Focus on cell tropism of enveloped viruses is regulated by interactions
June 5, 2017
Focus on cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission dissemination and replication within the host. that this C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2. In this study we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells effectively transinfect adjacent individual liver cells. Computer virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. contamination albeit inefficient of main hepatocytes and hepatoma cells A66 has been documented (12-14). The presence of extrahepatic reservoirs of HCV is usually suggested by the detection of viral RNA in serum and peripheral blood mononuclear cells of HCV+ individuals (15-17). Both B A66 and T lymphocytes appear to be infected contamination of B and T cell lines (7 8 18 One study however shows that replicating forms of HCV RNA are restricted to hepatocytes whereas only nonreplicating forms are present in B lymphocytes and none are in T lymphocytes (6). HCV envelope glycoproteins E1 and E2 mediate access into target cells. We as well as others recently exhibited that unmodified E1E2 heterodimers reach the cell surface and are incorporated into retroviral pseudoparticles which can infect main hepatocytes and some hepatoma cell lines (19-22). Use of the soluble E2 ectodomain as a surrogate model for studying HCV interactions with cell-surface molecules has identified several potential HCV access receptors including CD81 scavenger receptor class B type 1 low-density lipoprotein receptor and glycosaminoglycans (22-24). Several groups including ours have shown that CD81 is necessary but not sufficient for HCV pseudovirus access into target cells (19 25 26 Furthermore we recently demonstrated that CD81 functions as a postattachment access coreceptor (26). Mobile factors that act in collaboration with Compact disc81 to mediate HCV entry and binding remain to become discovered. Engagement of particular receptors is necessary for viral fusion and entrance but adsorption of viral contaminants towards the cell surface area may appear through envelope glycoprotein connections with other substances (27-33). The C type lectins DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-getting nonintegrin; Compact disc209) and L-SIGN (DC-SIGNR; lymph and liver node-specific; Compact disc209L) work as catch receptors for many infections including HIV type 1 (HIV-1) (34) Ebola pathogen (35) cytomegalovirus (36) and dengue pathogen (37). Both A66 L-SIGN and DC-SIGN come with an extracellular C-terminal area which has a calcium-dependent carbohydrate identification area (CRD) and a Ace2 membrane-proximal heptad-repeat area very important to oligomerization (38-41). Catch of viral contaminants is mediated with the CRD and promotes infections of focus on cells both in cis and in trans (34 35 42 43 DC-SIGN also identifies intercellular adhesion substances 2 and 3 A66 which work as cell-adhesion receptors that regulate transendothelial migration of dendritic cells (DC) from bloodstream to tissues aswell as DC-T cell connections. We yet others possess lately confirmed that recombinant soluble E2 patient-derived HCV virions and retroviruses pseudotyped with HCV envelope glycoproteins particularly bind to L-SIGN and DC-SIGN (44-46). HCV catch by SIGN substances depends on the current presence of the CRD indicating that identification of high mannose oligosaccharides in the viral envelope glycoproteins is crucial for binding. The specificity of the interaction is certainly underscored by observations that (was utilized expressing HCV envelope glycoproteins. Sequences encoding the full-length E1 and E2 (proteins 132-746) you start with the final 60 proteins from the capsid (ΔC) had been PCR amplified A66 from p90/HCV FL-long pU composed of the genome of infectious HCV isolate H77 (48) and subcloned into pcDNA3.1 (Invitrogen). Putative splice acceptor sites had been modified by conventional mutagenesis as defined (21). mAbs 507D 604 and 612X spotting the CRD of DC-SIGN L-SIGN or both lectins respectively had been bought fromR&D Systems. Anti-HCV E2 mAb 091b-5 was bought from Austral Biological. Anti-CD81 mAb JS-81 was extracted from Pharmingen. Mannan and Chloroquine were purchased from Sigma. Cell Lines. Unless usually specified cells had been purchased in the American Type Lifestyle Collection and expanded in DMEM supplemented with 10% fetal bovine serum/1% penicillin/streptomycin/2 mM glutamine. HeLa cells stably expressing L-SIGN or DC-SIGN had been generated as defined (44) and preserved in DMEM.