RAS from Litopenaeus vannamei Interacts with WSSV-VP26 in vitro

Abstract:

White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp farming, which has great impacts by causing economic losses in aquaculture industry. It belongs to the Whispovirus genus of the Nimaviridae family. However, how the virus enter the cell is not very clear. Interaction between the virus and host is important for viral infection. Recently, some researchers found that GTP-binding proteins, such as Rab5, Rab6, and Rab7 interacted with infectious hypodermol and hematopoietic necrosis virus (IHHNV) and WSSV. However, the studies did not reveal the interaction between RAS protein and WSSV. The RAS protein belongs to the GTP-binding proteins and exists in eukaryotes from yeast to humans. Being a membrane protein, RAS becomes post-translationally modified after synthesis and is transported via the endoplasmic reticulum and Golgi complex route. Thus, in this study, the RAS of Litopenaeus vannamei was cloned. Then, it was ligated with prokaryotic expression vector pBAD/gⅢA using T4 DNA ligase, transformed into E.coli TOP10, and induced with L-arabinose. Pure RAS protein was acquired using Co2+ affinity chromatography purification. Mass spectrometry analysis showed the recombinant protein was RAS of L. vannamei. Far-western and ELISA assays were applied to determine the interaction of RAS with WSSV-VP26, WSSV-VP28N, and WSSV-VP37. The Far-western assay showed that the RAS protein interacted with VP26. The ELISA assay indicated that the interaction between RAS and VP26 grew stronger with the increasing concentration of RAS. Taken together, the RAS protein was responsible for WSSV infection. This study could provide the basis for research on the mechanism of WSSV invasion.