E2 amino acid mutations, CD81 expression, and HCV core antigen in patients with mixed cryoglobulinemia. (A) Sequence alignment of combined E2 aa residues 386 to 412 (HVR1) and 468 to 555 (including HVR2/CD81 binding sites) of isolates of HCV-1a- and HCV-1b-infected patients (numbering according to HCV-J) and aa residues 468 to 556 of HCV-3a-infected patients (numbering according to HCV-K3a) (n = 43). HVR1 was amplified by nested reverse transcriptase-polymerase chain reaction in all patients (n = 58), and HVR2/CD81 binding sites were amplified in 14 of 14 patients with MC and 29 of 44 patients without MC. Residues are indicated by standard single-letter codes. Dashes indicate residues identical to the consensus sequence calculated from all isolates investigated (cons-1a, cons-1b, and cons-3a). Vertical lines indicate the HVR2; uncolored boxes, the CD81 binding sites CD81-1 and CD81-2; and MC+, patients with mixed cryoglobulinemia type II. The prototype sequences for HCV-1a (HCVH77), HCV-1b (HCV-J), and HCV-3a (HCVK3a) subtypes are shown as reference sequences.| indicates deletion at this position. Prediction of solvent accessibility (exposed [e], buried [b], unknown [gap]) shown on the basis of complete E2 aa sequence of HCV prototype HCV H77, HCV-J, and HCV K3a (www.embl-heidelberg.de/predictprotein). Gray boxes indicate aa positions identified by statistical learning classification algorithms that allow prediction of MC (www.csie.ntu.edu.tw/~cjlin/libsvm, and Durbin et al⁶ ), and prediction based on HVR1 aa sequences (n = 58) and HVR2/CD81 binding site sequences (n = 43) of the present study. There were 21 HVR1 sequences of MC-positive patients⁷  found in the European Molecular Biology Laboratory database added for analyses (accession nos. AJ406073-AJ406149). (B) CD81 expression on CD19⁺ B lymphocytes in relative fluorescence units (mean and upper SD error bar) measured by FACS analyses shown for all patients with (MC+) and without (MC-) mixed cryoglobulinemia, chronic hepatitis B (n = 7), and healthy controls (n = 10). ^*One-tailed P value. (C) Total HCV core antigen detection on PBMCs (pg/mL) measured by enzyme-linked immunosorbent assay (Quantitative Ortho trak-C assay; Ortho-Diagnostics, Neckargemünd, Germany) shown for MC-positive (MC+) and MC-negative (MC-) patients. Error bars represent means and upper SD.