Antiviral effects against influenza A virus infection by a short hairpin RNA targeting the non-coding terminal region of the viral nucleoprotein gene.

RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.

Comparison of the antiviral potential among soluble forms of herpes simplex virus type-2 glycoprotein D receptors, herpes virus entry mediator A, nectin-1 and nectin-2, in transgenic mice.

Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-2Ig. In a transgenic mouse line with high expression of nectin-1Ig, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.

Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection.

The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml-1 showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice.

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh.

In mammals, the cornified cell envelope forms beneath the plasma membrane in epithelia and provides a vital physical barrier consisting of insoluble proteins cross-linked by transglutaminase (TGase). In the horseshoe crab Tachypleus tridentatus, TGase is stored in hemocytes and secreted in response to the simulation of bacterial lipopolysaccharides. Here we characterized a TGase substrate designated as caraxin that was identified in horseshoe crab cuticle. One of the homologs, caraxin-1, possessed a unique domain structure consisting of N-and C-terminal heptad repeats and a central domain with a tandem-repeated structure of a pentapeptide. Western blotting showed the specific localization of caraxin-1 in sub-cuticular epidermis. Moreover, we identified the pentapeptide motif to be a chitin-binding unit. Analytical ultracentrifugation revealed that caraxin-1 exists as an oligomer with 310-350 kDa, which is approximately 20-mer based on the molecular mass of the monomer. The oligomers were cross-linked by TGase to form an elaborate mesh with honeycomb structures, which was electron-microscopically found to be different from the clotting mesh triggered by lipopolysaccharide-induced hemocyte exocytosis. We determined several cross-linking sites in the N-and C-terminal domains of caraxin-1. The replacements of Leu to Pro at positions 36 and 118 in caraxin-1 reduced the alpha-helix content, which destroyed the TGase-dependent mesh, thus indicating the importance of the N-and C-terminal domains for the proper mesh formation. In arthropods, TGase-dependent protein cross-linking may be involved in the initial stage of host defense at the sub-cuticular epidermis, as in the case of mammalian skin.