Identification of the Host Interactors of wsv112 of WSSV by Yeast Two-Hybrid

Abstract:

The ORF wsv112 of white spot syndrome virus (WSSV) encodes a dUTPases. It plays an essential role in nucleotide biosynthesis. Hydrolysis of dUTP by UTPase produces dUMP, required for the de novo synthesis of dTTP, and maintains low cellular ratios of dUTP/dTTP, thus preventing the mis-incorporation of uracil into chromosomal DNA. In order to identify the host interactors of wsv112，wsv112 was cloned into the bait vector pGBKT7 and used to screen an intestine cDNA library of Litopenaeus vannamei, which had previously been constructed by yeast two-hybrid sequencing transformation. The positive clone was identified through different culture media, color change, polymerase chain reaction (PCR), and sequencing. The result showed that the bait plasmid pGBKT7-112 showed no virulence or self-activating effect on yeast strain Y2H Gold. A total of 526 blue clones were screened, which were analyzed by PCR and homology analysis using the BLAST in NCBI, and 6 possible interaction proteins of Litopenaeus vannamei were obtained. Then through the Yeast two-hybrid reply hybridization experiment, only two gene interactions were confirmed with the wsv112. They were identified as lectin C gene of Marsupenaeus japonicas (AGW27416.1) and 40S ribosomal protein S20 gene of Procambarus clarkia (ALE99171.1) with 37% and 98% identity. This study may provide a theoretical basis for further study of the wsv112 interaction mechanisms.