Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024

Orf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024.

The yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database.

In this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay.

Our findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.