Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C͞hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein͞LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of 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 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. H epatitis 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 (1-3) 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 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 in situ 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 upregulated 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 a receptor for HCV and other members of the family of viruses Flaviviridae. Materials and Methods

Type II cryoglobulinemia is strongly associated with concomitant HCV infection and a high rate of false negative serologic tests. HCV virions and HCV antigen-antibody complexes are concentrated in the cryoprecipitates, most commonly in association with the WA type of rheumatoid factor, suggesting a role for HCV in the pathogenesis of mixed cryoglobulinemia.

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Through the use of absorbed idiotypic antisera prepared against single isolated monoclonal IgM anti-γ-globulins, partial cross-idiotypic specificity was demonstrated with other IgM anti-γ-globulins. Such antisera classified these proteins into at least three groups. The major group which included 60% of the anti-γ-globulins was particularly homogeneous. The anti-γ-globulin specific antigens were detected best in hemagglutination and hemagglutination inhibition systems. They were not found in monoclonal IgM proteins that lacked anti-γ-globulin activity although related antigens were detected at low concentrations in pooled immunoglobulin preparations as well as in heterogeneous anti-Rh antibodies. Several lines of evidence were obtained indicating that the antibody combining site was involved in the specific determinants. Attempts were made to analyze the fine specificity of each anti-γ-globulin for the Fc fragment of different subclasses of human immunoglobulins as well as those of other species. Differences were observed but these were not readily related to the cross-specificity antigens. The anti-γ-globulin specific antigens were very analogous to those previously described for monoclonal IgM cold agglutinins. Although each protein could be distinguished from all the others on the basis of individual idiotypic antigens, the antigens common to the specific groups of proteins with each of these activities were prominent and readily detected with multiple antisera. The results indicate basic similarities between proteins of a given activity even in unrelated individuals.

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The specificities of anti-polynucleotide antibodies found in human sera were studied using several immunological procedures. Anti-native DNA (NDNA) antibodies and certain anti-double-stranded RNA (DSRNA) antibodies were found to react with single-stranded DNA (SDNA), and anti-NDNA antibodies were observed to react more avidly with SDNA than with NDNA in most sera tested. Antibodies to NDNA showed no preferential reactivity with NDNA or SDNA derived from mammalian tissue, bacterial, or viral sources. Precipitating antibodies reactive with individual bases, with common determinants of bases, and with common determinants of SDNA and NDNA were detected utilizing synthetic polydeoxyribonucleotides. Antibodies to DSRNA were also heterogeneous and reactive with both Poly A · Poly U and Poly I · Poly C in addition to reactivity with Poly A and SDNA. In contrast, antibodies to a ribonucleo-protein determined by hemagglutination and by precipitation showed no reaction with NDNA, SDNA, or DSRNA. Serial studies of serum specimens from patients with systemic lupus erythematosus (SLE) indicated that anti-NDNA antibodies were closely associated with disease activity. Titers of antibodies to SDNA or DSRNA were also frequently increased during these periods but in addition showed peaks during quiescent periods. Anti-NDNA antibodies were detected in most patients' sera at sometime during the course of the disease. Three patients were observed with active SLE, who did not develop anti-NDNA antibodies, even in the presence of severe renal disease. Evidence that other antigen-antibody systems may also play a role in the pathogenesis of the renal disease was particularly apparent in these patients. Anti-ribonucleoprotein antibodies were not well correlated with the peaks of antibody activity of other polynucleotide antibodies, suggesting that an independent immunogen was responsible for induction of these antibodies. The close association of certain populations of anti-polynucleotide antibodies during the course of active SLE, the presence of cross-reacting antigenic determinants of SDNA, NDNA, and DSRNA, the preferential avidity of anti-NDNA antibodies for SDNA, and the frequent increase of anti-SDNA antibodies in SLE and other diseases associated with active tissue destruction suggest that SDNA is a ubiquitous antigen that may stimulate the formation of antibodies reactive with a variety of polynucleotides.

The strong association of HCV infection with MC-II and the selective concentration of the virus with the WA mRF in the cryoglobulins are compelling suggestions that the virus is directly involved in production of the mRF and the pathophysiology of MC-II. There is, however, only limited data on HCV involvement in both processes. In cutaneous vasculitis, which is the most prevalent clinical feature of the disease, there is evidence that complexes of HCV, mRF and IgG are formed in situ from components of the cryoglobulins that are present in the blood in a dissociated state. It is postulated that local factors, cooling and stasis predispose to formation of these lesions in the lower limbs. However, since cutaneous vasculitis does not correlate with cryoglobulin levels and may not be induced by cold challenge, other factors may be involved. In particular, the conditions which activate the vascular endothelial cells, leading to the leukocytoclastic vasculitis, require delineation. In contrast to cutaneous vasculitis, HCV RNA has not been prominently detected in immune complexes in MPGN lesions and has not been detected at all in the peripheral neuropathy lesions. These preliminary observations suggest that different pathophysiological processes are involved in for these lesions than in cutaneous vasculitis. From the correlation of remission of disease with decreased cryoglobulinemia and viremia in treated patients with MC-II, and from immunohistological data on the hepatitic lymphoid follicles in MC-II (see chapter 7), it appears that an antigen-driven benign proliferation of B cells is responsible for production off mRF and cryoglobulinemia. New findings have suggested that one mechanism for developing mixed cryoglobulinemia may be that HCV-VLDL complexes that contain apo E2 are poorly endocytosed by the LDLR, which may be a major route of entry of the virus to the cell; persistence of the complexes in the circulation may then stimulate mRF production. This new hypothesis is based only on initial in vitro observations and require independent confirmation and validation in vivo. From indirect clinical evidence it has also been postulated that mRF in some patients may limit the cytopathology in MC-II, resulting in a lower prevalence of cirrhosis in these patients. These findings suggested another hypothesis, which is that the mRF prevents spread of infection to hepatocytes and other permissive and nonpermissive cells by blocking endocytosis of HCV-VLDL complexes by the LDLR. Furthermore, data on the composition of cryoglobulins, the molecular composition of WA mRF and the characterization of monoclonal B cells in the liver of patients with MC-II (see chapter 7) suggest that a specific population of B cells may be involved in the host response to HCV infection. These are B cells that proliferate with little or no somatic mutations of the immunoglobulin genes, are self-replicating, are stimulated by self antigens in a T cell-independent manner and bear the CD5 marker. The proliferation of this B cell population may be th...