Dengue virus core protein attenuates the host IFN response. In order to examine the effect that DENV-C protein has on induction of type I IFN, expression vectors were constructed containing the DENV-C gene. After transfection of HEK293 cells with the DENV-C expression vector, cells were treated with poly (I:C) and subsequently monitored for induction of IFN by qRT-PCR. Cells expressing the DENV-C protein showed decreased levels of IFN-beta expression when compared to mock transfected controls. A similar phenotype was observed using other cell types Huh7 and A549. In addition, overexpression of DENV-C decreased AV activity as measured by viral titers after treatment with poly (I:C). Components of the IFN-beta enhanceosome were examined by western blot analysis. No observable differences were detected in either the amount or phosphorylation of IRF3, NF-kappa B or c-Jun, indicating that these proteins were not responsible for the observed phenotype Dengue virus core protein interacts with host proteins. Possible interaction partners for DENV-C were identified by co-immunoprecipitation (Co-IP), followed by mass spectrometry (MS). MS identified a number of possible host interaction partners. Results were then verified by Co-IP and reverse Co-IP followed by western blot analysis. Virus-host protein interactions were also verified by examining co-localization by confocal microscopy. The cellular protein NF45was determined to interact with DENV-C. Mutations in the DENV-C gene affect its ability to abrogate the host IFN response. A number of mutations were introduced into the DENV-C gene using site directed mutagenesis. The DENV-C mutants were then examined for their ability to abrogate induction of type I IFN after treatment with Poly(I:C). Mutations in the N-terminus appear to affect the observed phenotype of DENV-C. In addition, localization of DENV-C and mutants were observed by confocal microscopy. Mutations to the N-terminus of the protein affect the nuclear localization of DENV-C, indicating that the N-terminus is important for the affect that DENV-C has on induction of type I IFN either through mediating interactions with specific host proteins, or by influencing the localization of the DENV-C protein.
| Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar et al. (2013) Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles. J Virol 87:13094-106 |
