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1.
J Biol Chem ; 287(37): 31242-57, 2012 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-22767607

RESUMEN

Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.


Asunto(s)
Apolipoproteínas E/metabolismo , Hepacivirus/fisiología , Receptores Depuradores de Clase B/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Apolipoproteínas E/genética , Línea Celular Tumoral , Humanos , Ratones , Estructura Terciaria de Proteína , Ratas , Receptores Depuradores de Clase B/genética , Proteínas del Envoltorio Viral/genética
2.
PLoS Pathog ; 7(7): e1002144, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21814513

RESUMEN

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.


Asunto(s)
Retículo Endoplásmico/metabolismo , Hepacivirus/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus/fisiología , Línea Celular , Retículo Endoplásmico/genética , Retículo Endoplásmico/virología , Humanos , Proteínas no Estructurales Virales/genética , Proteínas Virales/genética
3.
J Biol Chem ; 286(27): 23865-76, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21555519

RESUMEN

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.


Asunto(s)
Hepacivirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Secuencias de Aminoácidos , Línea Celular , Hepacivirus/genética , Humanos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/genética
4.
J Virol ; 83(12): 6048-66, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19357170

RESUMEN

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.


Asunto(s)
Adenovirus Humanos/genética , Baculoviridae/genética , Vectores Genéticos , Receptores Virales/genética , Transducción Genética , Adenovirus Humanos/ultraestructura , Animales , Baculoviridae/ultraestructura , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Recombinantes de Fusión/genética
5.
Am J Respir Cell Mol Biol ; 37(6): 631-9, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17641299

RESUMEN

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.


Asunto(s)
Adenoviridae/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Tráquea/citología , Tráquea/metabolismo , Adenoviridae/genética , Adulto , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Activación del Canal Iónico , Microscopía Confocal , Proteínas Mutantes/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Mucosa Respiratoria/ultraestructura , Tráquea/ultraestructura , Transducción Genética
6.
Hum Gene Ther ; 21(3): 251-69, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19788389

RESUMEN

In vivo gene transfer to the human respiratory tract by adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response, and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on CFTR function. This negative interference did not result from a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype that uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared with Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from patients with CF (CF-HAE) via the apical surface, restored chloride channel function at relatively low vector doses, and showed relatively stable expression of GFP-CFTR for several weeks.


Asunto(s)
Adenoviridae/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/terapia , Terapia Genética , Vectores Genéticos , Mucosa Respiratoria/metabolismo , Adulto , Western Blotting , Proteínas de la Cápside/fisiología , Células Cultivadas , Cloruros/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Endocitosis , Técnicas de Transferencia de Gen , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Integrinas/metabolismo , Mutación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mucosa Respiratoria/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes/fisiología
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