Hepatitis C virus envelope proteins bind lactoferrin.
Hepatitis C virus (HCV) has two envelope proteins, E1 and E2, which form a heterooligomer. During dissection of interacting regions of HCV E1 and E2, we found the presence of an interfering compound or compounds in skim milk. Here we report that human as well as bovine lactoferrin, a multifunctional immunomodulator, binds two HCV envelope proteins. As determined by far-Western blotting, the bacterially expressed E1 and E2 could bind lactoferrin in human milk directly separated or immunopurified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bindings of lactoferrin and HCV envelope proteins in vitro were confirmed by another method, the pull-down assay, with immunoprecipitated lactoferrin-bound protein A resin. By the same assay, mammal-expressed recombinant E1 and E2 were also demonstrated to bind human lactoferrin efficiently in vitro. Direct interaction between E2 and lactoferrin was proved in vivo, since anti-human lactoferrin antibody efficiently coimmunoprecipitated with secreted and intracellular forms of the E2 protein, but not glutathione S-transferase (GST), from lysates of HepG2 cells transiently cotransfected with the expression plasmids of human lactoferrin and gE2t-GST (the N-terminal two-thirds of E2 fused to GST) or GST. The N-terminal loop of lactoferrin, the region important for the antibacterial activity, has only a little role in the binding ability to HCV E2 but affected the secretion or stability of lactoferrin. Taken together, these results indicate the specific interaction between lactoferrin and HCV envelope proteins in vivo and in vitro.
Yi
M
M
Department of Molecular Biology, Medical School, Kanazawa University, Takara-Machi, Japan.
Kaneko
S
S
Yu
D Y
DY
Murakami
S
S
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism*
C087035:E1 protein, Hepatitis C virus; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D007781:Lactoferrin