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The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein–protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, β3, and β4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.
Afsaneh Sadremomtaz; Zayana M. Al-Dahmani; Angel J. Ruiz-Moreno; Alessandra Monti; Chao Wang; Taha Azad; John C. Bell; Nunzianna Doti; Marco A. Velasco-Velázquez; Debora de Jong; Jørgen de Jonge; Jolanda Smit; Alexander Dömling; Harry van Goor; Matthew R. Groves. Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein. Journal of Medicinal Chemistry 2021, 1 .
AMA StyleAfsaneh Sadremomtaz, Zayana M. Al-Dahmani, Angel J. Ruiz-Moreno, Alessandra Monti, Chao Wang, Taha Azad, John C. Bell, Nunzianna Doti, Marco A. Velasco-Velázquez, Debora de Jong, Jørgen de Jonge, Jolanda Smit, Alexander Dömling, Harry van Goor, Matthew R. Groves. Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein. Journal of Medicinal Chemistry. 2021; ():1.
Chicago/Turabian StyleAfsaneh Sadremomtaz; Zayana M. Al-Dahmani; Angel J. Ruiz-Moreno; Alessandra Monti; Chao Wang; Taha Azad; John C. Bell; Nunzianna Doti; Marco A. Velasco-Velázquez; Debora de Jong; Jørgen de Jonge; Jolanda Smit; Alexander Dömling; Harry van Goor; Matthew R. Groves. 2021. "Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein." Journal of Medicinal Chemistry , no. : 1.
The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air–liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.
Bram ter Ellen; Nilima Dinesh Kumar; Ellen Bouma; Berit Troost; Denise van de Pol; Heidi van der Ende-Metselaar; Leonie Apperloo; Djoke van Gosliga; Maarten Van Den Berge; Martijn Nawijn; Peter van der Voort; Jill Moser; Izabela Rodenhuis-Zybert; Jolanda Smit. Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air–Liquid Interface Cultured Human Primary Bronchial Epithelial Cells. Viruses 2021, 13, 1335 .
AMA StyleBram ter Ellen, Nilima Dinesh Kumar, Ellen Bouma, Berit Troost, Denise van de Pol, Heidi van der Ende-Metselaar, Leonie Apperloo, Djoke van Gosliga, Maarten Van Den Berge, Martijn Nawijn, Peter van der Voort, Jill Moser, Izabela Rodenhuis-Zybert, Jolanda Smit. Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air–Liquid Interface Cultured Human Primary Bronchial Epithelial Cells. Viruses. 2021; 13 (7):1335.
Chicago/Turabian StyleBram ter Ellen; Nilima Dinesh Kumar; Ellen Bouma; Berit Troost; Denise van de Pol; Heidi van der Ende-Metselaar; Leonie Apperloo; Djoke van Gosliga; Maarten Van Den Berge; Martijn Nawijn; Peter van der Voort; Jill Moser; Izabela Rodenhuis-Zybert; Jolanda Smit. 2021. "Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air–Liquid Interface Cultured Human Primary Bronchial Epithelial Cells." Viruses 13, no. 7: 1335.
Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. In April 2021, the World Health Organization stated, however, the following: “the current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive”. It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to head comparison of the antiviral activity of ivermectin and a structurally related, but metabolically more stable, moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that the compounds predominantly act on a step after virus cell entry. Surprisingly, however, in human airway-derived cell models, moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at a concentration of 10 µM. These disappointing results calls for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on Vero cells. Altogether, these findings suggest that, even by using a high-dose regimen of ivermectin or switching to another drug in the same class are unlikely to be useful for treatment against SARS-CoV-2 in humans.
Nilima Dinesh Kumar; Bram M. ter Ellen; Ellen M. Bouma; Berit Troost; Denise P. I. van de Pol; Heidi H. van der Ende-Metselaar; Djoke van Gosliga; Leonie Apperloo; Orestes A. Carpaij; Maarten Van Den Berge; Martijn C. Nawijn; Ymkje Stienstra; Izabela A Rodenhuis-Zybert; Jolanda M. Smit. Moxidectin and ivermectin inhibit SARS-CoV-2 replication in Vero E6 cells but not in human primary airway epithelium cells. 2021, 1 .
AMA StyleNilima Dinesh Kumar, Bram M. ter Ellen, Ellen M. Bouma, Berit Troost, Denise P. I. van de Pol, Heidi H. van der Ende-Metselaar, Djoke van Gosliga, Leonie Apperloo, Orestes A. Carpaij, Maarten Van Den Berge, Martijn C. Nawijn, Ymkje Stienstra, Izabela A Rodenhuis-Zybert, Jolanda M. Smit. Moxidectin and ivermectin inhibit SARS-CoV-2 replication in Vero E6 cells but not in human primary airway epithelium cells. . 2021; ():1.
Chicago/Turabian StyleNilima Dinesh Kumar; Bram M. ter Ellen; Ellen M. Bouma; Berit Troost; Denise P. I. van de Pol; Heidi H. van der Ende-Metselaar; Djoke van Gosliga; Leonie Apperloo; Orestes A. Carpaij; Maarten Van Den Berge; Martijn C. Nawijn; Ymkje Stienstra; Izabela A Rodenhuis-Zybert; Jolanda M. Smit. 2021. "Moxidectin and ivermectin inhibit SARS-CoV-2 replication in Vero E6 cells but not in human primary airway epithelium cells." , no. : 1.
The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory and anti-oxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. Resveratrol and pterostilbene showed antiviral activity in African green monkey kidney cells and in human primary bronchial epithelial cells cultured in an air-liquid interface system. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly 5 rounds of replication, demonstrating the long-lasting effect of these compounds. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to treat SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether or not these drugs are advantageous for COVID-19 treatment.
Bram M. ter Ellen; Nilima Dinesh Kumar; Ellen M. Bouma; Berit Troost; Denise P.I. van de Pol; Heidi H. van der Ende-Metselaar; Leonie Apperloo; Djoke van Gosliga; Maarten Van Den Berge; Martijn C. Nawijn; Peter H.J. van der Voort; Jill Moser; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. Resveratrol And Pterostilbene Potently Inhibit SARS-CoV-2 Replication In Vitro. 2020, 1 .
AMA StyleBram M. ter Ellen, Nilima Dinesh Kumar, Ellen M. Bouma, Berit Troost, Denise P.I. van de Pol, Heidi H. van der Ende-Metselaar, Leonie Apperloo, Djoke van Gosliga, Maarten Van Den Berge, Martijn C. Nawijn, Peter H.J. van der Voort, Jill Moser, Izabela A. Rodenhuis-Zybert, Jolanda M. Smit. Resveratrol And Pterostilbene Potently Inhibit SARS-CoV-2 Replication In Vitro. . 2020; ():1.
Chicago/Turabian StyleBram M. ter Ellen; Nilima Dinesh Kumar; Ellen M. Bouma; Berit Troost; Denise P.I. van de Pol; Heidi H. van der Ende-Metselaar; Leonie Apperloo; Djoke van Gosliga; Maarten Van Den Berge; Martijn C. Nawijn; Peter H.J. van der Voort; Jill Moser; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Resveratrol And Pterostilbene Potently Inhibit SARS-CoV-2 Replication In Vitro." , no. : 1.
Many intra-cellular processes rely on transport by endosomes. Recent experimental techniques have provided insights into organelle maturation and its specific role in, for instance, the ability of a virus to escape an endosome and release its genetic material in the cytoplasm. Endosome maturation and dynamics depend on GTPases called Rabs, found on their membrane. Here, we introduce a mathematical framework, combining coagulation and fragmentation of endosomes with two variables internal to each organelle, to model endosomes as intra-cellular compartments characterised by their levels of (active) Rab5 and Rab7. The key element in our framework is the “per-cell endosomal distribution” and its its dynamical equation or Boltzmann equation. The Boltzmann equation, then, allows one to deduce simple equations for the total number of endosomes in a cell, and for the mean and standard deviation of the Rab5 and Rab7 levels. We compare our solutions with experimental data sets of Dengue viral escape from endosomes. The relationship between endosomal Rab levels and pH suggests a mechanism which can account for the observed variability in viral escape times, which in turn regulate the viability of a viral intra-cellular infection. Author summary Endosomes are intra-cellular receptacle-like organelles, which transport endocytosed cargo upon internalisation from the plasma membrane. These early endosomes, also known as sorting endosomes, mature to late endosomes, with a lower pH than early ones, as a consequence of the intricate dynamics of a family of molecules called Rabs. Viruses exploit this endosomal pH drop to their advantage. Here we bring together experimental data on Dengue viral escape times from endosomes and a novel mathematical framework inspired by the theory of droplet coalescence, to improve our understanding of endosome maturation, and in turn to quantify the large variability of viral escape times. This mathematical framework can easily be generalised to model the dynamics of other intra-cellular organelles, such as mitochondria or the endoplasmic reticulum.
Mario Castro; Grant Lythe; Jolanda M. Smit; Carmen Molina-París. Fusion and fission events regulate endosome maturation and viral escape. 2020, 1 .
AMA StyleMario Castro, Grant Lythe, Jolanda M. Smit, Carmen Molina-París. Fusion and fission events regulate endosome maturation and viral escape. . 2020; ():1.
Chicago/Turabian StyleMario Castro; Grant Lythe; Jolanda M. Smit; Carmen Molina-París. 2020. "Fusion and fission events regulate endosome maturation and viral escape." , no. : 1.
Despite the high disease burden of dengue virus, there is no approved antiviral treatment or broadly applicable vaccine to treat or prevent dengue virus infection. In the last decade, many antiviral compounds have been identified but only few have been further evaluated in pre-clinical or clinical trials. This review will give an overview of the direct-acting and host-directed antivirals identified to date. Furthermore, important parameters for further development that is, drug properties including efficacy, specificity and stability, pre-clinical animal testing, and combinational drug therapy will be discussed.
Berit Troost; Jolanda M Smit. Recent advances in antiviral drug development towards dengue virus. Current Opinion in Virology 2020, 43, 9 -21.
AMA StyleBerit Troost, Jolanda M Smit. Recent advances in antiviral drug development towards dengue virus. Current Opinion in Virology. 2020; 43 ():9-21.
Chicago/Turabian StyleBerit Troost; Jolanda M Smit. 2020. "Recent advances in antiviral drug development towards dengue virus." Current Opinion in Virology 43, no. : 9-21.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating. Chikungunya virus (CHIKV) is an alphavirus that is transmitted to humans by infected mosquitoes. Disease symptoms can include fever, rash, myalgia, and long-lasting debilitating joint pains. Unfortunately, there is currently no licensed vaccine or antiviral treatment available to combat CHIKV. Understanding the virus:host interactions during the replication cycle of the virus is crucial for the development of effective antiviral therapies. In this study we elucidated the trafficking behavior of CHIKV particles early in infection. During cell entry, CHIKV virions require an intact microtubule network for efficient delivery of the viral genome into the host cell thereby increasing the chance to productively infect a cell.
Tabitha E. Hoornweg; Ellen M. Bouma; Denise P.I. Van De Pol; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. Chikungunya virus requires an intact microtubule network for efficient viral genome delivery. PLOS Neglected Tropical Diseases 2020, 14, e0008469 .
AMA StyleTabitha E. Hoornweg, Ellen M. Bouma, Denise P.I. Van De Pol, Izabela A. Rodenhuis-Zybert, Jolanda M. Smit. Chikungunya virus requires an intact microtubule network for efficient viral genome delivery. PLOS Neglected Tropical Diseases. 2020; 14 (8):e0008469.
Chicago/Turabian StyleTabitha E. Hoornweg; Ellen M. Bouma; Denise P.I. Van De Pol; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Chikungunya virus requires an intact microtubule network for efficient viral genome delivery." PLOS Neglected Tropical Diseases 14, no. 8: e0008469.
The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.
Ellen M. Bouma; Denise P. I. van de Pol; Ilson D. Sanders; Izabela Rodenhuis-Zybert; Jolanda M. Smit. Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway. Journal of Virology 2020, 94, 1 .
AMA StyleEllen M. Bouma, Denise P. I. van de Pol, Ilson D. Sanders, Izabela Rodenhuis-Zybert, Jolanda M. Smit. Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway. Journal of Virology. 2020; 94 (13):1.
Chicago/Turabian StyleEllen M. Bouma; Denise P. I. van de Pol; Ilson D. Sanders; Izabela Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway." Journal of Virology 94, no. 13: 1.
In recent decades, chikungunya virus (CHIKV) has re-emerged, leading to outbreaks of chikungunya fever in Africa, Asia and Central and South America. The disease is characterized by a rapid onset febrile illness with (poly)arthralgia, myalgia, rashes, headaches and nausea. In 30 to 40% of the cases, CHIKV infection causes persistent (poly)arthralgia, lasting for months or even years after initial infection. Despite the drastic re-emergence and clinical impact there is no vaccine nor antiviral compound available to prevent or control CHIKV infection. Here, we evaluated the antiviral potential of tomatidine towards CHIKV infection. We demonstrate that tomatidine potently inhibits virus particle production of multiple CHIKV strains. Time-of -addition experiments in Huh7 cells revealed that tomatidine acts at a post-entry step of the virus replication cycle. Furthermore, a marked decrease in the number of CHIKV-infected cells was seen, suggesting that tomatidine predominantly acts early in infection yet after virus attachment and cell entry. Antiviral activity was still detected at 24 hours post-infection, indicating that tomatidine controls multiple rounds of CHIKV replication. Solasodine and sarsasapogenin, two structural derivatives of tomatidine, also showed strong albeit less potent antiviral activity towards CHIKV. In conclusion, this study identifies tomatidine as a novel compound to combat CHIKV infection in vitro.
Berit Troost; Lianne Mulder; Mayra Diosa-Toro; Denise Van De Pol; Izabela Rodenhuis-Zybert; Jolanda M. Smit. Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro. Scientific Reports 2020, 10, 1 -12.
AMA StyleBerit Troost, Lianne Mulder, Mayra Diosa-Toro, Denise Van De Pol, Izabela Rodenhuis-Zybert, Jolanda M. Smit. Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro. Scientific Reports. 2020; 10 (1):1-12.
Chicago/Turabian StyleBerit Troost; Lianne Mulder; Mayra Diosa-Toro; Denise Van De Pol; Izabela Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro." Scientific Reports 10, no. 1: 1-12.
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating.Author SummaryChikungunya virus (CHIKV) is an alphavirus that is transmitted to humans by infected mosquitoes. Disease symptoms can include fever, rash, myalgia, and long-lasting debilitating joint pains. Unfortunately, there is currently no licensed vaccine or antiviral treatment available to combat CHIKV. Understanding the virus:host interactions during the replication cycle of the virus is crucial for the development of effective antiviral therapies. In this study we elucidated the trafficking behavior of CHIKV particles early in infection. During cell entry, CHIKV virions require an intact microtubule network for efficient delivery of the viral genome into the host cell thereby increasing the chance to productively infect a cell.
Tabitha E. Hoornweg; Ellen M. Bouma; Denise P.I. Van De Pol; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. Chikungunya virus requires an intact microtubule network for efficient viral genome delivery. 2020, 1 .
AMA StyleTabitha E. Hoornweg, Ellen M. Bouma, Denise P.I. Van De Pol, Izabela A. Rodenhuis-Zybert, Jolanda M. Smit. Chikungunya virus requires an intact microtubule network for efficient viral genome delivery. . 2020; ():1.
Chicago/Turabian StyleTabitha E. Hoornweg; Ellen M. Bouma; Denise P.I. Van De Pol; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Chikungunya virus requires an intact microtubule network for efficient viral genome delivery." , no. : 1.
Chikungunya virus (CHIKV) is an important re-emerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug, 5-nonyloxytryptamine (5-NT), was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle; at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and gRNA release into the cell cytosol. Additionally, we show that the serotonin receptor antagonist MM also has antiviral properties towards CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.ImportanceThe rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including Chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus:host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound and elucidated the mechanism of action and propose serotonergic drugs as potential host-directed antivirals for CHIKV.
Ellen M. Bouma; Denise P.I. Van De Pol; Ilson D. Sanders; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. Serotonergic drugs inhibit CHIKV infection at different stages of the cell entry pathway. 2020, 1 .
AMA StyleEllen M. Bouma, Denise P.I. Van De Pol, Ilson D. Sanders, Izabela A. Rodenhuis-Zybert, Jolanda M. Smit. Serotonergic drugs inhibit CHIKV infection at different stages of the cell entry pathway. . 2020; ():1.
Chicago/Turabian StyleEllen M. Bouma; Denise P.I. Van De Pol; Ilson D. Sanders; Izabela A. Rodenhuis-Zybert; Jolanda M. Smit. 2020. "Serotonergic drugs inhibit CHIKV infection at different stages of the cell entry pathway." , no. : 1.
Chikungunya virus (CHIKV) presents a major burden on healthcare systems worldwide, but specific treatment remains unavailable. Attachment and fusion of CHIKV to the host cell membrane is mediated by the E1/E2 protein spikes. We used an in vitro single-particle fusion assay to study the effect of the potent, neutralizing antibody CHK-152 on CHIKV binding and fusion. We find that CHK-152 shields the virions, inhibiting interaction with the target membrane and inhibiting fusion. Analysis of the ratio of bound antibodies to epitopes implied that CHIKV fusion is a highly cooperative process. Further, dissociation of the antibody at lower pH results in a finely balanced kinetic competition between inhibition and fusion, suggesting a window of opportunity for the spike proteins to act and mediate fusion even in the presence of antibody.
Jelle S. Blijleven; Ellen M. Bouma; Mareike K.S. Van Duijl; Jolanda M. Smit; Antoine M. Van Oijen. Cooperative Chikungunya virus membrane fusion and its sub-stoichiometric inhibition by CHK-152 antibody. 2020, 1 .
AMA StyleJelle S. Blijleven, Ellen M. Bouma, Mareike K.S. Van Duijl, Jolanda M. Smit, Antoine M. Van Oijen. Cooperative Chikungunya virus membrane fusion and its sub-stoichiometric inhibition by CHK-152 antibody. . 2020; ():1.
Chicago/Turabian StyleJelle S. Blijleven; Ellen M. Bouma; Mareike K.S. Van Duijl; Jolanda M. Smit; Antoine M. Van Oijen. 2020. "Cooperative Chikungunya virus membrane fusion and its sub-stoichiometric inhibition by CHK-152 antibody." , no. : 1.
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause a debilitating disease that is primarily characterized by persistent joint pain. CHIKV has been emerging globally, while neither a vaccine nor antiviral medication is available. The anti-parasitic drug suramin was previously shown to inhibit CHIKV replication. In this study we aimed to obtain more detailed insight into its mechanism of action. We found that suramin interacts with virions and can inhibit virus binding to cells. It also appeared to inhibit post-attachment steps of the infection process, likely by preventing conformational changes of the envelope glycoproteins required for fusion and the progression of infection. Suramin-resistant CHIKV strains were selected and genotyping and reverse genetics experiments indicated that mutations in E2 were responsible for resistance. The substitutions N5R and H18Q were reverse engineered in the E2 glycoprotein in order to understand their role in resistance. The binding of suramin-resistant viruses with these two E2 mutations was inhibited by suramin like that of wild-type virus, but they appeared to be able to overcome the post-attachment inhibitory effect of suramin. Conversely, a virus with a G82R mutation in E2 (implicated in attenuation of vaccine strain 181/25), which renders it dependent on the interaction with heparan sulfate for entry, was more sensitive to suramin than wild-type virus. Using molecular modelling studies, we predicted the potential suramin binding sites on the mature spikes of the chikungunya virion. We conclude that suramin interferes with CHIKV entry by interacting with the E2 envelope protein, which inhibits attachment and also interferes with conformational changes required for fusion.
Irina C. Albulescu; Leonie White-Scholten; Ali Tas; Tabitha E. Hoornweg; Salvatore Ferla; Kristina Kovacikova; Jolanda M. Smit; Andrea Brancale; Eric J. Snijder; Martijn J. Van Hemert. Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection. Viruses 2020, 12, 314 .
AMA StyleIrina C. Albulescu, Leonie White-Scholten, Ali Tas, Tabitha E. Hoornweg, Salvatore Ferla, Kristina Kovacikova, Jolanda M. Smit, Andrea Brancale, Eric J. Snijder, Martijn J. Van Hemert. Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection. Viruses. 2020; 12 (3):314.
Chicago/Turabian StyleIrina C. Albulescu; Leonie White-Scholten; Ali Tas; Tabitha E. Hoornweg; Salvatore Ferla; Kristina Kovacikova; Jolanda M. Smit; Andrea Brancale; Eric J. Snijder; Martijn J. Van Hemert. 2020. "Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection." Viruses 12, no. 3: 314.
Autophagy, originally described as a conserved bulk degradation pathway important to maintain cellular homeostasis during starvation, has also been implicated in playing a central role in multiple physiological processes. For example, autophagy is part of our innate immunity by targeting intracellular pathogens to lysosomes for degradation in a process called xenophagy. Coevolution and adaptation between viruses and autophagy have armed viruses with a multitude of strategies to counteract the antiviral functions of the autophagy pathway. In addition, some viruses have acquired mechanisms to exploit specific functions of either autophagy or the key components of this process, the autophagy-related (ATG) proteins, to promote viral replication and pathogenesis. In this chapter, we describe several examples where the strategy employed by a virus to subvert autophagy has been described with molecular detail. Their stratagems positively or negatively target practically all the steps of autophagy, including the signaling pathways regulating this process. This highlights the intricate relationship between autophagy and viruses and how by commandeering autophagy, viruses have devised ways to fine-tune their replication.
Nilima Dinesh Kumar; Jolanda M. Smit; Fulvio Reggiori. Strategies employed by viruses to manipulate autophagy. Progress in Molecular Biology and Translational Science 2020, 172, 203 -237.
AMA StyleNilima Dinesh Kumar, Jolanda M. Smit, Fulvio Reggiori. Strategies employed by viruses to manipulate autophagy. Progress in Molecular Biology and Translational Science. 2020; 172 ():203-237.
Chicago/Turabian StyleNilima Dinesh Kumar; Jolanda M. Smit; Fulvio Reggiori. 2020. "Strategies employed by viruses to manipulate autophagy." Progress in Molecular Biology and Translational Science 172, no. : 203-237.
Arboviruses that are transmitted to humans by mosquitoes represent one of the most important causes of febrile illness worldwide. In recent decades, we have witnessed a dramatic re-emergence of several mosquito-borne arboviruses, including dengue virus (DENV), West Nile virus (WNV), chikungunya virus (CHIKV) and Zika virus (ZIKV). DENV is currently the most common mosquito-borne arbovirus, with an estimated 390 million infections worldwide annually. Despite a global effort, no specific therapeutic strategies are available to combat the diseases caused by these viruses. Multiple cellular pathways modulate the outcome of infection by either promoting or hampering viral replication and/or pathogenesis, and autophagy appears to be one of them. Autophagy is a degradative pathway generally induced to counteract viral infection. Viruses, however, have evolved strategies to subvert this pathway and to hijack autophagy components for their own benefit. In this review, we will focus on the role of autophagy in mosquito-borne arboviruses with emphasis on DENV, CHIKV, WNV and ZIKV, due to their epidemiological importance and high disease burden.
Liliana Echavarria-Consuegra; Jolanda M. Smit; Fulvio Reggiori. Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses. Open Biology 2019, 9, 190009 .
AMA StyleLiliana Echavarria-Consuegra, Jolanda M. Smit, Fulvio Reggiori. Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses. Open Biology. 2019; 9 (3):190009.
Chicago/Turabian StyleLiliana Echavarria-Consuegra; Jolanda M. Smit; Fulvio Reggiori. 2019. "Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses." Open Biology 9, no. 3: 190009.
Dengue is the most common arboviral disease worldwide with 96 million symptomatic cases annually. Despite its major impact on global human health and huge economic burden there is no antiviral drug available to treat the disease. The first tetravalent dengue virus vaccine was licensed in 2015 for individuals aged 9 to 45, however, most cases are reported in infants and young children. This, together with the limited efficacy of the vaccine to dengue virus (DENV) serotype 2, stresses the need to continue the search for compounds with antiviral activity to DENV. In this report, we describe tomatidine as a novel compound with potent antiviral properties towards all DENV serotypes and the related Zika virus. The strongest effect was observed for DENV-2 with an EC50 and EC90 value of 0.82 and 1.61 μM, respectively, following infection of Huh7 cells at multiplicity of infection of 1. The selectivity index is 97.7. Time-of-drug-addition experiments revealed that tomatidine inhibits virus particle production when added pre, during and up to 12 h post-infection. Subsequent experiments show that tomatidine predominantly acts at a step after virus-cell binding and membrane fusion but prior to the secretion of progeny virions. Tomatidine was found to control the expression of the cellular protein activating transcription factor 4 (ATF4), yet, this protein is not solely responsible for the observed antiviral effect. Here, we propose tomatidine as a candidate for the treatment of dengue given its potent antiviral activity.
Mayra Diosa-Toro; Berit Troost; Denise van de Pol; Alexander Heberle; Silvio Urcuqui-Inchima; Kathrin Thedieck; Jolanda M. Smit. Tomatidine, a novel antiviral compound towards dengue virus. Antiviral Research 2018, 161, 90 -99.
AMA StyleMayra Diosa-Toro, Berit Troost, Denise van de Pol, Alexander Heberle, Silvio Urcuqui-Inchima, Kathrin Thedieck, Jolanda M. Smit. Tomatidine, a novel antiviral compound towards dengue virus. Antiviral Research. 2018; 161 ():90-99.
Chicago/Turabian StyleMayra Diosa-Toro; Berit Troost; Denise van de Pol; Alexander Heberle; Silvio Urcuqui-Inchima; Kathrin Thedieck; Jolanda M. Smit. 2018. "Tomatidine, a novel antiviral compound towards dengue virus." Antiviral Research 161, no. : 90-99.
Due to the high burden of dengue disease worldwide, a better understanding of the interactions between dengue virus (DENV) and its human host cells is of the outmost importance. Although microRNAs modulate the outcome of several viral infections, their contribution to DENV replication is poorly understood. We investigated the microRNA expression profile of primary human macrophages challenged with DENV and deciphered the contribution of microRNAs to infection. To this end, human primary macrophages were challenged with GFP-expressing DENV and sorted to differentiate between truly infected cells (DENV-positive) and DENV-exposed but non-infected cells (DENV-negative cells). The miRNAome was determined by small RNA-Seq analysis and the effect of differentially expressed microRNAs on DENV yield was examined. Five microRNAs were differentially expressed in human macrophages challenged with DENV. Of these, miR-3614-5p was found upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. The cellular targets of miR-3614-5p were identified by liquid chromatography/mass spectrometry and western blot. Adenosine deaminase acting on RNA 1 (ADAR1) was identified as one of the targets of miR-3614-5p and was shown to promote DENV infectivity at early time points post-infection. Overall, miRNAs appear to play a limited role in DENV replication in primary human macrophages. The miRNAs that were found upregulated in DENV-infected cells did not control the production of infectious virus particles. On the other hand, miR-3614-5p, which was upregulated in DENV-negative macrophages, reduced DENV infectivity and regulated ADAR1 expression, a protein that facilitates viral replication. Dengue is the most common mosquito-borne disease worldwide and it is an increasing global concern for public health as its etiological agent, dengue virus (DENV), keeps spreading around the globe. Currently there are no specific antiviral therapies available to treat disease. Macrophages are important target cells during natural DENV infection of humans. Here, we unraveled the importance of miRNAs in DENV replication in human primary macrophages. The expression profile of miRNAs was determined in truly DENV-infected cells and cells that were exposed but not productively infected by the virus by RNA sequencing. We revealed that only five miRNAs are regulated in primary macrophages challenged with DENV. These results show that miRNAs do not play a major role in DENV replication. Unexpectedly, we did identify a miRNA with moderate yet significant antiviral properties to DENV. Moreover, miRNA-3614-5p was found to not only decrease DENV but also West Nile virus infectivity. Mass spectrometry and bioinformatics analysis identified adenosine deaminase acting on RNA 1 (ADAR1) as one of the targets. Moreover, ADAR1 was observed to promote the early stages of DENV replication. Collectively, our study broadens the knowledge of the contribution of human miRNAs in shaping the network of interactions between DENV and its human host cells.
Mayra Diosa-Toro; Liliana Echavarría-Consuegra; Jacky Flipse; Geysson Javier Fernandez; Joost Kluiver; Anke Van Den Berg; Silvio Urcuqui-Inchima; Jolanda M. Smit. MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression. PLOS Neglected Tropical Diseases 2017, 11, e0005981 .
AMA StyleMayra Diosa-Toro, Liliana Echavarría-Consuegra, Jacky Flipse, Geysson Javier Fernandez, Joost Kluiver, Anke Van Den Berg, Silvio Urcuqui-Inchima, Jolanda M. Smit. MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression. PLOS Neglected Tropical Diseases. 2017; 11 (10):e0005981.
Chicago/Turabian StyleMayra Diosa-Toro; Liliana Echavarría-Consuegra; Jacky Flipse; Geysson Javier Fernandez; Joost Kluiver; Anke Van Den Berg; Silvio Urcuqui-Inchima; Jolanda M. Smit. 2017. "MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression." PLOS Neglected Tropical Diseases 11, no. 10: e0005981.
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.
Nilda Vanesa Ayala Nunez; Tabitha E. Hoornweg; Denise P. I. Van De Pol; Klaas A. Sjollema; Jacky Flipse; Hilde M. Van Der Schaar; Jolanda M. Smit. How antibodies alter the cell entry pathway of dengue virus particles in macrophages. Scientific Reports 2016, 6, 28768 .
AMA StyleNilda Vanesa Ayala Nunez, Tabitha E. Hoornweg, Denise P. I. Van De Pol, Klaas A. Sjollema, Jacky Flipse, Hilde M. Van Der Schaar, Jolanda M. Smit. How antibodies alter the cell entry pathway of dengue virus particles in macrophages. Scientific Reports. 2016; 6 (1):28768.
Chicago/Turabian StyleNilda Vanesa Ayala Nunez; Tabitha E. Hoornweg; Denise P. I. Van De Pol; Klaas A. Sjollema; Jacky Flipse; Hilde M. Van Der Schaar; Jolanda M. Smit. 2016. "How antibodies alter the cell entry pathway of dengue virus particles in macrophages." Scientific Reports 6, no. 1: 28768.
The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts’ antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection.
Jacky Flipse; Mayra Diosa-Toro; Tabitha E. Hoornweg; Denise P. I. Van De Pol; Silvio Urcuqui-Inchima; Jolanda M. Smit. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses. Scientific Reports 2016, 6, 29201 .
AMA StyleJacky Flipse, Mayra Diosa-Toro, Tabitha E. Hoornweg, Denise P. I. Van De Pol, Silvio Urcuqui-Inchima, Jolanda M. Smit. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses. Scientific Reports. 2016; 6 (1):29201.
Chicago/Turabian StyleJacky Flipse; Mayra Diosa-Toro; Tabitha E. Hoornweg; Denise P. I. Van De Pol; Silvio Urcuqui-Inchima; Jolanda M. Smit. 2016. "Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses." Scientific Reports 6, no. 1: 29201.
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.
Tabitha E. Hoornweg; Mareike K. S. van Duijl-Richter; Nilda Vanesa Ayala Nunez; Irina C. Albulescu; Martijn J. van Hemert; Jolanda M. Smit. Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells. Journal of Virology 2016, 90, 4745 -4756.
AMA StyleTabitha E. Hoornweg, Mareike K. S. van Duijl-Richter, Nilda Vanesa Ayala Nunez, Irina C. Albulescu, Martijn J. van Hemert, Jolanda M. Smit. Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells. Journal of Virology. 2016; 90 (9):4745-4756.
Chicago/Turabian StyleTabitha E. Hoornweg; Mareike K. S. van Duijl-Richter; Nilda Vanesa Ayala Nunez; Irina C. Albulescu; Martijn J. van Hemert; Jolanda M. Smit. 2016. "Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells." Journal of Virology 90, no. 9: 4745-4756.