仲屋 友喜

Background: Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. Methods: The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. Results: NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. Conclusions: NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Abstract Chronic hepatitis B virus (HBV) infection is a major medical concern worldwide. Current treatments for HBV infection effectively inhibit virus replication; however, these treatments cannot cure HBV and novel treatment-strategies should be necessary. In this study, we identified tripartite motif-containing protein 26 (TRIM26) could be a supportive factor for HBV replication. Small interfering RNA-mediated TRIM26 knockdown (KD) modestly attenuated HBV replication in human hepatocytes. Endogenous TRIM26 physically interacted with HBV core protein (HBc), but not polymerase and HBx, through the TRIM26 SPRY domain. Unexpectedly, TRIM26 inhibited HBc ubiquitination even though TRIM26 is an E3 ligase. HBc was degraded by TRIM26 KD in Huh-7 cells, whereas the reduction was restored by a proteasome inhibitor. RING domain-deleted TRIM26 mutant (TRIM26ΔR), a dominant negative form of TRIM26, sequestered TRIM26 from HBc, resulting in promoting HBc degradation. Taking together, this study demonstrated that HBV utilizes TRIM26 to avoid the proteasome-dependent HBc degradation. The interaction between TRIM26 and HBc might be a novel therapeutic target against HBV infection.

Abstract Background Although conventional polymerase chain reaction (PCR) methods are widely used in diagnosis, the titer of the pathogenic virus is difficult to determine based on the PCR. In our prior report, a long-range reverse-transcription quantitative PCR (LR-RT-qPCR) assay was developed to assess the titer of UV-irradiated influenza A virus (IAV) rapidly. In this research, we focused on whether the LR-RT-qPCR assay could evaluate the titer of IAV inactivated by other methods. Methods IAV was inactivated by: heating at 100 °C for periods ranging from 1 to 15 min, treating with 0.12% sodium hypochlorite for periods ranging from 3 to 30 min, or treating with 70% ethanol for periods ranging from 10 to 30 min. Fifty percent tissue culture infectious dose (TCID₅₀) assay was performed to confirm the efficacy of the inactivation methods, followed by LR-RT-qPCR to investigate the correlation between infectivity and copy number. Results One minute heating, 3 min sodium hypochlorite treatment, or 10 min ethanol treatment was sufficient to deactivate IAV. Changes before and after the inactivations in the copy numbers on LR-RT-qPCR were significantly different among the inactivation methods. Heat-inactivation drastically decreased the copy number to below the cutoff value around 5 copies/μL after 5 min treatment. The inactivation time of heating estimated using LR-RT-qPCR was marginally higher than that determined using TCID₅₀. However, the treatments with sodium hypochlorite or ethanol moderately and minimally affected the copy numbers obtained using LR-RT-qPCR (~ 1 digit or no copy number decrease), respectively. Conclusions In addition to good applicability in UV-irradiation previously reported, the LR-RT-qPCR method is suitable for evaluating the effect of heat-inactivation on IAV infectivity. However, minor modifications may be made and investigated in the future to reduce the time intervals with TCID₅₀. Although this method is not applicable for the ethanol inactivation, rapid evaluation of the effects of chlorination on IAV can be determined by comparing copy numbers before and after treatment using the LR-RT-qPCR method.

Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog.

RD-114 virus is a replication-competent feline endogenous retrovirus. RD-114 virus contaminates several feline and canine live attenuated vaccines and the issue of contamination of RD-114 virus in vaccines should be solved. To date, three infectious molecular clones (pSc3c, pCRT1, and pRD-UCL) have been reported. In this study, we sequenced the entire nucleotide sequence of pRD-UCL and compared the nucleotide sequences of the three infectious molecular clones. As a result, these three infectious clones were nearly identical with each other in gag-pol and env coding regions. These data support the notion that the active locus of infectious RD-114 virus is single in the feline genome. The length of long terminal repeat (LTR) of pCRT1 was 47 bp shorter than those of pSc3c and pRD-UCL. The 47-bp sequence named direct repeat A (DR-A) was duplicated in the U3 region in pSc3c and pRD-UCL. Although several potential enhancer binding sites are present in the DR-A, there was no significant difference in promoter activities between the LTRs of pRD-UCL and pCRT1 in two human cell lines. We also analyzed the splicing pattern of the RD-114 virus by reverse transcription-polymerase chain reaction and confirmed that RD-114 virus is a simple retrovirus. The data presented here will provide basic information about RD-114 virus to solve the contamination issue in live attenuated vaccines.