Description: Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced, HBV gene expression and replication in hepatoma cells. Mechanistically, the SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on the HBV genome (nucleotides [nt] 3009 to 3019) was responsible for the inhibition of the SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with the HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms. IMPORTANCE HBV is a major human pathogen whose replication is regulated by host factors. Liver-enriched transcription factors are critical for many liver functions, including metabolism, development, and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this study, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.

Description: We engineered a disulfide-stabilized influenza virus hemagglutinin (HA) trimer, termed HA 3 -SS, by introducing cysteine residues into the HA stem to covalently bridge the three protomers. HA 3 -SS has increased thermostability compared to wild-type HA, and binding of head- and stem-targeted antibodies (Abs) is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza virus HA immunogens.

Description: An accessory gene between the S and E gene loci is contained in all coronaviruses (CoVs), and its function has been studied in some coronaviruses. This gene locus in human coronavirus OC43 (HCoV-OC43) encodes the ns12.9 accessory protein; however, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein (HCoV-OC43-ns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-ns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo . Intriguingly, exogenous ns12.9 and heterologous viroporins with ion channel activity could compensate for the production of HCoV-OC43-ns12.9, indicating that the ion channel activity of ns12.9 plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication revealed that ns12.9 protein had no measurable effect on virus entry, subgenomic mRNA (sgmRNA) synthesis, and protein expression. Further characterization revealed that HCoV-OC43-ns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus (HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response, and virulence in HCoV-OC43-ns12.9-infected mice were observed compared to the levels for HCoV-OC43-WT-infected mice. Taken together, our results demonstrated that the ns12.9 accessory protein functions as a viroporin and is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection. IMPORTANCE HCoV-OC43 was isolated in the 1960s and is a major agent of the common cold. The functions of HCoV-OC43 structural proteins have been well studied, but few studies have focused on its accessory proteins. In the present study, we demonstrated that the ns12.9 protein is a newly recognized viroporin, and the ns12.9 gene knockout virus (HCoV-OC43-ns12.9) presents a growth defect in vitro and in vivo . We identified the important functions of the ns12.9 viroporin in virion morphogenesis during HCoV-OC43 infection. Furthermore, mice infected with HCoV-OC43-ns12.9 exhibited reduced inflammation and virulence accompanied by a lower titer in the brain than that of wild-type-infected mice, suggesting the ns12.9 viroporin influences virus pathogenesis. Therefore, our findings revealed that the ns12.9 viroporin facilitates virion morphogenesis to enhance viral production, and these results provided a deeper understanding of HCoV-OC43 pathogenesis.

Description: Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno , where ndhA and ndhB overlap by 4 bp and are ~26 kb away from pno . As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.

Description: There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

Description: Two ferret-adapted H5N1 viruses capable of respiratory droplet transmission have been reported with mutations in the hemagglutinin receptor-binding site and stalk domains. Glycan microarray analysis reveals that both viruses exhibit a strong shift toward binding to "human-type" α2-6 sialosides but with notable differences in fine specificity. Crystal structure analysis further shows that the stalk mutation causes no obvious perturbation of the receptor-binding pocket, consistent with its impact on hemagglutinin stability without affecting receptor specificity.

Description: In this study, we attempted to adopt the auxotrophic mevalonate synthase mutant ( mvaS mutant) of Staphylococcus aureus to study whether a nongrowing but viable cell population is tolerant to bactericidal antibiotics. The mevalonate-depleted nongrowing mvaS mutant was found tolerant to antibiotics. Surprisingly, after prolonged cultivation, we obtained stable mvaS variants that were able to grow without mevalonate, which suggested unknown mechanisms for compensating undecaprenyl pyrophosphate production without mevalonate in S. aureus .

Description: To compare the in vitro antibacterial efficacies and resistance profiles of rifampin-based combinations against methicillin-resistant Staphylococcus aureus (MRSA) in a biofilm model, the antibacterial activities of vancomycin, teicoplanin, daptomycin, minocycline, linezolid, fusidic acid, fosfomycin, and tigecycline alone or in combination with rifampin against biofilm-embedded MRSA were measured. The rifampin-resistant mutation frequencies were evaluated. Of the rifampin-based combinations, rifampin enhances the antibacterial activities of and even synergizes with fusidic acid, tigecycline, and, to a lesser extent, linezolid, fosfomycin, and minocycline against biofilm-embedded MRSA. Such combinations with weaker rifampin resistance induction activities may provide a therapeutic advantage in MRSA biofilm-related infections.

Description: Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab' fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.

Description: Virus replication is mediated by interactions between the virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78-〉Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in the production of infectious virus particles. M2-K78R mutant progeny contained more hemagglutinin (HA) proteins, less viral RNAs, and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination by electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with the M1 protein to a lesser extent than for the wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal levels of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that the M2-K78R mutant virus induced autophagy and apoptosis earlier than did the wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating the efficient packaging of the viral genome into virus particles and the timing of virus-induced cell death. IMPORTANCE Annual epidemics and recurring pandemics of influenza viruses represent very high global health and economic burdens. The influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in virus entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N terminus of M2 proteins. However, viruses that are resistant to rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.