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Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with higher transmission potential have been emerging globally, including SARS-CoV-2 variants from the United Kingdom and South Africa. We report 4 travelers from Brazil to Japan in January 2021 infected with a novel SARS-CoV-2 variant with an additional set of mutations.

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection. In human cells, invading pathogens trigger an innate immune response that helps prevent viral replication and spread. Here, the authors reveal a mechanism of innate immunity that selectively leads to the autophagic degradation of hepatitis B virus core protein.

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 pointmutation 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

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild infectious disease that can, however, occasionally lead to severe neurological impairments. These two studies, by Nishimura et al . and Yamayoshi et al ., independently identify two different receptors for EV71—P-selectin glycoprotein ligand-1 ((PSGL-1) and scavenger receptor class B, member 2 (SCARB2) ( pages 728–729 ) and ( pages 798–801 ). Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), a common febrile disease occurring mainly in young children. Although clinical manifestations of HFMD are usually mild and self limiting, a severe EV71 outbreak can lead to a diverse array of neurological diseases. Identification of the specific cellular receptors is crucial for elucidating the mechanism of early virus-host interactions and the pathogenesis of enteroviruses 1 . Here we identify human P-selectin glycoprotein ligand-1 (PSGL-1; CD162), a sialomucin membrane protein expressed on leukocytes that has a major role in early stages of inflammation 2 , 3 , 4 , as a functional receptor for EV71 using an expression cloning method by panning 5 . The N-terminal region of PSGL-1 binds specifically to EV71. Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible mouse L929 cells. Five out of eight EV71 strains bound soluble PSGL-1 and used intact PSGL-1 as the primary receptor for infection of Jurkat T cells. Three other EV71 strains did not use PSGL-1, suggesting the presence of strain-specific replication of EV71 in leukocytes. EV71 replicated in nonleukocyte cell lines in a PSGL-1–independent manner, indicating the presence of alternative receptor(s) for EV71. The identification of PSGL-1 as a receptor for EV71 sheds new light on a role for PSGL-1–positive leukocytes in cell tropism and pathogenesis during the course of HFMD and other EV71-mediated diseases.

BackgroundHepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro.MethodsWe used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase.ResultsWe screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs.ConclusionsWe report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5'-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro.

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER) 1 , 2 , 3 . These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases 4 . HCV capsid protein (Core) associates with the LD 5 , envelope proteins E1 and E2 reside in the ER lumen 6 , and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.

Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.

Chronic hepatitis B virus (HBV) infections represent a major global health burden requiring effective therapeutic interventions. This study investigates the antiviral potential of microRNAs (miRNAs) targeting the HBV entry receptor, sodium-taurocholate cotransporting polypeptide (NTCP). Using an experimental model of primary human hepatocytes (PHHs), we highlighted a set of candidate antiviral miRNAs induced by interferon (IFN) alpha analog treatment. Notably, predictive analysis identified miR-29b-1-5p as interacting with the 3ʹ-untranslated region (3ʹ-UTR) of NTCP, suggesting a post-transcriptional regulatory mechanism. Functional analysis indicated that miR-29b-1-5p directly targeted the NTCP 3ʹ-UTR, leading to significant inhibition of NTCP transcripts. Consistently, hepatocytes overexpressing miR-29b-1-5p showed a remarkable reduction in HBV genome levels after infection. A rescue assay demonstrated that miR-29b-1-5p anti-HBV effect was specifically mediated by NTCP targeting. In summary, these findings underscore the therapeutic potential of miR-29b-1-5p against HBV, advocating for further exploration of miRNA-based therapies in the treatment of human viral infections.

Although numerous sarbecoviruses have been identified in bats, but most lack the ability to infect human cells. Some barriers limit coronavirus zoonosis, including susceptibility to host proteases. Here, we investigated whether exogenous protease treatment can circumvent host restrictions in two severe acute respiratory syndrome (SARS)-related bat coronaviruses. We found that the spike proteins of RaTG13 and Khosta-2, which are sarbecoviruses obtained from horseshoe bats in China and Russia, respectively, facilitated the ACE2-mediated entry of pseudotyped viruses into VeroE6/TMPRSS2 cells following elastase treatment. In contrast, trypsin and thermolysin exhibited no effects. Elastase-enhanced infectivity correlated with increased fusogenicity driven by the cleavage of spike proteins. This process was TMPRSS2-dependent and was inhibited by nafamostat, a TMPRSS2 inhibitor. Additionally, mutation of residue 809 within the S2 subunit of the RaTG13 spike protein (S809D) impaired elastase-induced cleavage and infectivity. Hence, proteolytic processing of the spike protein serves as a restriction to RaTG13 and Khosta-2 infections, which can be overcome by elastase. This suggests that elastase secreted in inflamed tissues during viral infection may increase the zoonotic potential of sarbecoviruses by facilitating human cell entry.