Hypervariable region 1 (HVR1) of envelope protein 2 (E2) of hepatitis C virus (HCV) serves important yet undefined roles in the viral life cycle. to parental viruses, scavenger receptor class B type I (SR-BI) dependency was decreased for H77HVR1/N476D/S733F, Aniracetam IC50 H77N476D/S733F, S52HVR1/A369V, and S52A369V, but not for J6HVR1. Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77N476D/S733F and S52A369V. Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served ApoE-independent but HVR1-dependent functions in HCV entry. INTRODUCTION Approximately 180 million people worldwide are chronically infected with hepatitis C virus (HCV) with an increased risk of developing liver cirrhosis and hepatocellular carcinoma (1). HCV is an enveloped positive-strand RNA virus of the family with a 9.6-kb genome consisting of 5 and 3 untranslated regions (UTRs) flanking an open reading frame (ORF) that encodes a single polyprotein. This polyprotein is processed into structural proteins (Core and envelope proteins E1 and E2), p7, and six nonstructural proteins (NS2 to NS5B) (2). HCV is a highly diverse virus, and isolates are divided into seven major genotypes, most containing multiple subtypes and differing by 30% and 20%, respectively, at the nucleotide and amino acid levels (2). Previous studies have shown genotype or isolate differences when analyzing HCV neutralization and in reverse genetics studies of Aniracetam IC50 HCV proteins (3,C5). This highlights the importance of including several isolates, preferably of diverse genotypes, in functional studies. While the process of HCV entry into the human hepatocyte remains incompletely understood, it is known to be a complex multistep process involving several receptors acting at (i) initial attachment, (ii) cell surface transport, and (iii) cellular uptake and infection initiation (6). Both the low-density lipoprotein receptor (LDLr) and scavenger receptor class B type I (SR-BI) are believed to be involved in early interactions between the cell and the virion, possibly priming conformational changes that allow further interactions with the late-stage receptor CD81 or entry factors Claudin I and Occludin (7,C10). Apparently, E2 interacts directly with CD81, and it has recently been suggested that CD81 and Claudin I are endocytosed with the virus particle in a clathrin-dependent manner (11, 12). The initial cell interactions have been proposed to occur through the association of the virus with apolipoproteins B and especially E (ApoB and ApoE) (13,C16). ApoE has been implicated in virus attachment to the host cell (17) by interaction with heparan sulfate proteoglycans (HSPGs) (18), whereas others have found recombinant E1 and E2 to interact directly with liver-derived HSPGs (19). However, a recent study demonstrated that virus-associated ApoE is responsible for interactions mediating attachment between the cell-associated HSPG syndecan 1 and HCV (20). In addition, there is indirect evidence suggesting that ApoE is responsible for HCV interactions with LDLr (14, 21). However, a recent study showed that HCV internalization through Aniracetam IC50 Slco2a1 LDLr does Aniracetam IC50 not lead to infection of the cell, suggesting that the ApoE-LDLr interaction might not mediate productive uptake of HCV (22). Thus, LDLr might primarily mediate cell attachment, possibly through an interaction with virus-associated ApoE (23). SR-BI has also been reported to interact with ApoE on the surface of the HCV particle and to interact with the E2 protein motif hypervariable region 1 (HVR1) (16, 24, 25). The latter finding was supported by the loss of SR-BI dependency of an HVR1-deleted genotype 2a virus, Jc1 (26). HVR1-deleted viruses have been shown to be infectious in both the chimpanzee and the human liver chimeric mouse model (3, 27), but so far, only a few studies have addressed how the deletion might affect the HCV life cycle. In this study, we first analyzed which step of the HCV life cycle was affected by HVR1 deletion and the adaptive mutations acquired by HVR1-deleted viruses. Using antibody blocking and receptor silencing, we explored the lipoprotein receptor dependency of Aniracetam IC50 parental and HVR1-deleted HCV. Interestingly, HVR1 deletion conferred decreased dependency on the LDLr, while decreased SR-BI dependency seemed to be linked to HVR1-related envelope mutations required to rescue the infectivity of some HVR1-deleted viruses. Finally, we found LDLr to be important at the entry.