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Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.

Hepatitis C virus (HCV) is a global disease burden, and a preventive vaccine is needed to control or eradicate the virus. Despite the advent of effective antiviral therapy, this treatment is not accessible to many patients and does not prevent reinfection, making chronic hepatitis C an ongoing global health problem. Thus, development of a prophylactic vaccine will represent a significant step toward global eradication of HCV. HCV exhibits high genetic variability, which leads frequently to immune escape. However, a considerable challenge faced in HCV vaccine development is designing an antigen that elicits broadly neutralizing antibodies. Here, we characterized the immunogenicity of a vaccine based on a soluble, secreted form of the E1E2 envelope heterodimer (sE1E2.LZ). Sera from mice immunized with sE1E2.LZ exhibited an anti-E1E2-specific response comparable to mice immunized with membrane-bound E1E2 (mbE1E2) or a soluble E2 ectodomain (sE2). In competition-inhibition ELISA using antigenic domain-specific neutralizing and nonneutralizing antibodies, sera from sE1E2.LZ-immunized mice showed nearly identical or stronger competition toward neutralizing antibodies when compared with mbE1E2. In contrast, sera from mice immunized with sE2, and to a lesser extent mbE1E2, competed more effectively with nonneutralizing antibodies. An assessment of neutralization activity using both HCV pseudoparticles and cell culture-derived infectious HCV showed that immunization with sE1E2.LZ elicited the broadest neutralization activity of the three antigens, and sE1E2.LZ induced neutralization activity against all genotypes. These results indicate that our native-like soluble glycoprotein design, sE1E2.LZ, induces broadly neutralizing antibodies and serves as a promising vaccine candidate for further development. [ABSTRACT FROM AUTHOR]

The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human--but not murine--cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse--but not human--cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain-- at least in part--the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses. [ABSTRACT FROM AUTHOR]

Background: The global control of hepatitis C virus (HCV) infection remains a great burden, owing to the high prices and potential drug resistance of the new direct-acting antivirals (DAAs), as well as the risk of reinfection in DAA-cured patients. Thus, a prophylactic vaccine for HCV is of great importance. We previously reported that a single recombinant soluble E2 (sE2) vaccine produced in insect cells was able to induce broadly neutralizing antibodies (NAbs) and prevent HCV infection in mice. Here the sE2 vaccine was evaluated in non-human primates.Methods: Rhesus macaques were immunized with sE2 vaccine in combination with different adjuvants. Vaccine-induced NAbs in antisera were tested for neutralization activities against a panel of cell culture-derived HCV (HCVcc), while T-cell responses were evaluated in splenocytes, peripheral blood mononuclear cells, and hepatic lymphocytes.Results: sE2 is able to elicit NAbs against HCVcc harboring structural proteins from multiple HCV genotypes in rhesus macaques. Moreover, sE2-immunized macaques developed systemic and intrahepatic memory T cells specific for E2. A significant correlation between the sE2-specific immunoglobulin G titers and neutralization spectrum was observed, highlighting the essential role of sE2 immunogenicity on achieving broad NAbs.Conclusions: sE2 is a promising HCV vaccine candidate that warrants further preclinical and clinical development. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals. [ABSTRACT FROM AUTHOR]

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. [ABSTRACT FROM AUTHOR]

Modification of RNA with N 6 -methyladenosine (m 6 A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m 6 A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m 6 A writers Mettl3 and Mettl14 , nor the m 6 A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro . To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling., Competing Interests: Competing interests All authors declare they have no competing interests.