HBV DNA polymerase upregulates the transcription of PD-L1 and suppresses T cell activity in hepatocellular carcinoma.

BACKGROUND: In HBV-associated HCC, T cells often exhibit a state of functional exhaustion, which prevents the immune response from rejecting the tumor and allows HCC to progress. Moreover, polymerase-specific T cells exhibit more severe T-cell exhaustion compared to core-specific T cells. However, whether HBV DNA polymerase drives HBV-specific CD8+ T cell exhaustion in HBV-related HCC remains unclear. METHODS: We constructed a Huh7 cell line stably expressing HA-HBV-DNA-Pol and applied co-culture systems to clarify its effect on immune cell function. We also examined how HBV-DNA-Pol modulated PD-L1 expression in HCC cells. In addition, HBV-DNA-Pol transgenic mice were used to elucidate the underlying mechanism of HBV-DNA-Pol/PD-L1 axis-induced T cell exhaustion. RESULTS: Biochemical analysis showed that Huh7 cells overexpressing HBV-DNA-Pol inhibited the proliferation, activation, and cytokine secretion of Jurkat cells and that this effect was dependent on their direct contact. A similar inhibitory effect was observed in an HCC mouse model. PD-L1 was brought to our attention during screening. Our results showed that the overexpression of HBV-DNA-Pol upregulated PD-L1 mRNA and protein expression. PD-L1 antibody blockade reversed the inhibitory effect of Huh7 cells overexpressing HBV-DNA-Pol on Jurkat cells. Mechanistically, HBV-DNA-Pol interacts with PARP1, thereby inhibiting the nuclear translocation of PARP1 and further upregulating PD-L1 expression. CONCLUSIONS: Our findings suggest that HBV-DNA-Pol can act as a regulator of PD-L1 in HCC, thereby directing anti-cancer immune evasion, which further provides a new idea for the clinical treatment of liver cancer.

Hepatitis B Virus DNA Polymerase Displays an Anti-Apoptotic Effect by Interacting with Elongation Factor-1 Alpha-2 in Hepatoma Cells.

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

Study on 2-arylthio-5-iodo pyrimidine derivatives as novel nonnucleoside inhibitors against hepatitis B virus DNA replication.

BACKGROUND: Novel nonnucleoside hepatitis B virus inhibitors have been recently developed for the reason of drug-resistant mutations and adverse effects of nucleoside analogs. In this study, two series of 2-arylthio-5-iodo pyrimidine analogs were firstly reported as potential anti-HBV agents. METHODOLOGY: Target compounds were prepared according to two high-yielded synthetic routes, and their anti-HBV activities were evaluated on Hep2.2.15 and HepAD38 cell lines, respectively. To probe the mechanism of active agents, a cell-based (Huh-7) study of biochemical markers (e.g., HBeAg, HBsAg, intracellular HBV DNA and pgRNA) was performed. Furthermore, the pharmacophore models were constructed for future optimization of lead compounds. CONCLUSION: 2-Arylthio-5-iodo pyrimidine derivatives firstly proved to be effective against HBV, which paves the way for future development of nonnucleoside anti-HBV agents.

Current developments in nucleoside/nucleotide analogues for hepatitis B.

There had been remarkable development in nucleos(t)ide analogues (NAs) and evolution in treatment strategies in last 15 years. Currently, there are five NAs available for chronic hepatitis B treatment, namely lamivudine, telbivudine and entecavir (nucleoside analogues), adefovir dipivoxil and tenofovir disoproxil fumarate (nucleotide analogues). The advantages of relatively infrequent side effects and easy administration per oral make NAs popular treatment options. The major drawback of earlier generation NAs is the risk of emergence of drug resistance. Current international guidelines recommend the use of more potent agents with high genetic barriers to resistance including entecavir and tenofovir as first line chronic hepatitis B treatment. However, there is no consensus regarding the subsequent treatment regimens in patients with suboptimal responses to NAs. De novo combination therapy of two NAs, response-guided therapy and roadmap concept in NAs with subsequent switch or add-on therapy can also potentially improve treatment efficacy and avoid resistance.

Properties of Hepatitis B Virus Associated DNA Polymerase

The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.

Emergence of YMDD Motif Mutant Hepatitis B Virus during Short-erm Lamivudine Therapy / 대한간학회지

BACKGROUND/AIMS: The emergence of lamivudine-resistant mutant hepatitis B virus (HBV), with aminoacid substitution in the YMDD motif of DNA polymerase, has been reported in the long-term lamivudine use group. However there is no report about the emergence of mutant viruses during the short-term lamivudine therapy. The objective of this study was to investigate the emergence of YMDD mutant HBV during short-term lamivudine therapy. METHODS: We evaluated twenty-eight chronic hepatitis B patients who were HBeAg and HBV DNA positive and treated with lamivudine 100mg p.o. daily for 12 weeks. First, we investigated the emergence of YMDD mutants by nested polymerase chain reaction (PCR) method developed by Chayama et al in 19 patients who lost HBV DNA during lamivudine therapy but showed HBV DNA re-emergence 2 weeks after the end of therapy. Second, DNA subcloning and sequencing of HBV DNA polymerase including YMDD motif was undertaken in one patient's serial blood samples at 0, 8, 12 weeks to confirm the results of nested PCR. RESULTS: YMDD motif mutation was detected in 17(90%) out of 19 patients at the end of therapy and the type of mutations were YIDD only. At the end of therapy, mutant was predominant in 5 patients, both mutant and wild type were similar in proportion in 3 patients, and wild type was predominant in 9 patients. When we carried out nested PCR serially with samples of 0, 2, 4, 8, 12, 14 weeks after initiation of therapy in 5 patients who were mutant predominant at 12 weeks, YIDD mutant began to be detected from 2 weeks in 4 patients and from 4 weeks in one patient. However, rapid turnover from mutant to wild type happened after the end of therapy, so only wild type was detected in 3 patients and wild type became predominant in 2 patients at 2 weeks after the end of therapy. All the sequencing results of serial blood samples in one patient were consistent with nested PCR data. CONCLUSIONS: The presence of YMDD motif HBV polymerase mutant may be possible before administration of lamivudine in Korean chronic hepatitis B patients. Nested PCR assay would be an useful method to detect YMDD mutant.

Terminal Protein-specific scFv Production by Phage Display

BACKGROUND: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the 65th residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. METHODS: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains 57~80th amino acid residues of TP domain. After isolation of mRNA of heavy-variable region (VH) and light-chain variable region (VL) from the spleen of the immunized mouse, DNA of VH and VL were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. ScFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. RESULTS: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. CONCLUSION: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

Anti-HBc IgM and anti-delta screening by EIA method

The clinical value of an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-HBc IgM was evaluated by testing 202 sera from acute viral hepatitis B (AVHB), hepatitis B (HB), chronic hepatitis (CAH), chronic liver disease (CLD), cirrhosis, primary hepatoma, HBsAg carrier, acute viral hepatitis A (AVHA), hepatitis A (HA), non-A, non-B (NANB) hepatitis and miscellaneous conditions other than hepatic disease, and 19 additional various hepatic disease cases were examined for anti-delta. In clinical situations the accurate diagnosis of HB is not always possible and the differential diagnosis seems to be very important especially in making decisions of treatment and estimation of prognosis. In overall cases the highest positive rate of anti-HBc IgM was found in AVHB as shown as 74.3% (26/35) comparing to other conditions in which the positive rate was extremely low (2.1%). The anti-HBc IgM appeared to be highly specific to AVHB (83.9%) as compared to the other. The positive rate of HBsAg was high in AVHB, CAH and HBsAg carrier (100.0%) followed by CLD, cirrhosis and HB (up to 70.8%). The ALT activities and ALPalb fractions were significantly high in AVHB (p less than 0.005). The correlation between the positivity of anti-HBc IgM and highly abnormal ALT appeared be high. AVHB was confined mostly to 10-20 age group and the male to female ratio was about 6 to 1. Subgroup of AVHB II with positive anti-HBc IgM appeared to have a greater chance being positive for HBsAg and ALPalb. The S/N ratio of anti-HBc IgM was as high as 20 which was unique to AVHB.