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

The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modified in situ hybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive-and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reversetranscription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was l4 ؎ 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive-and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed. (HEPATOLOGY 1998;28:573-584.)Extensive in situ hybridization (ISH) and immunohistochemical studies of chronic hepatitis C in humans have found that only small percentages of hepatocytes are infected. A study of two chimpanzees in which chronic hepatitis C developed after acute infection (Dr. Robert Purcell, personal communication) demonstrated, using ISH, that RNA was present in most hepatocytes within 2 days of infection but was no longer detectable at 20 weeks. 1 Studies of chronic hepatitis C in humans using ISH for detection of hepatitis C virus (HCV) RNA or immunohistochemical techniques for detection of HCV antigens in the liver produced some conflicting results with both techniques, but the findings in most of the studies using these two different methodologies were similar, with approximately 50% and 70% of liver specimens positive and less than 5% and 10% of hepatocytes positive by ISH and antigen detection, respectively. 2 Recent electron-microscopic studies for HCV were also consistent with previous in situ studies; HCV particles were rarely found in acute hepatitis C in chimpanzees and not at all in chronic hepatitis C in humans. 3 The finding of only small percentages of hepatocytes positive for HCV in chronic hepatitis C is puzzling because the most prominent pathological effect of HCV infection is hepatitis. In preliminar...

A study of drug use locations In Hartford, CT, Is designed to understand the environmental and social conditions within ·hlgh risk sues"where drug users inject drugs or smoke crack, In order to develop AIDS preventIon models that build upon the physical and social organization of these locations. The study assesses high-risk sites characterized on the basis of type of location or structure, presence and strength ofgatekeepers, and presence and strength of HIV prevention opportunities and pressures. A combInation of ethnographic, epidemiological, and social network methods ara used to document the characteristics, social organization, natural history, and dynamics ofthese sites, the network relations of site users, and the various opportunitIes for, or barriers to, on-site social-level HIV prevention Intervention. This paper provides an overview of the study and presents preliminary findings, Including the degree to which drug injectors and crack smokers use specific types of sites In Hartford. Thepaperalso discusses the ways these findings Inform development ofon-site, type-specific and peer-led or structural HIV-pravention Interventions.The AIDS epidemic among impoverished, urban drug users presents unique challenges to health researchers, treatment providers, and policy makers.

The posttranscriptional regulatory mechanism(s) underlying thymidine kinase (TK) mRNA accumulation was investigated in BALB/c 3T3 cells during their progression from Go into S phase of the cell cycle. Very little TK mRNA could be detected in either the nuclear or the cytoplasmic compartment from cells harvested in Go or G1. At the onset of S phase, however, the level of nuclear TK mRNA precursors and mature TK mRNAs increased dramatically. The high molecular weight TK heterogeneous nuclear RNA species detected in the nuclei of S-phase cells were polyadenylylated and hybridized to intron sequences derived from the TK gene. A series of high molecular weight precursors could be chased to lower molecular weight species in the presence of actinomycin D, suggesting an ordered removal of intron sequences with the kinetics of a precursor-product relation-ship. These results demonstrate a striking change in the nuclear posttranscriptional processing of TK heterogeneous nuclear RNA at the G1-S boundary and, furthermore, define a model system for the examination of RNA-processing events in vivo.Proliferation of mammalian cells is a stringently controlled process that is responsive to the presence of exogenous growth factors (1, 2). Although much information has accumulated concerning the induction ofgenes in early G1 that are expressed when cells emerge from quiescence (3-6), very little is known about the regulatory events in late G1 that control the onset ofDNA synthesis (7). Concomitant with the entry of cells into S phase, a number of DNA biosynthetic enzymes, including thymidine kinase (TK) (8, 9), thymidylate synthase (10), dihydrofolate reductase (11), and the histone proteins (12), are induced. The cloning of the genes for most ofthese enzymes has now provided several molecular models for investigating cellular regulatory events at the G1-S boundary.The induction of TK enzyme activity (9) and the onset of DNA synthesis (13,14) are both regulated by a labile protein in BALB/c 3T3 cells. Moreover, the coordinate induction of these events at the G1-S boundary requires insulin-like growth factor 1 (15, 16), suggesting control by some common regulatory signal. The steady-state level of total TK mRNA increases "40-fold as serum-stimulated 3T3 cells progress from G1 into S phase and is due to transcriptional and posttranscriptional mechanisms (17). Since the rate of TK gene transcription is stimulated only 2-to 4-fold in S-phase cells (17, 18), most of the increase is attributed to posttranscriptional regulatory processes.In the present study we have examined the steady-state levels of nuclear and cytoplasmic TK mRNAs after restimulation of quiescent cells with calf serum. Go cells were found to have very little mature TK mRNA in either the nuclear or cytoplasmic compartments. At the G1-S boundary, however, a dramatic change in the processing of TK heterogeneous nuclear RNA (hnRNA) occurred, resulting in the appearance ofTK mRNA precursors and the accumulation of mature TK mRNA in the nucleus. These data suggest t...