Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

ABSTRACT The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis. IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.

Download Full-text

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Cells ◽

10.3390/cells10071828 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1828

Author(s):

Jared Kirui ◽

Yara Abidine ◽

Annasara Lenman ◽

Koushikul Islam ◽

Yong-Dae Gwon ◽

...

Keyword(s):

Chikungunya Virus ◽

Molecular Mechanisms ◽

Rna Virus ◽

Ectopic Expression ◽

Point Mutations ◽

Cell Entry ◽

Target Cells ◽

Human Hepatoma Cells ◽

Chikv Infection ◽

Phosphatidylserine Receptor

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

Download Full-text

First Imported Case of Chikungunya Virus Infection in a Travelling Canadian Returning from the Caribbean

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2016/2980297 ◽

2016 ◽

Vol 2016 ◽

pp. 1-5

Author(s):

Christian Therrien ◽

Guillaume Jourdan ◽

Kimberly Holloway ◽

Cécile Tremblay ◽

Michael A. Drebot

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Neutralizing Antibodies ◽

Chikungunya Virus ◽

Joint Stiffness ◽

Clinical Feature ◽

Chikv Infection ◽

Imported Case ◽

The Caribbean ◽

Hands And Feet

This is the first Canadian case of Chikungunya virus (CHIKV) infection reported in a traveller returning from the Caribbean. Following multiple mosquito bites in Martinique Island in January 2014, the patient presented with high fever, headaches, arthralgia on both hands and feet, and a rash on the trunk upon his return to Canada. Initial serological testing for dengue virus infection was negative. Support therapy with nonsteroidal anti-inflammatory drugs was administered. The symptoms gradually improved 4 weeks after onset with residual arthralgia and morning joint stiffness. This clinical feature prompted the clinician to request CHIKV virus serology which was found to be positive for the presence of IgM and neutralizing antibodies. In 2014, over four hundred confirmed CHIKV infection cases were diagnosed in Canadian travellers returning from the Caribbean and Central America. Clinical suspicion of CHIKV or dengue virus infections should be considered in febrile patients with arthralgia returning from the recently CHIKV endemic countries of the Americas.

Download Full-text

PP2A Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication by Deactivating irf3 and Limiting Type I Interferon Production

Viruses ◽

10.3390/v11100948 ◽

2019 ◽

Vol 11 (10) ◽

pp. 948 ◽

Cited By ~ 1

Author(s):

Jiayu Xu ◽

Lu Zhang ◽

Yunfei Xu ◽

He Zhang ◽

Junxin Gao ◽

...

Keyword(s):

Virus Infection ◽

Therapeutic Target ◽

Type I Interferon ◽

Negative Regulator ◽

Host Cells ◽

Respiratory Syndrome Virus ◽

Target Cells ◽

Type I ◽

Dependent Manner ◽

Pp2a Activity

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase in mammalian cells, is known to regulate the kinase-driven intracellular signaling pathways. Emerging evidences have shown that the PP2A phosphatase functions as a bona-fide therapeutic target for anticancer therapy, but it is unclear whether PP2A affects a porcine reproductive and respiratory syndrome virus infection. In the present study, we demonstrated for the first time that inhibition of PP2A activity by either inhibitor or small interfering RNA duplexes in target cells significantly reduced their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) infection. Further analysis revealed that inhibition of PP2A function resulted in augmented production of type I interferon (IFN). The mechanism is that inhibition of PP2A activity enhances the levels of phosphorylated interferon regulatory factor 3, which activates the transcription of IFN-stimulated genes. Moreover, inhibition of PP2A activity mainly blocked PRRSV replication in the early stage of viral life cycle, after virus entry but before virus release. Using type I IFN receptor 2 specific siRNA in combination with PP2A inhibitor, we confirmed that the effect of PP2A on viral replication within target cells was an interferon-dependent manner. Taken together, these findings demonstrate that PP2A serves as a negative regulator of host cells antiviral responses and provides a novel therapeutic target for virus infection.

Download Full-text

Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

mBio ◽

10.1128/mbio.01956-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 32

Author(s):

Maria Dolores Fernandez-Garcia ◽

Laurent Meertens ◽

Maxime Chazal ◽

Mohamed Lamine Hafirassou ◽

Ophélie Dejarnac ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Virus Entry ◽

Fever Virus ◽

Host Cells ◽

Target Cells ◽

Wild Type ◽

E Protein ◽

Virulence Attenuation ◽

Molecular Determinants

ABSTRACTThe live attenuated yellow fever virus (YFV) vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation.IMPORTANCEThe yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.

Download Full-text

Cathepsin B Protease Facilitates Chikungunya Virus Envelope Protein-Mediated Infection Via Endocytosis or Macropinocytosis

Viruses ◽

10.3390/v12070722 ◽

2020 ◽

Vol 12 (7) ◽

pp. 722

Author(s):

Mai Izumida ◽

Hideki Hayashi ◽

Atsushi Tanaka ◽

Yoshinao Kubo

Keyword(s):

Cathepsin B ◽

Chikungunya Virus ◽

Ebola Virus ◽

Leukemia Virus ◽

Host Cells ◽

Virus Envelope ◽

Chikv Infection ◽

Enveloped Virus ◽

Entry Pathway ◽

Virus Envelope Protein

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.

Download Full-text

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein

Journal of Virology ◽

10.1128/jvi.00232-16 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5256-5269 ◽

Cited By ~ 12

Author(s):

Anne-Laure Favier ◽

Evelyne Gout ◽

Olivier Reynard ◽

Olivier Ferraris ◽

Jean-Philippe Kleman ◽

...

Keyword(s):

Virus Infection ◽

Ebola Virus ◽

Mannose Binding Lectin ◽

Innate Immune ◽

Host Cells ◽

Target Cells ◽

Viral Glycoprotein ◽

Mannose Binding ◽

Ebov Infection ◽

Binding Lectin

ABSTRACTEbola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system.IMPORTANCEA specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response.

Download Full-text

Identification of a Putative Coreceptor on Vero Cells That Participates in Dengue 4 Virus Infection

Journal of Virology ◽

10.1128/jvi.75.17.7818-7827.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7818-7827 ◽

Cited By ~ 77

Author(s):

José de Jesús Martı́nez-Barragán ◽

Rosa M. del Angel

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Vero Cells ◽

Host Cells ◽

Cell Surface Receptor ◽

Target Cells ◽

Virus Binding ◽

Dengue Virus Infection ◽

Surface Receptor ◽

A Cell

ABSTRACT Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (α-mannose orN-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.

Download Full-text

Differential Proteome Analysis of Chikungunya Virus Infection on Host Cells

PLoS ONE ◽

10.1371/journal.pone.0061444 ◽

2013 ◽

Vol 8 (4) ◽

pp. e61444 ◽

Cited By ~ 29

Author(s):

Christina Li-Ping Thio ◽

Rohana Yusof ◽

Puteri Shafinaz Akmar Abdul-Rahman ◽

Saiful Anuar Karsani

Keyword(s):

Virus Infection ◽

Chikungunya Virus ◽

Proteome Analysis ◽

Host Cells

Download Full-text

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection

Journal of Virology ◽

10.1128/jvi.02160-18 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 6

Author(s):

Rutger D. Luteijn ◽

Ferdy van Diemen ◽

Vincent A. Blomen ◽

Ingrid G. J. Boer ◽

Saravanan Manikam Sadasivam ◽

...

Keyword(s):

Virus Infection ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Host Cell ◽

Host Factors ◽

Virus Infectivity ◽

Sequencing Analysis ◽

Vaccinia Virus Infection ◽

Genome Wide ◽

A Genome

ABSTRACTVaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCEPoxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

Download Full-text

Organometallic Complex Strongly Impairs Chikungunya Virus Entry to the Host Cells

Frontiers in Microbiology ◽

10.3389/fmicb.2020.608924 ◽

2020 ◽

Vol 11 ◽

Author(s):

Débora Moraes de Oliveira ◽

Igor de Andrade Santos ◽

Daniel Oliveira Silva Martins ◽

Yasmim Garcia Gonçalves ◽

Léia Cardoso-Sousa ◽

...

Keyword(s):

Chikungunya Virus ◽

Hydrophobic Interactions ◽

Therapeutic Potential ◽

Virus Entry ◽

Antiviral Treatment ◽

Host Cells ◽

Virus Infectivity ◽

Chloride Salt ◽

Organometallic Complex ◽

Chikv Infection

Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV) that is transmitted by the bite of the female of Aedes sp. mosquito. The symptoms include fever, muscle aches, skin rash, and severe joint pains. The disease may develop into a chronic condition and joint pain for months or years. Currently, there is no effective antiviral treatment against CHIKV infection. Treatments based on natural compounds have been widely studied, as many drugs were produced by using natural molecules and their derivatives. Alpha-phellandrene (α-Phe) is a naturally occurring organic compound that is a ligand for ruthenium, forming the organometallic complex [Ru2Cl4(p-cymene)2] (RcP). Organometallic complexes have shown promising as candidate molecules to a new generation of compounds that presented relevant biological properties, however, there is a lack of knowledge concerning the anti-CHIKV activity of these complexes. The present work evaluated the effects of the RcP and its precursors, the hydrate ruthenium(III) chloride salt (RuCl3⋅xH2O) (Ru) and α-Phe, on CHIKV infection in vitro. To this, BHK-21 cells were infected with CHIKV-nanoluciferase (CHIKV-nanoluc), a viral construct harboring the nanoluciferase reporter gene, at the presence or absence of the compounds for 16 h. Cytotoxicity and impact on infectivity were analyzed. The results demonstrated that RcP exhibited a strong therapeutic potential judged by the selective index > 40. Antiviral effects of RcP on different stages of the CHIKV replicative cycle were investigated; the results showed that it affected early stages of virus infection reducing virus replication by 77% at non-cytotoxic concentrations. Further assays demonstrated the virucidal activity of the compound that completely blocked virus infectivity. In silico molecular docking calculations suggested different binding interactions between aromatic rings of RcP and the loop of amino acids of the E2 envelope CHIKV glycoprotein mainly through hydrophobic interactions. Additionally, infrared spectroscopy spectral analysis indicated interactions of RcP with CHIKV glycoproteins. These data suggest that RcP may act on CHIKV particles, disrupting virus entry to the host cells. Therefore, RcP may represent a strong candidate for the development of anti-CHIKV drugs.

Download Full-text