The Mechanism Of Inhibition On WSSV Infection By TRIM32 In Cherax Quadricarinatus

Tripartite motif-containing(TRIM)proteins are highly conserved molecules that participate in a variety of biological processes such as regulation of development,apoptosis,and innate immunity in vertebrates.However,the functions of these proteins are still poorly understood in crustaceans even in invertebrate.Previously,we found that the transcript of a TRIM32 homolog(named CqTrim32 like gene)was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue(Hpt)cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome infection.To further characterize the CqTRIM32 and its role in WSSV-host interaction in a crustacean,the full-length cDNA of CqTrim32 was identified with 975 bp which contains a predicted open reading frame encoding 195 amino acids with a putative molecular mass of 21.45 kDa protein.Multiple amino acid alignment showed that CqTRIM32 contained a conserved RING-finger domain but without B-BOX domain and coiled-coil region,which was different from the traditional TRIMs family.The phylogenetic tree construction showed that CqTrim32 was grouped into an independent branch without closely relative species.The transcript distribution analysis revealed a ubiquitous presence of CqTrim32 in the tissues selected with the highest expression in muscle,relatively abundant in Hpt and the lowest presence in eyestalk.Interestingly,the expression of CqTrim32 was significantly up-regulated at both 24h and 48h after WSSV challenge in vivo in crayfish Hpt tissue.Meanwhile,the expression of CqTrim32 was significantly up-regulated at both 12 h and 24h after WSSV challenge in vitro in Hpt cells too.Furthermore,the Hpt cells exhibited significantly lower ubiquitination activity when silenced with CqTrim32,in which the transcription of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly upregulated,suggesting that the increase of WSSV replication was likely to be directly caused by the loss-of-function of CqTrim32 via its regulation on the cellular ubiquitination activity.Meanwhile,protein transfection showed that rCqTRIM32 can inhibit the the expression of VP28.And pulldown assay implies that the reason why rCqTRIM32 can inhibit the the expression of VP28 is that rCqTRIM32 can directly interact with VP28,an envelope protein.Importantly,CqTRIM32 is found to interact with viral envelope protein component of VP28,and participates in process of ubiquitination.Taken together,CqTRIM32 immediately interacts with VP28 to inhibit the replication of WSSV through ubiquitination mediated by CqTRIM32.