Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3 0 -5 0 order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.

Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1–NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP–EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV–NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV–NTCP attachment to the viral invasion beyond the host cell membrane.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs.

Edited by Ivan SadowskiKeywords: KRAB domain Zinc finger protein TRIM28 SETDB1 HP1-gamma HIV-1 LTR a b s t r a c t Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed P50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >10 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 10 HEV genome RNA copies (10 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products.

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The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti‐HBV reagents; it has a very low resistance rate and is used as the first‐line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV‐refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare‐up) and assessed ETV resistance using replication‐competent 1.38‐fold HBV genome‐length molecular clones. The full genome sequences of infected HBVs in ETV‐refractory patients were determined. The HBV molecular clones were generated with the patient‐derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core‐particle‐associated HBV DNA using Southern blotting and real‐time polymerase chain reaction. Among these cases, ETV‐resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV‐resistant mutation was detected in the other cases. The identified ETV‐resistant mutations did not confer resistance to tenofovir disoproxil fumarate. Conclusion: The HBV replication model with patient‐derived sequences is useful for assessing replication efficiency, susceptibility to anti‐HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment‐failure cases of chronic hepatitis B. (Hepatology Communications 2017;1:110‐121)