An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

Expression of the readthrough transcript CiDRE in alveolar macrophages boosts SARS-CoV-2 susceptibility and promotes COVID-19 severity.

Lung infection during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin-I-converting enzyme 2 (ACE2) receptor induces a cytokine storm. However, the precise mechanisms involved in severe COVID-19 pneumonia are unknown. Here, we showed that interleukin-10 (IL-10) induced the expression of ACE2 in normal alveolar macrophages, causing them to become vectors for SARS-CoV-2. The inhibition of this system in hamster models attenuated SARS-CoV-2 pathogenicity. Genome-wide association and quantitative trait locus analyses identified a IFNAR2-IL10RB readthrough transcript, COVID-19 infectivity-enhancing dual receptor (CiDRE), which was highly expressed in patients harboring COVID-19 risk variants at the IFNAR2 locus. We showed that CiDRE exerted synergistic effects via the IL-10-ACE2 axis in alveolar macrophages and functioned as a decoy receptor for type I interferons. Collectively, our data show that high IL-10 and CiDRE expression are potential risk factors for severe COVID-19. Thus, IL-10R and CiDRE inhibitors might be useful COVID-19 therapies.

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses.

Many patients with severe COVID-19 suffer from pneumonia and the elucidation of the mechanisms underlying the development of this severe condition is important. The in vivo function of the ORF8 protein secreted by SARS-CoV-2 is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2 and found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrated that the serum ORF8 protein levels are well-correlated with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a SARS-CoV-2 viral cytokine involved in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

Ubiquitin specific peptidase 32 acts as an oncogene in epithelial ovarian cancer by deubiquitylating farnesyl-diphosphate farnesyltransferase 1.

Epithelial ovarian cancer (EOC) is the seventh most common cancer worldwide and the deadliest gynecological malignancy because of its aggressiveness and high recurrence rate. To discover new therapeutic targets for EOC, we combined public EOC microarray datasets with our previous in vivo shRNA screening dataset. The top-ranked gene ubiquitin specific peptidase 32 (USP32), coding a deubiquitinating enzyme, is a component of the ubiquitin proteasome system. Clinically, USP32 is expressed in primary ovarian cancer, especially in metastatic peritoneal tumors, and negatively impacts the survival outcome. USP32 regulates proliferative and epithelial mesenchymal transition capacities that are associated with EOC progression. Proteomic analysis identified farnesyl-diphosphate farnesyltransferase 1 (FDFT1) as a novel substrate of USP32 that is an enzyme in the mevalonate pathway, essentially associated with cell proliferation and stemness. USP32 and FDFT1 expression was higher in tumor spheres than in adherent cells. Inhibition of USP32, FDFT1, or mevalonate pathway considerably suppressed tumor sphere formation, which was restored by adding squalene, a downstream product of FDFT1. These findings suggested that USP32-FDFT1 axis contributes to EOC progression, and could be novel therapeutic targets for EOC treatment.

A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Deneddylation by SENP8 restricts hepatitis B virus propagation.

Proteins newly synthesized from messenger RNA undergo Posttranslational modifications (PTMs) such as phosphorylation, glycosylation, methylation, and ubiquitination. These PTMs have important roles in protein stability, localization, and conformation and have been reported to be involved in hepatitis B virus (HBV) propagation. Although ubiquitination plays an essential role in HBV life cycles, the involvement of ubiquitin-like proteins (UBLs) in HBV life cycles has been understudied. Through comprehensive gain- and loss-of-function screening of UBLs, we observed that neddylation, a PTM in which neural precursor cell, expressed developmentally downregulated 8 (NEDD8) is conjugated to substrate proteins, was required for efficient HBV propagation. We also found that overexpression of sentrin-specific protease 8 (SENP8), which cleaves conjugated NEDD8, suppressed HBV propagation. Further, the catalytic activity of SENP8 was required for the suppression of HBV propagation. These results indicated that the reduction of neddylation negatively regulated HBV propagation. In addition, we demonstrated that suppression of HBV propagation via SENP8 overexpression was independent of hepatitis B protein X (HBx) and HBV promoter activity. Therefore, our data suggested that neddylation plays an important role in the late stages of HBV life cycles.

Quantitative and qualitative lipid improvement with chronic hepatitis C virus eradication using direct-acting antivirals.

AIM: Direct-acting antivirals have revolutionized hepatitis C virus (HCV) therapy by providing a high sustained virological response (SVR) rate and subsequent favorable lipid increases. Proprotein convertase subtilisin-kexin like-9 (PCSK9) plays an important role in regulating quantitative lipid levels. This study examined the interactions between quantitative PCSK9 and lipid changes, as well as qualitative lipid changes in terms of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligand containing apolipoprotein B (LAB) and high-density lipoprotein (HDL) cholesterol uptake capacity (HDL-CUC). METHODS: Patients with chronic HCV infection (N = 231) who achieved an SVR by direct-acting antivirals without lipid-lowering therapy were included for comparisons of PCSK9, LAB, HDL-CUC, and other clinical indices between pretreatment and SVR12 time points. RESULTS: LDL (LDL) cholesterol and HDL cholesterol levels were quantitatively increased at SVR12, along with higher PCSK9 (all p < 0.0001). PCSK9 was significantly correlated with LDL cholesterol (r = 0.244, p = 0.0003) and apolipoprotein B (r = 0.222, p = 0.0009) at SVR12. Regarding qualitative LDL changes, LAB was significantly decreased and LAB/LDL cholesterol and LAB/apolipoprotein B proportions were improved at SVR12 (all p < 0.0001). In terms of qualitative HDL changes, HDL-CUC was significantly ameliorated, along with HDL-CUC/HDL cholesterol, HDL-CUC/ apolipoprotein A1, and HDL-CUC/ apolipoprotein A2 at SVR12 (all p < 0.0001). CONCLUSIONS: HCV eradication by direct-acting antivirals may produce quantitative lipid profile changes, along with PCSK9 production recovery in addition to qualitative lipid improvement, which possibly confers the additional secondary benefits of atherosclerosis improvement and cardiovascular disease event reduction.

In Vivo Dynamics of Reporter Flaviviridae Viruses.

Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

Peroxiredoxin 1, a Novel HBx-Interacting Protein, Interacts with Exosome Component 5 and Negatively Regulates Hepatitis B Virus (HBV) Propagation through Degradation of HBV RNA.

Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here, we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Coimmunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acids 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens, and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1, and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA coimmunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was coimmunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection.IMPORTANCE Hepatitis B virus (HBV) infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to downregulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.

USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.

Infection with flaviviruses requires BCLXL for cell survival.

BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.