It might not be determined from the studies performed whether the low level of endocytosis of HDL compared with those of VLDL and LDL, as shown by cytometric studies with DiI-labeled lipoproteins, was responsible for the absence of detectable levels of HDL-mediated endocytosis of HCV

It might not be determined from the studies performed whether the low level of endocytosis of HDL compared with those of VLDL and LDL, as shown by cytometric studies with DiI-labeled lipoproteins, was responsible for the absence of detectable levels of HDL-mediated endocytosis of HCV. The complete inhibition by anti-LDL receptor antibody of HCV endocytosis by a variety of cells suggests that the LDL receptor may be the main mechanism for entry of HCV into cells. the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses Ibutamoren (MK-677) indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors. Hepatitis C virus (HCV), the principal viral cause of chronic hepatitis, is not readily replicated in cell culture systems, and, as yet, no information on cell receptors for the virus is available. However, several observations from studies on the role of HCV in mixed cryoglobulinemia (1C3) have provided some insights to HCV entry into cells. Mixed cryoglobulinemia is a systemic vasculitis associated with cold-precipitable immunoglobulins in the blood. During the past 5 years, a strong association of HCV Ibutamoren (MK-677) infection with mixed cryoglobulins has been established (4), and the specific concentration of HCV in type II mixed cryoglobulins that consist of polyclonal IgG and monoclonal IgM has been demonstrated (1). It also was shown that very low density lipoprotein (VLDL) is selectively associated with HCV in type II cryoglobulins (2). In studies on the cutaneous vasculitic lesions in type II cryoglobulinemia using hybridization (ISH), the HCV RNA virion form (positive strand) but not the putative replicative form (negative strand) of the virus was detected in keratinocytes in lesions but not normal skin of the same patients (3). Furthermore, it was demonstrated that LDL receptors were up-regulated on keratinocytes in cutaneous vasculitis lesions compared with normal skin (3). These observations and the finding that anti- lipoprotein precipitates HCV from infected serum (5) suggested that the low density lipoprotein (LDL) receptor also may be a receptor for HCV complexed to VLDL or LDL. This hypothesis led to this study to determine whether the LDL receptor is also Rabbit Polyclonal to OR2T11 a receptor for HCV and other members of the family of viruses Flaviviridae. Materials and Methods Reagents, Antisera Virus Stocks, and Cell Lines. Cyclohexanedione, phenylarsine oxide (PAO), heparin sulfate, and EGTA were purchased from Sigma; 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine iodine (DiI) was purchased from Molecular Probes. Purified IgG 2a mouse monoclonal anti-LDL receptor antibody (C7 clone) was obtained from Oncogene Scientific Products (Cambridge, MA). Anti-bovine viral diarrhea virus (BVDV) envelope antibody bovine serum, 49, was provided by Marc S. Collett (Viro Pharma, Malvern, PA). Mouse monoclonal IgG 2a anti-CD-16, anti-CD-19, and anti-transferrin (CD71) were purchased from Immunotech (Hialeah, FL). Anti- was purchased from Jackson ImmunoResearch. Anti-apolipoprotein (apo) E and apo A-I were purchased from Cortex Pharmaceuticals (San Leandro, CA); apo B was purchased from Sigma. Purified mouse monoclonal IgG apo E (1D7), apo A-I (3G10), and apo B (4G3) were Ibutamoren (MK-677) purchased from the University of Ottawa Heart Institute. F(ab)2, an antibody fragment with two antigen-combining sites, preparations of mouse IgG were prepared by treating the mouse monoclonal antibodies from 30 minutes to 10 hours with 3% pepsin (Sigma) (pH 3.5) at 37C. The F(ab)2 fragments were isolated by column chromatography using a HR 10/30 Superose 12 column (Amersham Pharmacia). Bovine viral diarrhea virus-free donor calf serum was purchased from Boyt Veterinary Laboratory (Neosho, MO). Potassium bromide density gradient ultracentrifugation was used for preparation of VLDL, LDL, and high density lipoprotein (HDL) from normal sera, and these lipoproteins complexed to HCV from infected sera. The VLDL band [density (d) = 0.95C1.006 g/ml], the LDL band (d = 1.019C1.063), the HDL band (d = 1.063C1.21 g/ml), and HCV free of lipoproteins (d > 1.21) were isolated by aspiration and then dialyzed against Hanks balanced salt solution (Sigma) containing 0.01% EDTA. Isolated HCV-VLDL was dissociated to HCV and VLDL by treatment with deoxycholate and fractionated by sucrose density gradient ultracentrifugation as described (6). The high density HCV fraction, free of lipoproteins, was further fractionated by column chromatography on a lecithin pretreated Superose 6 (Amersham Pharmacia). The peak of HCV present in the void volume was contaminated with small amounts of immunoglobulins that were removed by using immobilized rProtein A (Repligen). Immoblotting (dot blots) to detect small amounts of protein was performed as described (7). Sensitivity of the assay was 100 pg for IgG and IgM and 200 pg for apolipoproteins B and E. Lipoproteins were quantitated by Lowry.