Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha colocalized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.antivirals ͉ membranous web ͉ PI4K-IIIa ͉ PIK4CA ͉ RNAi H CV depends on cellular membrane-trafficking pathways at each step of its life cycle, including clathrin-mediated endocytosis, lipid droplet formation for virion assembly, and lipoprotein secretory pathways for egress (1-3). Like all positivestranded RNA viruses, HCV reorganizes intracellular membranes to establish sites of viral replication (4). HCV proteins are synthesized first at the rough endoplasmic reticulum (ER). Then the nonstructural proteins form viral replication complexes. HCV replication is thought to be intimately associated with modified cellular membranes that have protease-and nucleaseresistant properties (5, 6). Expression of the entire HCV polyprotein or the nonstructural protein NS4B alone results in formation of a unique multi-vesiculated structure termed the ''membranous web'' thought to be derived, at least in part, from ER membrane (6-8). Studies show that nonstructural proteins and viral RNA synthesis are associated with membranous webs (7,9). For these reasons they are implicated as the sites of active viral replication, although the precise origin and composition of the web-associated vesicles remains unclear.Cytosolic membrane rearrangements are a con...
Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection. Hepatitis C virus (HCV)3 entry into permissive cells is initiated through its E1 and E2 glycoprotein interactions with at least four cellular co-factors as follows: CD81; scavenger receptor class B, type I (SR-BI); and two tight junction-resident proteins, claudin-1 and occludin (1-6). CD81 belongs to the tetraspanin family and thus contains two extracellular loops, of which the second larger loop (ECL2) specifically binds to the HCV E2 glycoprotein (7, 8). SR-BI, which is normally involved in the regulation of lipoprotein metabolism and cholesterol trafficking (9), plays a role in early steps of HCV entry (10 -12); however, its direct interaction with the virus has not been unambiguously demonstrated. Claudin-1 and occludin also consist of four transmembrane domains but are functionally distinct from tetraspanins. The HCV entry determinants of claudin-1 and occludin have been mapped to the first and the second extracellular loops of these proteins, respectively (4,13,14). Considering that all four HCV entry co-factors are expressed in non-liver tissues (1, 5), the narrow tropism of this virus is surprising. It is thus likely that the relative expression levels of entry co-factors, their spatial organization, and/or interactions with each other render a cell permissive to infection (15). An alternative possibility suggested in a rece...
The fluoroquinolones (FQ) are used in the treatment of Mycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQ R ) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQsensitive M. smegmatis by its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and in M. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of 14 C-labeled CIP or SPX. The open reading frame responsible for the increase in FQ R , mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M. tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M. smegmatis mutant strain lacking an intact mfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQ R in M. smegmatis.Although the fluoroquinolones (FQ) are used against multidrug-resistant Mycobacterium tuberculosis (1), initial experience with ciprofloxacin (CIP) and ofloxacin (OFX) resulted in the rapid development of resistance (27,34) and the use of the more effective sparfloxacin (SPX) is limited by toxicity (24). Nevertheless, newer drugs, such as moxifloxacin and gatifloxacin, have higher therapeutic ratios (8) and appear promising. Therefore, continued study of the mechanisms by which FQ resistance (FQ R ) develops in mycobacteria seems warranted. Other bacteria, such as Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus, develop FQ R in a stepwise process, principally involving alterations in the A and B subunits of both gyrase and topoisomerase IV (30). However, there appear to be other elements involved in FQ R , because a very high level of resistance may not be completely explained by mutations in topoisomerase genes and strains with a low level of resistance may lack mutations in these genes (39). The only other mechanism that has been implicated in FQ R is the increased expression of FQ-transporting efflux pumps (29), which could play a role in both low-level and very high-level resistance (25).Mycobacteria do not contain topoisomerase IV (7), and strains with moderate levels of resistance to CIP, OFX, or levofloxacin (LVX) (Ͼ3 g) or SPX (Ͼ1 g) all contain gyrA mutations (36 MATERIALS AND METHODSBacterial strains and growth conditions. Wild-type (WT), FQ-sensitive M. smegmatis strain mc 2 155 was used for genetic selections (14). Some plasmids were also tested for the ability to confer resistance in M. bovis BCG Pasteur. Mycobacteria were grown in 7H9 oleic acid-albumin-dextrose-0.05% Tween 80 (OAD-TW) liquid or on 7H10 ...
The molecular characterization of partial-length genomic segment 2 of porcine picobirnavirus (PBV) strains and the development of a specific reverse transcription-PCR (RT-PCR) assay for detection of virus in feces are reported. Phylogenetic analysis indicated that the studied porcine isolates were more closely related (>85% identity) to human PBV belonging to genogroup I than to the other porcine PBV described so far. Analysis by RT-PCR and polyacrylamide gel electrophoresis of fecal samples collected in Venezuela and Argentina showed that PBV circulate at high frequencies in piglets.
The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5' NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.
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