Microtubule associated protein 9 inhibits liver tumorigenesis by suppressing ERCC3.

BACKGROUND: Chromosomal instability plays an important part in cancer, but its genetic basis in liver tumorigenesis remains largely unclear. We aimed to characterize the mechanistic significance and clinical implication of mitotic regulator microtubule-associated protein 9 (MAP9) in hepatocellular carcinoma (HCC). METHODS: The biological functions of MAP9 were determined by in vitro tumorigenicity assays. Systematic MAP9 knockout mouse (MAP9∆/∆) and hepatocyte-specific MAP9 knockout mouse (MAP9∆/∆hep) were generated to confirm the role of MAP9 in HCC. The clinical impact of MAP9 was assessed in primary HCC tissue samples. FINDINGS: We found that MAP9 was frequently silenced in HCC tissue samples. The transcriptional silence of MAP9 in liver cancer cell lines and tissue samples was mediated by its promoter hypermethylation. MAP9 promoter hypermethylation or downregulation was associated with poor survival and recurrence in patients with HCC. Mechanistically, ectopic expression of MAP9 in LO2 and HepG2 cell lines impaired cell proliferation, colony formation, migration and invasion, and induced cell apoptosis and cycle arrest, whereas knockdown of MAP9 in Miha cell line showed the opposite effects. We found that MAP9∆/∆ mice spontaneously developed a liver hyperplastic nodule and MAP9∆/∆hep accelerated diethylnitrosamine-induced HCC formation. The tumour suppressive effect of MAP9 in HCC was mediated by downregulating excision repair cross-complementation group 3 (ERCC3), a nucleotide excision repair gene. Restoration of ERCC3 expression possessed an oncogenic potency and abrogated the tumour suppressive effects of MAP9. INTERPRETATION: MAP9 is a novel tumour suppressor in HCC by inhibiting ERCC3 expression, and serves as a prognostic factor in HCC patients.

Recombinant influenza A virus hemagglutinin HA2 subunit protects mice against influenza A(H7N9) virus infection.

A novel avian influenza A(H7N9) virus has emerged to infect humans in eastern China since 2013. An effective vaccine is needed because of the high mortality despite antiviral treatment and intensive care. We sought to develop an effective vaccine for A(H7N9) virus. The HA2 subunit was chosen as the vaccine antigen because it is highly conserved among the human A(H7N9) virus strains. Moreover, in silico analysis predicted two immunogenic regions within the HA2 subunit that may contain potential human B-cell epitopes. The HA2 fragment was readily expressed in Escherichia coli. In BALB/c mice, intraperitoneal immunization with two doses of HA2 with imiquimod (2-dose-imiquimod) elicited the highest geometric mean titer (GMT) of anti-HA2 IgG (12699), which was greater than that of two doses of HA2 without imiquimod (2-dose-no-adjuvant) (6350), one dose of HA2 with imiquimod (1-dose-imiquimod) (2000) and one dose of HA2 without imiquimod (1-dose-no-adjuvant) (794). The titer of anti-HA2 IgG was significantly higher in the 1-dose-imiquimod group than the 1-dose-no-adjuvant group. Although both hemagglutination inhibition titers and microneutralization titers were below 10, serum from immunized mice showed neutralizing activity in a fluorescent focus microneutralization assay. In a viral challenge experiment, the 2-dose-imiquimod group had the best survival rate (100 %), followed by the 2-dose-no-adjuvant group (90 %), the 1-dose-imiquimod group (70 %) and the 1-dose-no-adjuvant group (40 %). The 2-dose-imiquimod group also had significantly lower mean pulmonary viral loads than the 1-dose-imiquimod, 1-dose-no-adjuvant and non-immunized groups. This recombinant A(H7N9)-HA2 vaccine should be investigated as a complement to egg- or cell-based live attenuated or subunit influenza vaccines.

Toll-like receptor 7 agonist imiquimod in combination with influenza vaccine expedites and augments humoral immune responses against influenza A(H1N1)pdm09 virus infection in BALB/c mice.

Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.

A cholesterol tag at the N terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo.

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG3) conjugated to the peptides' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG3-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide's insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.

Interaction domain of glycoproteins gB and gH of Marek's disease virus and identification of an antiviral peptide with dual functions.

Our previous study reported that both glycoproteins gB and gH of the herpesvirus Marek's disease virus (MDV) contain eleven potential heptad repeat domains. These domains overlap with α-helix-enriched hydrophobic regions, including the gH-derived HR1 (gHH1) and HR3 (gHH3) and gB-derived HR1 (gBH1) regions, which demonstrate effective antiviral activity, with 50% inhibitory concentrations (IC(50)) of less than 12 µM. Plaque formation and chicken embryo infection assays confirmed these results. In this study, biochemical and biophysical analyses detected potential interactions between these peptides. gHH1, gHH3, and gBH1 were found to interact with each other in pairs. The complex formed by gHH3 and gBH1 showed the most stable interaction at a molar ratio of 1:3, the binding between gHH1 and gBH1 was relatively weak, and no interaction was observed between the three HR peptides. These results indicate that gHH3 and gBH1 are likely the key contributors to the interaction between gB and gH. Furthermore, each HR peptide from herpesvirus glycoproteins did not effectively inhibit virus infection compared with peptides from a class I enveloped virus. In this report, the HR mimic peptide modified with a double glutamic acid (EE) or a double lysine (KK) at the non-interactive sites (i.e., solvent-accessible sites) did not noticeably affect the antiviral activity compared with the wild-type HR peptide, whereas tandem peptides from gH-derived gHH1 and gB-derived gBH1 (i.e., gBH1-Linker-gHH1) produced efficient antiviral effects, unlike the individual peptides. The proposed interpretation of inhibition of entry has been addressed. Our results support the hypothesis that the interaction domain between glycoproteins gH and gB is a critical target in the design of inhibitors of herpesvirus infection.

Characterisation and evaluation of antiviral recombinant peptides based on the heptad repeat regions of NDV and IBV fusion glycoproteins.

Mixed virus infections can cause livestock losses that are more devastating than those caused by single virus infections. Newcastle disease virus (NDV) and infectious bronchitis virus (IBV), serious threats to the poultry industry, can give rise to complex mixed infections that hinder diagnosis and prevention. In this study, we show that newly designed peptides, which are based on the heptad repeat (HR) region of the fusion glycoproteins from NDV and IBV, have more potent antiviral activity than the mother HR peptides. Plaque formation and chicken embryo infectivity assays confirmed these results. The novel peptides completely inhibited single virus infections and mixed infections caused by NDV and IBV. Furthermore, we assessed cell toxicity and possible targets for the peptides, thereby strengthening the notion that HR2 is an attractive site for therapeutic intervention. These results suggest the possibility of designing a relatively broad-spectrum class of antiviral peptides that can reduce the effects of mixed-infections.