Biochemical and functional characterization of Epstein-Barr virus-encoded BARF1 protein: interaction with human hTid1 protein facilitates its maturation and secretion.
EBV BARF1 gene encodes a secretory protein with transforming and mitogenic activities. In this report, the post-translational modification, folding, maturation and secretion of BARF1 are systematically studied by site-directed mutagenesis and overexpression of the protein in mammalian cells using the vaccinia/T7 system. The protein was shown to be post-translationally modified by N-linked glycosylation on the asparagine 95 residue. This modification was confirmed to be essential for the maturation and secretion of the protein. Analysis of the four cysteine residues by site-directed mutagenesis demonstrated that cysteine 146 and 201 were essential for proper folding and secretion of the protein. To search for human proteins involved in the maturation process of the protein, a yeast two-hybrid screening was carried out using the BARF1 sequence from amino acids 21-221 (BARF1Delta) as bait, leading to the identification of human hTid1 protein as a potential interacting protein. This interaction was subsequently confirmed by coimmunoprecipitation and dual immunofluorescent labeling of cells coexpressing BARF1 and hTid1, and the interaction domain in hTid1 was mapped to amino acids 149-320. Interestingly, coexpression of BARF1 with hTid1 demonstrated that hTid1 could promote secretion of BARF1, suggesting that hTid1 may act as a chaperone to facilitate the folding, processing and maturation of BARF1.
Wang
L
L
School of Biological Sciences, Nanyang Technological University, Proteos, Singapore.
Tam
J P
JP
Liu
D X
DX
D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
C086495:BARF1 protein, Human herpesvirus 4; C120664:DNAJA3 protein, human; D050956:HSP40 Heat-Shock Proteins; D014764:Viral Proteins