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1.
Sci Rep ; 6: 28768, 2016 07 07.
Article in English | MEDLINE | ID: mdl-27385443

ABSTRACT

Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact.


Subject(s)
Antibodies/physiology , Dengue Virus/physiology , Dengue/virology , Macrophages/virology , Actin Cytoskeleton/pathology , Animals , Antibody-Dependent Enhancement , Cell Line , Cell Membrane/pathology , Cell Membrane/virology , Culicidae , Endocytosis , Humans , Kinetics , Mice , Receptors, IgG/metabolism , Virus Internalization
2.
J Virol ; 90(9): 4745-4756, 2016 May.
Article in English | MEDLINE | ID: mdl-26912616

ABSTRACT

UNLABELLED: Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes. IMPORTANCE: Since its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, conflicting results with regard to the cell entry pathway used by CHIKV have been published. Here we applied a novel technology to visualize the entry behavior of single CHIKV particles in living cells. Our results show that CHIKV cell entry is extremely rapid and occurs via clathrin-mediated endocytosis. Membrane fusion from within acidic early endosomes is observed. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taking these findings together, this study provides detailed insight into the cell entry process of CHIKV.


Subject(s)
Chikungunya virus/physiology , Molecular Imaging , Virus Internalization , Virus Physiological Phenomena , Animals , Cell Line , Cells, Cultured , Chikungunya Fever/virology , Chlorocebus aethiops , Cholesterol/metabolism , Clathrin/metabolism , Endocytosis , Endosomes/metabolism , Endosomes/virology , Humans , Membrane Fusion , Microscopy, Fluorescence , Molecular Imaging/methods , Staining and Labeling
3.
Antiviral Res ; 100(1): 238-45, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23994499

ABSTRACT

Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier that SA-17, a doxorubicin derivative, efficiently inhibits the in vitro infection of DENV and yellow fever virus. Here we explored SA-17's mechanism of inhibition and investigated if the compound is active against ADE of DENV infection. Since enhanced infectivity stimulated by antibodies has been observed with standard and immature DENV, both types of virions were included in the study. We observed that SA-17 (i) inhibits DENV infection by preventing binding/entry to the cell and (ii) interferes with antibody-mediated infection of both standard and immature DENV2. SA-17 markedly reduced the infectivity of DENV2 in ADE conditions, with IC50s ranging from 0.26 to 2.89µM. The compound exerted its activity when added before, during, and after antibody-opsonization of standard and immature virus. Thus, molecules with the characteristics of SA-17 may be attractive antiviral agents since they can be used both to block DENV2 entry during primary and secondary infection and to inhibit ADE of standard and immature virus.


Subject(s)
Antibody-Dependent Enhancement/drug effects , Antiviral Agents/pharmacology , Dengue Virus/drug effects , Dengue/virology , Doxorubicin/pharmacology , Antibodies, Viral/immunology , Dengue/drug therapy , Dengue/immunology , Dengue Virus/physiology , Doxorubicin/analogs & derivatives , Humans , Virus Replication/drug effects
4.
PLoS One ; 7(5): e37244, 2012.
Article in English | MEDLINE | ID: mdl-22624001

ABSTRACT

There is an urgent need for potent inhibitors of dengue virus (DENV) replication for the treatment and/or prophylaxis of infections with this virus. We here report on an aglycon analogue of the antibiotic teicoplanin (code name LCTA-949) that inhibits DENV-induced cytopathic effect (CPE) in a dose-dependent manner. Virus infection was completely inhibited at concentrations that had no adverse effect on the host cells. These findings were corroborated by quantification of viral RNA levels in culture supernatant. Antiviral activity was also observed against other flaviviruses such as the yellow fever virus and the tick-borne encephalitis virus (TBEV). In particular, potent antiviral activity was observed against TBEV. Time-of-drug-addition experiments indicated that LCTA-949 inhibits an early stage in the DENV replication cycle; however, a virucidal effect was excluded. This observation was corroborated by the fact that LCTA-949 lacks activity on DENV subgenomic replicon (that does not encode structural proteins) replication. Using a microsopy-based binding and fusion assay employing DiD-labeled viruses, it was shown that LCTA-949 targets the early stage (binding/entry) of the infection. Moreover, LCTA-949 efficiently inhibits infectivity of DENV particles pre-opsonized with antibodies, thus potentially also inhibiting antibody-dependent enhancement (ADE). In conclusion, LCTA-949 exerts in vitro activity against several flaviviruses and does so (as shown for DENV) by interfering with an early step in the viral replication cycle.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Dengue/prevention & control , Teicoplanin/analogs & derivatives , Teicoplanin/pharmacology , Virus Internalization/drug effects , Virus Replication/drug effects , Animals , Chlorocebus aethiops , Dengue Virus/physiology , Dose-Response Relationship, Drug , Encephalitis Viruses, Tick-Borne/drug effects , Fluorescent Antibody Technique , In Vitro Techniques , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribavirin/pharmacology , Vero Cells
5.
Methods ; 55(2): 137-43, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21855634

ABSTRACT

A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper.


Subject(s)
Carbocyanines/chemistry , Dengue Virus/physiology , Fluorescent Dyes/chemistry , Virion/chemistry , Virus Internalization , Animals , Cells, Cultured , Endocytosis , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Microscopy, Video/methods , Staining and Labeling/methods , Virion/isolation & purification
6.
J Nanobiotechnology ; 8: 1, 2010 Jan 20.
Article in English | MEDLINE | ID: mdl-20145735

ABSTRACT

BACKGROUND: Silver nanoparticles have proven to exert antiviral activity against HIV-1 at non-cytotoxic concentrations, but the mechanism underlying their HIV-inhibitory activity has not been not fully elucidated. In this study, silver nanoparticles are evaluated to elucidate their mode of antiviral action against HIV-1 using a panel of different in vitro assays. RESULTS: Our data suggest that silver nanoparticles exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or as an inhibitor of viral entry. Silver nanoparticles bind to gp120 in a manner that prevents CD4-dependent virion binding, fusion, and infectivity, acting as an effective virucidal agent against cell-free virus (laboratory strains, clinical isolates, T and M tropic strains, and resistant strains) and cell-associated virus. Besides, silver nanoparticles inhibit post-entry stages of the HIV-1 life cycle. CONCLUSIONS: These properties make them a broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating HIV-1 strains.

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