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Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

Wünschmann, Sabina; Medh, Jheem D.; Klinzmann, Donna; Schmidt, Warren N.; Stapleton, Jack T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2000 Português
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Hepatitis C virus (HCV) or HCV–low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm3), intermediate-density particles (1.12 to 1.18 g/cm3), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4°C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of α-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of α-sarcin...

Infectious Epstein-Barr Virus Lacking Major Glycoprotein BLLF1 (gp350/220) Demonstrates the Existence of Additional Viral Ligands

Janz, Annette; Oezel, Muhsin; Kurzeder, Christian; Mautner, Josef; Pich, Dagmar; Kost, Manuela; Hammerschmidt, Wolfgang; Delecluse, Henri-Jacques
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2000 Português
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The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.

Wild-Type Herpes Simplex Virus 1 Blocks Programmed Cell Death and Release of Cytochrome c but Not the Translocation of Mitochondrial Apoptosis-Inducing Factor to the Nuclei of Human Embryonic Lung Fibroblasts

Zhou, Guoying; Roizman, Bernard
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2000 Português
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Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells...

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

Klasse, P. J.; Rosenkilde, Mette M.; Signoret, Nathalie; Pelchen-Matthews, Annegret; Schwartz, Thue W.; Marsh, Mark
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 Português
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Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1LAI to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SULAI protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1LAI infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100...

Adaptation of Sindbis Virus to BHK Cells Selects for Use of Heparan Sulfate as an Attachment Receptor

Klimstra, William B.; Ryman, Kate D.; Johnston, Robert E.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1998 Português
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Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981–1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast...

Expression and Characterization of a Single-Chain Polypeptide Analogue of the Human Immunodeficiency Virus Type 1 gp120-CD4 Receptor Complex

Fouts, Timothy R.; Tuskan, Robert; Godfrey, Karla; Reitz, Marvin; Hone, David; Lewis, George K.; DeVico, Anthony L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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The infection of CD4+ host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further...

Interaction of Rotaviruses with Hsc70 during Cell Entry Is Mediated by VP5

Zárate, Selene; Cuadras, Mariela A.; Espinosa, Rafaela; Romero, Pedro; Juárez, Karla O.; Camacho-Nuez, Minerva; Arias, Carlos F.; López, Susana
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2003 Português
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Rotavirus infection seems to be a multistep process in which the viruses are required to interact with several cell surface molecules to enter the cell. The virus spike protein VP4, which is cleaved by trypsin into two subunits, VP5 and VP8, is involved in some of these interactions. We have previously shown that the neuraminidase-sensitive rotavirus strain RRV initially attaches to a sialic acid-containing cell molecule through the VP8 subunit of VP4 and subsequently interacts with integrin α2β1 through VP5. After these initial contacts, the virus interacts with at least two additional proteins located at the cell surface, the integrin αvβ3 and the heat shock cognate protein Hsc70. In this work, we have shown that rotavirus RRV and its neuraminidase-resistant variant nar3 interact with Hsc70 through a VP5 domain located between amino acids 642 and 658 of the protein. This conclusion is based on the observation that a recombinant protein comprising the 300 carboxy-terminal amino acids of VP5 binds specifically to Hsc70 and a synthetic peptide containing amino acids 642 to 658 competes with the binding of the RRV and nar3 viruses to the heat shock protein. The VP5 peptide also competed with the binding to Hsc70 of the recombinant VP5 protein...

The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity

Jeetendra, E.; Ghosh, Kakoli; Odell, Derek; Li, Jin; Ghosh, Hara P.; Whitt, Michael A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2003 Português
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The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity...

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

Hahon, Nicholas; Cooke, Kenneth O.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1967 Português
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The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of pH dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.

DC-SIGN Facilitates Fusion of Dendritic Cells with Human T-Cell Leukemia Virus Type 1-Infected Cells

Ceccaldi, Pierre-Emmanuel; Delebecque, Frédéric; Prevost, Marie-Christine; Moris, Arnaud; Abastado, Jean-Pierre; Gessain, Antoine; Schwartz, Olivier; Ozden, Simona
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2006 Português
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Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether...

Time Frames for Neutralization during the Human Immunodeficiency Virus Type 1 Entry Phase, as Monitored in Synchronously Infected Cell Cultures▿

Haim, Hillel; Steiner, Israel; Panet, Amos
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry...

Sulfated Homologues of Heparin Inhibit Hepatitis C Virus Entry into Mammalian Cells▿

Basu, Arnab; Kanda, Tatsuo; Beyene, Aster; Saito, Kousuke; Meyer, Keith; Ray, Ranjit
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.

Generation and Characterization of a Recombinant Vesicular Stomatitis Virus Expressing the Glycoprotein of Borna Disease Virus▿

Perez, Mar; Clemente, Roberto; Robison, Clinton S.; Jeetendra, E.; Jayakar, Himangi R.; Whitt, Michael A.; de la Torre, Juan C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
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Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVΔG*/BDVG). Cells infected with rVSVΔG*/BDVG produced high titers (107 PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSVΔG*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSVΔG*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSVΔG*/BDVG-infected cells. Notably...

Replication-Competent Variants of Human Immunodeficiency Virus Type 2 Lacking the V3 Loop Exhibit Resistance to Chemokine Receptor Antagonists▿

Lin, George; Bertolotti-Ciarlet, Andrea; Haggarty, Beth; Romano, Josephine; Nolan, Katrina M.; Leslie, George J.; Jordan, Andrea P.-O.; Huang, Chih-chin; Kwong, Peter D.; Doms, Robert W.; Hoxie, James A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
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Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function...

An Alteration of Human Immunodeficiency Virus gp41 Leads to Reduced CCR5 Dependence and CD4 Independence▿

Taylor, Brian M.; Foulke, J. Scott; Flinko, Robin; Heredia, Alonso; DeVico, Anthony; Reitz, Marvin
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4, increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increased resistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41, including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicate that HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences that are not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure of the CCR5 binding site.

A Novel Cellular Protein, VPEF, Facilitates Vaccinia Virus Penetration into HeLa Cells through Fluid Phase Endocytosis▿ †

Huang, Cheng-Yen; Lu, Tsai-Yi; Bair, Chi-Horng; Chang, Yuan-Shau; Jwo, Jeng-Kuan; Chang, Wen
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin- or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor...

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer, Productive Infection, and Replication and Their Requirement for HCV Receptors

Liu, Ziqing; He, Johnny J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2013 Português
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Hepatitis C virus (HCV) infection is believed to begin with interactions between cell-free HCV and cell receptors that include CD81, scavenger receptor B1 (SR-B1), claudin-1 (CLDN1), and occludin (OCLN). In this study, we have demonstrated that HCV spreading from infected hepatocytes to uninfected hepatocytes leads to the transfer of HCV and the formation of infection foci and is cell density dependent. This cell-cell contact-mediated (CCCM) HCV transfer occurs readily and requires all these known HCV receptors and an intact actin cytoskeleton. With a fluorescently labeled replication-competent HCV system, the CCCM transfer process was further dissected by live-cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and posttransfer. Importantly, the CCCM HCV transfer leads to productive infection of target cells. Taken together, these results show that CCCM HCV transfer constitutes an important and effective route for HCV infection and dissemination. These findings will aid in the development of new and novel strategies for preventing and treating HCV infection.

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

Elbahesh, Husni; Cline, Troy; Baranovich, Tatiana; Govorkova, Elena A.; Schultz-Cherry, Stacey; Russell, Charles J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2014 Português
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Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK...

Role of Hypervariable Region 1 for the Interplay of Hepatitis C Virus with Entry Factors and Lipoproteins

Bankwitz, Dorothea; Vieyres, Gabrielle; Hueging, Kathrin; Bitzegeio, Julia; Doepke, Mandy; Chhatwal, Patrick; Haid, Sibylle; Catanese, Maria Teresa; Zeisel, Mirjam B.; Nicosia, Alfredo; Baumert, Thomas F.; Kaderali, Lars; Pietschmann, Thomas
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2014 Português
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Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus...

MicroRNA miR-155 Inhibits Bone Morphogenetic Protein (BMP) Signaling and BMP-Mediated Epstein-Barr Virus Reactivation▿ †

Yin, Qinyan; Wang, Xia; Fewell, Claire; Cameron, Jennifer; Zhu, Hanqing; Baddoo, Melody; Lin, Zhen; Flemington, Erik K.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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MicroRNA miR-155 is expressed at elevated levels in human cancers including cancers of the lung, breast, colon, and a subset of lymphoid malignancies. In B cells, miR-155 is induced by the oncogenic latency gene expression program of the human herpesvirus Epstein-Barr virus (EBV). Two other oncogenic herpesviruses, Kaposi's sarcoma-associated herpesvirus and Marek's disease virus, encode functional homologues of miR-155, suggesting a role for this microRNA in the biology and pathogenesis of these viruses. Bone morphogenetic protein (BMP) signaling is involved in an array of cellular processes, including differentiation, growth inhibition, and senescence, through context-dependent interactions with multiple signaling pathways. Alteration of this pathway contributes to a number of disease states including cancer. Here, we show that miR-155 targets the 3′ untranslated region of multiple components of the BMP signaling cascade, including SMAD1, SMAD5, HIVEP2, CEBPB, RUNX2, and MYO10. Targeting of these mediators results in the inhibition of BMP2-, BMP6-, and BMP7-induced ID3 expression as well as BMP-mediated EBV reactivation in the EBV-positive B-cell line, Mutu I. Further, miR-155 inhibits SMAD1 and SMAD5 expression in the lung epithelial cell line A549...