of hepatitis B virus (HBV) infection on the occurrence of liver damage, HBV reactivation (HBVr) and the influence of HBVr on the prognosis of patients with advanced hepatocellular carcinoma (HCC) receiving systemic therapy. Methods: The clinical data of 403 patients with HBV-related HCC at the Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University et al, from July 2018 to December 2020 were collected. The incidence of liver damage and HBVr during systematic therapy, and the influence of HBVr on survival prognosis were analyzed. Results: Of the 403 patients, 89.1% were male (n=359), with a median age of 51 years (51.5±12.1). Before propensity score matching (PSM), the proportion of patients with cirrhosis, TNM and advanced BCLC stage was higher in high HBV-DNA (baseline HBV-DNA>1000 U/ml, n=147) group comparing with the low HBV-DNA (baseline HBV DNA≤1000 u/ml, n=256) group (P<0.05). There was no significant difference in baseline indexes between the two groups after PSM. In 290 patients after PSM, there was no significant difference in the incidence of liver damage and HBVr between high HBV-DNA group and low HBV-DNA group (P>0.05). Survival analysis was performed on 169 patients with survival data, the median overall survival (OS) was found to be 11.49 months (95%CI: 7.77-12.89) and 16.65 months (95%CI: 10.54-21.99, P=0.008) in the high and low HBV-DNA groups, respectively. And median progression-free survival (PFS) was 7.41 months (95%CI: 5.06-8.67) and 10.55 months (95%CI: 6.72-13.54, P=0.038), respectively, with a statistically significant difference. There were no differences in overall survival (OS) and progression-free survival (PFS) between patients with and without HBVr and those with or without liver damage (P>0.05). Conclusions: HBV-DNA levels above 1 000 U/ml before systemic therapy do not increase the risk of liver damage or HBVr during systemic therapy in patients with HBV-related hepatocellular carcinoma, and such patients can safely receive systemic therapy.

Cytokines Expression Compared to the Determinants of Cellular Apoptosis Prominently Attributes to the Deleterious Effects of 'A' Determinant Surface Gene Mutations in HBV Transfected Hepatoma Cell Line.

BACKGROUND: Previous studies have explored the role of AKT protein in anti-apoptotic/proliferative activities. However, there has been a lack of information regarding the role of Akt in association with cytokines expression in HBV-related (wild type HBV and HBV with mutations of 'a' determinant region) studies either in the case of HBV infection or in transfected hepatoma cells. The present study tries to determine the role of Akt and cytokines expression in the presence of small surface gene mutants in the hepatoma cell line. METHODS: Mutations of 'a' determinant region, viz. sA128V and sG145R, were created in wild-type pHBV1.3 by site-directed mutagenesis and transfected in hepatoma cell line. Secretory levels of HBsAg in the wild type as well as in both the mutants were analyzed by ELISA. Apoptotic analysis of transfected cells was studied by flow cytometry. Expression analysis of Akt and cytokines (TNF-alpha, IL-6, and IFN-gamma) was done by qPCR. RESULTS: The presence of significantly more alive cells in sG145R than sA128V transfected cells may be due to the up-regulation of the Akt gene expression. Cytokines expression was nearly similar between sA128V and wild-type pHBV1.3 transfected cells. Presence of sG145R showed dramatically high cytokines expression than sA128V and wild-type pHBV1.3. CONCLUSION: Cytokines expression predominantly contributes to the detrimental effects associated with the 'a' determinant region mutations particularly sG145R mutant. It may also be inferred that mechanisms associated with cellular apoptosis apparently do not play any major role to assign the 'a' determinant small surface gene mutation(s) for their pathological outcome.

Catenulopyrizomicins, new anti-Hepatitis B virus compounds, from the rare actinomycete Catenuloplanes sp. MM782L-181F7.

Hepatitis B virus (HBV) causes chronic hepatitis in humans, and current antiviral therapies rarely treat viral infections. To improve the treatment efficacy, novel therapeutic agents, especially those with different mechanisms of action, need to be developed for use in combination with the current antivirals. Here, we isolated new anti-HBV compounds, named catenulopyrizomicins A-C, from the fermentation broth of rare actinomycete Catenuloplanes sp. MM782L-181F7. Structural analysis revealed that these compounds contained a structure that is composed of thiazolyl pyridine moiety. The catenulopyrizomicins reduced the amount of intracellular viral DNA in HepG2.2.15 cells with EC50 values ranging from 1.94 to 2.63 µM with small but notable selectivity. Mechanistic studies indicated that catenulopyrizomicin promotes the release of immature virion particles that fail to be enveloped through alterations in membrane permeability.

The premise of capsid assembly modulators towards eliminating HBV persistence.

INTRODUCTION: The burden of chronic hepatitis B virus (HBV) results in almost a million deaths per year. The most common treatment for chronic hepatitis B infection is long-term nucleoside analogs (NUC) or one-year interferon-alpha (pegylated or non-pegylated) therapy before or after NUC therapy. Unfortunately, these therapies rarely result in HBV functional cure because they do not eradicate HBV from the nucleus of the hepatocytes, where the covalently closed circular DNA (cccDNA) is formed and/or where the integrated HBV DNA persists in the host genome. Hence, the search continues for novel antiviral therapies that target different steps of the HBV replication cycle to cure chronically infected HBV individuals and eliminate HBV from the liver reservoirs. AREAS COVERED: The authors focus on capsid assembly modulators (CAMs). These molecules are unique because they impact not only one but several steps of HBV viral replication, including capsid assembly, capsid trafficking into the nucleus, reverse transcription, pre-genomic RNA (pgRNA), and polymerase protein co-packaging. EXPERT OPINION: Mono- or combination therapy, including CAMs with other HBV drugs, may potentially eliminate hepatitis B infections. Nevertheless, more data on their potential effect on HBV elimination is needed, especially when used daily for 6-12 months.

Developments in pharmacotherapeutic agents for hepatitis B - how close are we to a functional cure?

INTRODUCTION: Hepatitis B virus (HBV) remains a public health concern given its global prevalence and potential complications including hepatocellular carcinoma (HCC). Current therapies, including nucleos(t)ide analogs (NA) and interferons (IFN), are effective in chronic treatment of HBV but rarely provide a functional cure due to inadequate host response and the presence of viral DNA. Therefore, novel therapies that enhance the innate immune response while suppressing DNA transcription may provide definitive treatment of HBV. AREAS COVERED: In this review, the authors provide a brief overview of commonly used agents and their efficacy in treatment of HBV. Newer therapies with direct antiviral agents such as bepirovirsen (antisense oligonucleotide (ASO)) and entry inhibitors such as bulevirtide have shown efficacy in reducing viral load but demonstrate further reductions in conjunction with immune modulators such as therapeutic vaccines. EXPERT OPINION: Combination therapy is far superior to monotherapy alone, necessitating the need for both immunomodulators and direct antiviral agents in chronic treatment of HBV. Therapies that target covalently closed circular (cccDNA) with immunomodulators like therapeutic vaccines have shown promising results and may ultimately achieve functional cure. However, therapies need to be evaluated in the context of the patient, considering both financial and socioeconomic factors.

Chronic Hepatitis B Infection: Current and Emerging Therapeutic Strategies.

The chronic infection of the hepatitis B virus (CHB) represents a major public health problem worldwide. Despite the availability of an effective prophylactic vaccine, millions of hepatitis B patients are at increased risk of developing chronic liver disease. The currently available treatments for HBV infection include interferon and nucleos(t)ide analogues that are effective at suppressing viral load and preventing or delaying the progression of liver disease. However, these treatments offer somewhat unsatisfactory clinical cures due to the persistence of the intrahepatic pool of covalently closed circular DNA (cccDNA) that serves as a reservoir for viral progenies and a potential source of recurring infections. Elimination of viral cccDNA remains a challenge for scientists and pharmaceutical industries in order to achieve the eradication and control of HBV infection. This would involve a detailed understanding of the molecular mechanisms of cccDNA formation, its intracellular stability, and regulation during replication and transcription. Recent advances in drug therapy have heralded a new horizon of novel therapeutic approaches for CHB infection, with several promising antiviral and immunomodulatory agents currently in preclinical or clinical testing. However, approval of any new curative therapy would involve rigorous evaluation of the efficacy and safety of each treatment and defining correct endpoints associated with improved clinical outcomes. This article summarizes the current landscape of HBV treatments, and drugs in clinical trials and highlights the most recent anti-HBV small molecules designed to directly target HBV or to improve immune response during chronic infection.

Design and Synthesis of (3-Phenylisoxazol-5-yl)methanimine Derivatives as Hepatitis B Virus Inhibitors.

Series of (3-phenylisoxazol-5-yl)methanimine derivatives were synthesized, and evaluated for anti-hepatitis B virus (HBV) activity in vitro. Half of them more effectively inhibited HBsAg than 3TC, and more favor to inhibit secretion of HBeAg than to HBsAg. Part of the compounds with significant inhibition on HBeAg were also effectively inhibit replication of HBV DNA. Compound (E)-3-(4-fluorophenyl)-5-((2-phenylhydrazineylidene)methyl)isoxazole inhibited excellently HBeAg with IC50 in 0.65 µM (3TC(Lamivudine) in 189.90 µM), inhibited HBV DNA in 20.52 µM (3TC in 26.23 µM). Structures of compounds were determined by NMR and HRMS methods, and chlorination on phenyl ring of phenylisoxazol-5-yl was confirmed by X-ray diffraction analysis, and the structure-activity relationships (SARs) of the derivatives was discussed. This work provided a new class of potent non-nucleoside anti-HBV agents.

Long-term follow-up of treatment-naïve HBeAg-negative patients with chronic hepatitis B.

BACKGROUND/AIMS: We aimed to explore long-term results of oral antiviral agents in treatment-naïve "HBeAg negative chronic hepatitis B (CHB)" and determine the factors affecting the complete virological response. METHOD: Patients with HBeAg-negative CHB who used oral antiviral agents for at least 3 years were evaluated retrospectively. RESULTS: A total of 173 patients were recorded. The mean duration of treatment was 62.2 ± 28.9 months. Complete virological responses (CVR) were 82.8% (n = 53/64) in tenofovir disoproxil fumarate (TDF), 84.4% (n = 49/58) in lamivudine (LAM), 83.9% (n = 26/31) in entecavir (ETV), 95% in telbivudine (LdT) (n = 19/20) (p = 0.290). Multivariate analysis revealed age ≤ 40 (p = 0.012, 95%CI = 1.38-13.76, OR = 4.36) and baseline HBV DNA value (p = 0.003, 95%CI = 1.23-2.63, OR = 1.78) as independent factors for CVR. Virological breakthrough was detected in 29 (50%) patients on LAM therapy, two (6.4%) patients on ETV therapy, and two (10%) patients on LdT therapy. Treatment was switched to another antiviral agent due to osteoporosis in four patients in the TDF group, muscle pain in nine patients in the LDT group, and headache in one patient in the ETV group. Hepatocelluler cancer was detected in five patients. HBsAg seroclearance developed in two patients. Anti-HBs seroconversion was not detected. CONCLUSION: CVR was achieved at similar rates with all four antiviral agents, while younger age (≤ 40) and low baseline viral load were the main factors for virological response. However, drug resistance and virological breakthrough in the LAM group and side effects in the LdT group were detected during the long-term follow-up. Moreover, HBsAg seroclearance was achieved at very low rates with oral antiviral agents.

A carrier-free metal-organic hybrid nanoassembly with combination anti-viral and hepato-protective activity for hepatitis B treatment.

Hepatitis B represents a major global public health burden, which is caused by the hepatitis B virus (HBV) with a high infection rate. Although several anti-HBV drugs have been developed for clinical treatment of hepatitis B, the current therapeutic strategies still suffer from undeniable adverse effects, insufficient efficacy after systemic administration and chronic inflammation. Here, we develop a carrier-free metal-organic hybrid nanoassembly that is co-loaded with tenofovir (TFV), an anti-viral agent and phosphorylated glycyrrhetinic acid (GAP), an anti-inflammatory compound (TFV/GAP/NA) to enhance the anti-HBV effect and alleviate the inflammatory response for hepatitis B treatment. The nanoassembly is easily prepared through the ionic interactions between the anionic phosphonate/phosphate groups from TFV/GAP and the zirconium cation, which has a stable nanostructure and a high drug-loading capacity. The nanoassembly prolongs the circulation time with reduced drug leakage in the blood and elevates drug accumulation in the liver after intravascular administration. After internalization mediated by the GAP ligand-GA receptor interaction, TFV/GAP/NA disassembles by the phosphatase-triggered degradation of the phosphate ester bonds in GAP and releases TFV, GAP and GA within the HBV-positive hepatocytes. The released TFV interferes with the HBV polymerase to inhibit the viral DNA replication, while the released GAP and GA suppress the pro-inflammatory protein expression. In mouse models, treatment with TFV/GAP/NA inhibits HBV production and alleviates inflammation-mediated liver injury.

The efficacy and safety of tacrolimus and entecavir combination therapy in the treatment of hepatitis B virus-associated glomerulonephritis: a multi-center, placebo controlled, and single-blind randomized trial.

BACKGROUND: The proteinuria remission in hepatitis B virus-associated glomerulonephritis (HBV-GN) patients with massive proteinuria treated with antiviral therapy was low. Tacrolimus (TAC) is effective in primary nephropathy and can inhibit HBV infection by inhibiting HBV binding to sodium taurocholate cotransporting polypeptide on liver cells. This study evaluated the efficacy and safety of TAC combined with ETV compared with entecavir (ETV) monotherapy in HBV-GN. METHODS: Patients diagnosed with HBV-GN were recruited for this prospective, randomized, controlled, multicenter, single-blinded study in China. Patients were given TAC and ETV therapy (the TAC+ETV group) or placebo and ETV therapy (the ETV group) for 26 weeks. The efficacy endpoints included proteinuria remission, including complete and partial remission (CR and PR), the change of 24-hour proteinuria (24 h UP) and HBV DNA titer. The safety endpoints were the incidence of HBV virologic breakthrough and adverse events. RESULTS: There were 14 patients in the TAC+ETV group and 17 patients in the ETV group. In the intention-to-treat analyses, 64.3% (9/14) of patients in the TAC+ETV group and 58.8% (10/17) in the ETV group achieved PR or CR at 26 weeks (P=0.38). At week 14, 42.9% (6/14) and 41.2% (7/17) of patients in the TAC+ETV group and the ETV group, respectively, achieved PR or CR (P=0.23). At week 26, the 24 h UP had decreased by 2.63±6.33 g from baseline in the TAC+ETV group and 1.42±4.34 g in the ETV group (P=0.55). The serum albumin increased by 11.1±7.30 g/L from baseline in the TAC+ETV group and 3.81±5.09 g/L in the ETV group (P<0.001). Log10 HBV DNA decreased by 1.49±2.04 from baseline in the TAC+ETV group and 2.47±2.08 in the ETV group (P=0.37); 28.6% (4/14) patients had HBV DNA virologic breakthrough in the ETV group, while none in the TAC+ETV group (P=0.29). CONCLUSIONS: In adult HBV-GN patients, TAC and ETV combination therapy may significantly improve serum albumin levels without increasing the risk of HBV reactivation compared with entecavir monotherapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03062813.

[Effects of hepatitis B virus on Th17, Treg and Th17/Treg ratio in different alanine aminetransferase stages].

OBJECTIVE: To evaluate the effects of hepatitis B virus (HBV) on helper T lymphocytes 17 (Th17), regulatory T lymphocyte (Treg) and Th17/Treg ratio in chronic hepatitis B patients in different alanine aminetransferase (ALT) stages. METHODS: In the study, 336 chronic hepatitis B patients in the first hospital of Lanzhou University were analyzed. The hepatitis B antigen antibody parameters were measured by chemiluminescence immunoassay analyzer, the liver function parameters were measured by automatic biochemical analyzer, the HBV loads were measured by quantitative PCR, Th17, Treg and Th17/Treg ratios were detected by flow cytometry. Among them, 111 cases (ALT < 40 U/L) of ALT were normal hepatitis B, 108 cases of chronic hepatitis B with ALT above normal upper limit and < 2 times higher (40 U/L≤ALT < 80 U/L), and 117 cases of chronic hepatitis B with ALT above 2 times normal upper limit (80 U/L≤ALT). According to the viral load, they were divided into low replication group with HBV DNA < 4.0 lg copies/mL, medium replication group with 4.0 lg copies/mL≤HBV DNA < 6.0 lg copies/mL and high replication group with HBV DNA ≥ 6.0 lg copies / mL. Dunnett T3 variance analysis were used to analyze the effects of HBV on Th17, Treg and Th17/Treg ratio in the chronic hepatitis B patients in different ALT stages. The changes of virological and immunological indexes before and after treatment were observed for 24 weeks of antiviral therapy in the hepatitis B patients with ALT≥double upper limit of normal group. RESULTS: In the ALT normal group, different virus load HBV had minor effects on Th17, Treg and Th17/Treg ratio. In the ALT≥2 times upper limit of normal group, with the virus load increased, Th17 (3.18%±0.79% in low replication group, 3.78%±0.92% in medium replication group and 4.57%±1.15% in high replication group), Treg cells (5.52%±1.58% in low replication group, 5.89%±1.84% in medium replication group and 6.37%±2.35% in high replication group) and their ratio Th17/Treg (0.57±0.25 in low replication group, 0.65±0.29 in medium replication group and 0.73±0.36 in high replication group) were significantly increased (P < 0.05). After entecavir treatment 24 weeks, the patient' s HBV-DNA decreased significantly, Th17 (3.89%±1.02% vs. 2.06%±0.46%), Treg (6.02%±2.03% vs. 5.06%±1.25%), Th17/Treg ratio (0.65±0.28 vs. 0.41±0.14) decreased significantly (P < 0.05). CONCLUSION: Investigation on the effects of HBV on Th17 and Treg cells and their ratios in different ALT states can clarify the effects of HBV on the body from the immunological perspective and can further understand the ALT grouping for antiviral treatment theoretical significance, which is helpful for clinical treatment.

Molecular characterization of hepatitis B virus (HBV) isolated from a pediatric case of acute lymphoid leukemia, with a delayed response to antiviral treatment: a case report.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is effectively used as the first-line antiviral for chronic hepatitis B virus (HBV) infection in adults and children older than 12 years. To date, no confirmed case of virologic breakthrough (VBT) in a pediatric case has been reported. CASE PRESENTATION: Here we describe a case of a 5-year old, asymptomatically infected with HBV infection two months after chemotherapy for precursor B acute lymphoblastic leukemia (ALL). Although the 5-year old male is South African, his family originated from Guinea. At the end of the one-year follow-up, the infection progressed to chronic HBV infection, with a high viral load. At 36 weeks (8 months) post-treatment with lamivudine (LAM), there was a partial virologic response (PVR) and after 61 weeks (14 months), he was switched to TDF rescue monotherapy. Even with TDF treatment, he still experienced VBT and subsequent PVR. The full-length genome of HBV isolated 78 weeks after the switch to rescue TDF monotherapy was sequenced and belonged to genotype E. In addition to the LAM mutations (rtS256G and rtM267L), missense mutations in B-cell, T-cell, HLA class I and II-restricted epitopes emerged, which were to evade and escape host surveillance, leading to delayed viral clearance, persistence and disease progression. Two further events of VBT occurred between weeks 113 and 141 of TDF rescue-therapy. Viral loads and liver enzymes are normalizing progressively with long-term therapy. CONCLUSION: Although the host immune reconstitution may be delayed, prolonged TDF treatment was effective in treating this pediatric case of HBV infection with VBT and PVR.

Hepatitis B Reactivation in Patients Treated with Direct-Acting Antivirals for Hepatitis C.

INTRODUCTION: There is limited research about HBV reactivation (HBVr) due to direct-acting antivirals (DAA) for HCV and most are limited by short duration of follow-up, small sample size, and absence of baseline HBV DNA. We aimed to determine the incidence and clinical course of HBVr in HBsAg and/or anti-HBcIgG positive patients treated with DAA for HCV. METHODS: Seven centers retrospectively analyzed their database on HCV patients treated with DAA between 2015 and 2019. Patients with HBV coinfection or resolved HBV infection were enrolled. Serum transaminases, HBsAg, HBeAg, and HBV DNA were followed every 4 weeks during DAA treatment and every 12 weeks 1 year after treatment. Entecavir or tenofovir disoproxil fumarate was started in case of HBVr. The development of HBVr, HBV flare, liver failure, and mortality were determined. RESULTS: 852 patients received DAA treatment for HCV. Among them, 35 (4.1%) had HBV coinfection and 246 (28.9%) had resolved HBV infection. 257 patients (53.3% male, mean age: 63 ± 9) constituted the study group (29 with coinfection and 228 with resolved infection). Three patients with coinfection were HBV DNA positive. HBVr developed in 10 (34.5%) HBsAg positive patients, either during (n = 3) or 12-48 weeks after finishing DAA treatment. HBV flare and acute liver failure developed in 1 patient (3.4%), each. Two patients with resolved infection developed HBVr (0.87%) and one (0.44%) had HBV flare. Overall, none of the patients died or underwent liver transplantation due to HBVr. CONCLUSION: Patients with HBV/HCV coinfection have a high risk of HBVr after DAA treatment and should receive antiviral prophylaxis. Patients with resolved infection have a low risk of HBVr and can be monitored by serial ALT measurements.

IFN-α inhibits HBV transcription and replication by promoting HDAC3-mediated de-2-hydroxyisobutyrylation of histone H4K8 on HBV cccDNA minichromosome in liver.

The epigenetic modification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a crucial role in cccDNA transcription and viral persistence. Interferon-α (IFN-α) is a pivotal agent against HBV cccDNA. However, the mechanism by which IFN-α modulates the epigenetic regulation of cccDNA remains poorly understood. In this study, we report that IFN-α2b enhances the histone deacetylase 3 (HDAC3)-mediated de-2-hydroxyisobutyrylation of histone H4 lysine 8 (H4K8) on HBV cccDNA minichromosome to restrict the cccDNA transcription in liver. By screening acetyltransferases and deacetylases, we identified that HDAC3 was an effective restrictor of HBV transcription and replication. Moreover, we found that HDAC3 was able to mediate the de-2-hydroxyisobutyrylation of H4K8 in HBV-expressing hepatoma cells. Then, the 2-hydroxyisobutyrylation of histone H4K8 (H4K8hib) was identified on the HBV cccDNA minichromosome, promoting the HBV transcription and replication. The H4K8hib was regulated by HDAC3 depending on its deacetylase domain in the system. The low level of HDAC3 and high level of H4K8hib were observed in the liver tissues from HBV-infected human liver-chimeric mice. The levels of H4K8hib on HBV cccDNA minichromosome were significantly elevated in the liver biopsy specimens from clinical hepatitis B patients, which was consistent with the high transcriptional activity of cccDNA. Strikingly, IFN-α2b effectively facilitated the histone H4K8 de-2-hydroxyisobutyrylation mediated by HDAC3 on the HBV cccDNA minichromosome in primary human hepatocytes and hepatoma cells, leading to the inhibition of HBV transcription and replication. Our finding provides new insights into the mechanism by which IFN-α modulates the epigenetic regulation of HBV cccDNA minichromosome.

Hepatocyte steatosis inhibits hepatitis B virus secretion via induction of endoplasmic reticulum stress.

The effects of hepatocyte steatosis on hepatitis B virus (HBV) DNA replication and HBV-related antigen secretion are incompletely understood. The aims of this study are to explore the effects and mechanism of hepatocyte steatosis on HBV replication and secretion. Stearic acid (SA) and oleic acid (OA) were used to induce HepG2.2.15 cell steatosis in this study. The expressions of glucose-regulated protein 78 (GRP78), phosphorylation of protein kinase R-like endoplasmic reticulum (ER) kinase (p-PERK), and eukaryotic translation initiation factor 2α (p-eIF2α) were detected by Western blotting (WB). HBV DNA, HBsAg, and HBeAg in the supernatant were determined by real-time fluorescent polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. Intracellular HBV DNA, HBsAg level, and HBV RNA were measured by real-time fluorescent PCR, WB, and real-time quantitative reverse transcriptase-PCR, respectively. The results showed that SA and OA significantly increased intracellular lipid droplets and triglyceride levels. SA and OA significantly induced GRP78, p-PERK, and p-eIF2α expressions from 24 to 72 h. 4-phenylbutyric acid (PBA) alleviated ER stress induced by SA. SA promoted intracellular HBsAg and HBV DNA accumulation; however, it inhibited the transcript of HBV 3.5 kb mRNA and S mRNA. The secretion of HBsAg and HBV DNA inhibited by SA or OA could be partially restored by pretreatment with PBA but not by inhibiting GRP78 expression with siRNA. Hepatocyte steatosis inhibits HBsAg and HBV DNA secretion via induction of ER stress in hepatocytes, but not via induction of GRP78.

Differential Effects of Viral Nucleic Acid Sensor Signaling Pathways on Testicular Sertoli and Leydig Cells.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon ß promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.

Analysis of Global Transcriptome Change in Mouse Embryonic Fibroblasts After dsDNA and dsRNA Viral Mimic Stimulation.

The activation of innate immunity by viral nucleic acids present in the cytoplasm plays an essential role in controlling viral infection in both immune and non-immune cells. The dsDNA and dsRNA viral mimics can stimulate the cytosolic nucleic acids sensors and activate the antiviral innate immunity. In this study, taking advantage of dsDNA and dsRNA viral mimics, we investigated the global transcriptome changes after the antiviral immunity activation in mouse embryonic fibroblasts. Results from our data identified a positive feedback up-regulation of sensors (e.g., Tlr2, Tlr3, Ddx58, cGAS), transducers (e.g., Traf2, Tbk1) and transcription factors (e.g., Irf7, Jun, Stat1, Stat2) in multiple pathways involved in detecting viral or microbial infections upon viral mimic stimulation. A group of genes involved in DNA damage response and DNA repair such as Parp9, Dtx3l, Rad52 were also up-regulated, implying the involvement of these genes in antiviral immunity. Molecular function analysis further showed that groups of helicase genes (e.g., Dhx58, Helz2), nuclease genes (e.g., Dnase1l3, Rsph10b), methyltransferase genes (e.g., histone methyltransferase Prdm9, Setdb2; RNA methyltransferase Mettl3, Mttl14), and protein ubiquitin-ligase genes (e.g., Trim genes and Rnf genes) were up-regulated upon antiviral immunity activation. In contrast, viral mimic stimulation down-regulated genes involved in a broad range of general biological processes (e.g., cell division, metabolism), cellular components (e.g., mitochondria and ribosome), and molecular functions (e.g., cell-cell adhesion, microtubule binding). In summary, our study provides valuable information about the global transcriptome changes upon antiviral immunity activation. The identification of novel groups of genes up-regulated upon antiviral immunity activation serves as useful resource for mining new antiviral sensors and effectors.

Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 µm Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 µm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.