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1.
Virology ; 366(1): 40-50, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17498767

ABSTRACT

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.


Subject(s)
Anions/pharmacology , Antiviral Agents/pharmacology , HIV/physiology , Hepacivirus/physiology , Animals , Carbohydrate Metabolism , Cell Line, Tumor , HIV/drug effects , HIV-1/drug effects , HIV-1/physiology , HIV-2/drug effects , HIV-2/physiology , Hepacivirus/drug effects , Humans , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Virus Replication
2.
J Gen Virol ; 87(Pt 9): 2571-2576, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16894195

ABSTRACT

L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans-infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans-infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.


Subject(s)
Cell Adhesion Molecules/physiology , Hepacivirus/pathogenicity , Hepatitis C/etiology , Lectins, C-Type/physiology , Receptors, Cell Surface/physiology , Receptors, Virus/physiology , Alleles , Carcinoma, Hepatocellular/virology , Cell Adhesion Molecules/genetics , HeLa Cells , Hepatitis C/genetics , Hepatitis C/virology , Hepatocytes/virology , Humans , Lectins, C-Type/genetics , Minisatellite Repeats , Protein Isoforms/genetics , Protein Isoforms/physiology , Receptors, Cell Surface/genetics , Receptors, Virus/genetics , Transfection , Viral Envelope Proteins/physiology
3.
Proc Natl Acad Sci U S A ; 101(39): 14067-72, 2004 Sep 28.
Article in English | MEDLINE | ID: mdl-15371595

ABSTRACT

Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host. Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry. However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry. Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells. We and others recently demonstrated that the 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 efficiently transinfect adjacent human liver cells. Virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells. Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection.


Subject(s)
Cell Adhesion Molecules/metabolism , Hepacivirus/metabolism , Hepatocytes/virology , Lectins, C-Type/metabolism , Receptors, Cell Surface/metabolism , Viral Envelope Proteins/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, CD/chemistry , Antigens, CD/immunology , Cell Line , Chloroquine/pharmacology , Dendritic Cells/metabolism , Dendritic Cells/virology , HeLa Cells , Hepacivirus/genetics , Hepacivirus/pathogenicity , Hepatocytes/metabolism , Humans , Liver/cytology , Liver/metabolism , Liver/virology , Mannans/chemistry , Mannans/immunology , Mannans/pharmacology , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Tetraspanin 28 , Transfection , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology
4.
Proc Natl Acad Sci U S A ; 101(19): 7270-4, 2004 May 11.
Article in English | MEDLINE | ID: mdl-15123813

ABSTRACT

Hepatitis C virus (HCV) envelope glycoproteins E1/E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a post-attachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.


Subject(s)
Antigens, CD/physiology , Hepacivirus/physiology , Receptors, Virus/physiology , Cell Line, Tumor , Humans , Membrane Fusion/physiology , Tetraspanin 28
5.
J Virol ; 77(24): 13418-24, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14645599

ABSTRACT

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.


Subject(s)
Cell Membrane/metabolism , Hepacivirus/pathogenicity , Introns , Viral Envelope Proteins/metabolism , Viral Structural Proteins/metabolism , Base Sequence , Dimerization , HeLa Cells , Hepacivirus/genetics , Hepacivirus/metabolism , Humans , Molecular Sequence Data , Sequence Deletion , Viral Envelope Proteins/genetics , Viral Structural Proteins/genetics
6.
J Virol ; 76(17): 8953-7, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12163614

ABSTRACT

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.


Subject(s)
HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/pathogenicity , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Receptors, CCR5/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Gene Deletion , HIV Envelope Protein gp120/genetics , Humans , Molecular Sequence Data , Peptide Fragments/genetics , Peptides/chemistry , Peptides/metabolism , Receptors, CCR5/chemistry , Recombinant Fusion Proteins , Sulfur/chemistry , Transfection
7.
J Virol ; 76(6): 3059-64, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11861874

ABSTRACT

CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.


Subject(s)
HIV-1/pathogenicity , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Amino Acid Sequence , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV Infections/virology , HIV-1/classification , HIV-1/genetics , HIV-1/isolation & purification , HIV-1/metabolism , Humans , Molecular Sequence Data , Mutation
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