An ELISA plate was coated with eE2 and probed with serum from patients chronically infected with either genotype 1, 2, or 3. Serum from a healthy donor was tested in parallel as a negative control. Anti-human HRP was used to quantify the result. Serum from the kinase inhibitor Temsirolimus three infected patients bound to J6 eE2 (genotype 2a) at similar titers regardless of infecting genotype, while the serum of the uninfected donor responded at background levels (Fig. (Fig.7A).7A). This illustrates the capacity of eE2 to be recognized by antibodies in patient sera, while also pointing out the maintenance of cross-reactive epitopes. FIG. 7. Functional analysis of eE2 and eE2-C656S. (A) ELISA plates were coated with eE2 and probed with a series of 10-fold dilutions of serum from patients infected with HCV (genotype 1, 2, or 3) or a healthy donor.
Antibodies in HCV-infected patient sera could … eE2 blocks HCVcc entry. It was shown previously that properly folded, purified E2 ectodomain from pestiviruses, bovine viral diarrhea virus, and classical swine fever virus was able to block viral infection (33, 49). In order to confirm correct folding and function of HCV eE2, we performed a similar assay using HCVcc. Recombinant human CD81 LEL, which has been shown to inhibit HCVcc infection, was used as a positive control for blocking infection (38). About 100 50% tissue culture infective doses of HCVcc were incubated with serial twofold dilutions of purified eE2, eE2-C656S, GST-human-CD81-LEL (hCD81), GST-mouse-CD81-LEL (mCD81), or GST.
eE2, eE2-C656S, and hCD81 reduced the number of focus-forming units in a concentration-dependent manner, while the mCD81 and GST proteins had no effect. This experiment yielded a 50% blocking efficiency in the range of 25 to 150 ��g/ml for eE2, eE2-C656S, and hCD8-LEL (Fig. (Fig.7B).7B). Thus, HCVcc infection can be effectively blocked by eE2 or eE2-C656S at concentrations similar to those of hCD81-LEL. In order to rule out the possibility that inhibition of viral infection was due to toxicity, we quantified cell death after incubation with purified protein using fluorescence-activated cell sorting analysis. Cells were incubated for 3 days with twofold dilutions of eE2, GST, and hCD81-LEL, followed by staining with Via-Probe to estimate viability. As shown in Fig. Fig.7C,7C, purified eE2 and hCD81 are not toxic at 200 ��g/ml (the concentration with the highest level of inhibition).
Inhibition of HCVcc entry by eE2 is thought to occur by sequestering of ce
Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent, cytoskeletal-associated GSK-3 protein kinase. The expression of DAPK is implicated in the sensitivity of cells to apoptotic effects of cytokines such as interferon (IFN)-��, tumor necrosis factor (TNF)��, and transforming growth factor-��.