Abstract
Hepatitis B virus (HBV) X protein (HBx) has been determined to play a crucial role in the replication and transcription of HBV, and its biological functions mainly depend on the interaction with other host proteins. This study aims at screening the proteins that bind to the key functional domain of HBx by integrated proteomics. Proteins that specifically bind to the transactivation domain of HBx were selected by comparing interactors of full-length HBx and HBx-D5 truncation determined by glutathione-S-transferase (GST) pull-down assay combined with mass spectrometry (MS). The function of HBx interactor Pin1 in HBV replication was further investigated by in vitro experiments. In this study, a total of 189 proteins were identified from HepG2 cells that specifically bind to the transactivation domain of HBx by GST pull-down and subsequent MS. After gene ontology (GO) analysis, Pin1 was selected as the protein with the highest score in the largest cluster functioning in protein binding, and also classified into the cluster of proteins with the function of structural molecule activity, which is of great potential to be involved in HBV life cycle. The interaction between Pin1 and HBx has been further confirmed by Ni2+-NTA pulldown assay, co-immunoprecipitation, and immunofluorescence microscopy. HBsAg and HBeAg levels significantly decreased in Pin1 expression inhibited HepG2.2.15 cells. Besides, the inhibition of Pin1 expression in HepG2 cells impeded the restored replication of HBx-deficient HBV repaired by ectopic HBx expression. In conclusion, our study identified Pin1 as an interactor binds to the transactivation domain of HBx, and suggested the potential association between Pin1 and the function of HBx in HBV replication.
Highlights
Hepatitis B virus (HBV) infection is one of the most important risk factors of hepatocellular carcinoma (HCC)[1,2,3]
Published studies have identified abundant proteins interact with HBx, such as jumonji C-domain-containing 5 (JMJD5)[24], focal adhesion protein[25], and H sp4026, which participated in the HBV replication and development of HBV related hepatocellular carcinoma
This study is to our knowledge the first study to select the proteins that interact with the transactivation domain of HBx, which can be regarded as the “key” host proteins that involved in the functions of HBx
Summary
Hepatitis B virus (HBV) infection is one of the most important risk factors of hepatocellular carcinoma (HCC)[1,2,3]. The viral genome is a 3.2 kb, partially double-stranded, circular DNA, which consist of 4 open reading frames (ORFs) named pre-C/C, pre-S/S, P, and X, respectively[5] Among these ORFs, the X ORF is responsible for encoding HBx p rotein[6], which is considered to be a multifunctional regulator and one of the most important factors that involved in the pathogenesis and carcinogenesis of H BV7. This study aims at screening the potential host proteins that may interact with the key function domain of HBx systematically. 189 proteins that bind to the transactivation domain of HBx were identified, among which we selected Pin[1] as an important host factor and further investigated the role of Pin[1] in HBV replication
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