However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application. bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated. The proteins were successfully expressed and purified in and reverse: codon bias. The fusion proteins containing hexahistidine tags were expressed in 0.01,* em P /em 0.05. (C) 2?3+ HCV replicon cells were treated with phosphate-buffered saline, Interferon (IFN, C25-140 100 U/mL), D1ECL1S+D2ECL2 or 6HV1 for 48 h. The core protein level was measured by Western blotting (upper), and the intracellular HCV RNA was determined by qRT-PCR (lower). The experiment was performed in triplicate, and the error bars represent the standard error of the means (SEM). (D) HCVcc were pretreated with 100 M D1ECL1S+D2ECL2 prior to inoculation and used to infect cells after 10-fold dilution of the inoculums (100C10). Infection with untreated virus was performed in parallel C25-140 in the presence of the D1ECL1S+D2ECL2 concentrations indicated. (E) HCVcc was mixed with phosphate-buffered saline, D1ECL1S+D2ECL2, 6HV1, or heparin (200 g/mL) and then added to precooled Huh7.5.1 cells and allowed for 3 hours incubation at 4C. Cells were then thoroughly washed in phosphate-buffered saline and subjected to RNA isolation. Cell surface bound HCV was quantified by qRT-PCR. *** em P /em 0.001,* em P /em 0.05. D1ECL1S+D2ECL2 impedes HCV cell entry by directly acting on the virus Next the mechanism whereby D1ECL1S+D2ECL2 inhibits HCV entry was determined. In theory, as the OCLN functional domain, D1ECL1S+D2ECL2 can inhibit HCV entry by acting directly on HCV particles. To test this possibility, HCV was pre-incubated with D1ECL1S+D2ECL2 (100 nM) for 1 h before inoculation, and then the mixture was diluted 10 times before adding to the cells (at a final concentration of 10 nM). Importantly, the MOI was kept constant under all of the conditions. As shown in Fig 4E, the inhibitory effect of D1ECL1S+D2ECL2 was more pronounced when the virus was pre-incubated with the protein prior to inoculation, demonstrating that D1ECL1S+D2ECL2 acted directly on the HCV particle. At the same time, a virion binding assay was performed at 4C. A well characterized HCV-cell binding inhibitor, Heparin, was used as a positive control. HCV virions pre-incubated with PBS, D1ECL1S+D2DECL2, heparin or 6HV1 were allowed to bind to Huh7.5.1 monolayers at 4C. Unbound virions were washed away and the bound virions were determined by quantifying HCV genomic SIR2L4 RNA. HCV binding to target cells is expressed as a percentage relative to the PBS vehicle-treated control. As shown in Fig 4F, D1ECL1S+D2ECL2 decreased the amount of HCV bound to the target cells. In summary, D1ECL1S+D2ECL2 appears to directly impair the ability of the virus itself to enter the target cells. Discussion TJ tetraspanin protein ECLs are responsible for TJ formation, signal sensing and even pathogen invasion as the cellular co-receptors. In this study, we demonstrated a novel strategy for expressing tetraspanin TJ proteins by fusing ECLs to an HIV-1 gp41 glycoprotein-derived -helical bundle scaffold. This method allows expression of either single loop or double loops of human OCLN. The recombinant fusion proteins C25-140 were normally enriched in the inclusion body but easily refolded and purified. Using HCV neutralizing assay, results showed that the double-loop constructs possessed significantly more biological activities than the single-loop constructs, suggesting that the structures of the fusion proteins are comparable to those of their native homologous proteins and both extracellular loops need to work synergistically. A model was established for multiple transmembrane proteins whose full-length proteins are difficult to express, providing an excellent tool for functional and.
However, most TJ proteins are four-time transmembrane proteins and are difficult to express in their correct soluble form, which limits their functional study and therapeutic application