scholarly journals The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1475
Author(s):  
Mizuki Yamamoto ◽  
Takeshi Ichinohe ◽  
Aya Watanabe ◽  
Ayako Kobayashi ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Renilla Luciferase ◽  
E Proteins ◽  
E Protein ◽  
Viral Lifecycle

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6–2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Morganna C. Lima ◽  
Elisa A. N. Azevedo ◽  
Clarice N. L. de Morais ◽  
Larissa I. O. de Sousa ◽  
Bruno M. Carvalho ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Virus Infection ◽  
Virus Replication ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Neurological Complications ◽  
Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

scholarly journals MAIT cells are activated in acute Dengue virus infection and after in vitro Zika virus infection

2018 ◽  
Vol 12 (1) ◽  
pp. e0006154 ◽  
Author(s):  
Dominic Paquin-Proulx ◽  
Vivian I. Avelino-Silva ◽  
Bianca A. N. Santos ◽  
Nathália Silveira Barsotti ◽  
Fabiana Siroma ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Zika Virus ◽  
Dengue Virus Infection ◽  
Mait Cells ◽  
Zika Virus Infection

scholarly journals In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein

2010 ◽  
Vol 84 (11) ◽  
pp. 5730-5740 ◽  
Author(s):  
Maofu Liao ◽  
Claudia Sánchez-San Martín ◽  
Aihua Zheng ◽  
Margaret Kielian
Keyword(s):  
Dengue Virus ◽  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Fusion Reaction ◽  
Low Ph ◽  
Viral Envelope ◽  
Interaction Sites ◽  
E Protein ◽  
Hairpin Formation

ABSTRACT The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.

scholarly journals Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb

2018 ◽  
Vol 115 (28) ◽  
pp. E6604-E6613 ◽  
Author(s):  
Ashish Vora ◽  
Wenshuo Zhou ◽  
Berlin Londono-Renteria ◽  
Michael Woodson ◽  
Michael B. Sherman ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Virus Transmission ◽  
Direct Interaction ◽  
Rnase A ◽  
Human Cells ◽  
Viral Rna ◽  
E Protein ◽  
In Vitro Cell Lines

Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.

scholarly journals Zika virus infection in human placental tissue explants is enhanced in the presence of dengue virus antibodies in-vitro

2018 ◽  
Vol 7 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Kyra Hermanns ◽  
Claudia Göhner ◽  
Anne Kopp ◽  
Andre Schmidt ◽  
Waltraut M. Merz ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Zika Virus ◽  
Placental Tissue ◽  
Tissue Explants ◽  
Zika Virus Infection ◽  
Human Placental Tissue

scholarly journals Zika Virus-Like Particles Bearing a Covalent Dimer of Envelope Protein Protect Mice from Lethal Challenge

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Giuditta De Lorenzo ◽  
Rapeepat Tandavanitj ◽  
Jennifer Doig ◽  
Chayanee Setthapramote ◽  
Monica Poggianella ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Wild Type ◽  
Lethal Challenge ◽  
E Protein ◽  
Virus Like Particle ◽  
High Level

ABSTRACT Zika virus (ZIKV) envelope (E) protein is the major target of neutralizing antibodies in infected hosts and thus represents a candidate of interest for vaccine design. However, a major concern in the development of vaccines against ZIKV and the related dengue virus is the induction of cross-reactive poorly neutralizing antibodies that can cause antibody-dependent enhancement (ADE) of infection. This risk necessitates particular care in vaccine design. Specifically, the engineered immunogens should have their cross-reactive epitopes masked, and they should be optimized for eliciting virus-specific strongly neutralizing antibodies upon vaccination. Here, we developed ZIKV subunit- and virus-like particle (VLP)-based vaccines displaying E in its wild-type form or E locked in a covalently linked dimeric (cvD) conformation to enhance the exposure of E dimers to the immune system. Compared with their wild-type derivatives, cvD immunogens elicited antibodies with a higher capacity to neutralize virus infection in cultured cells. More importantly, these immunogens protected animals from lethal challenge with both the African and Asian lineages of ZIKV, impairing virus dissemination to brain and sexual organs. Moreover, the locked conformation of E reduced the exposure of epitopes recognized by cross-reactive antibodies and therefore showed a lower potential to induce ADE in vitro. Our data demonstrated a higher efficacy of the VLPs in comparison with that of the soluble dimer and support VLP-cvD as a promising ZIKV vaccine. IMPORTANCE Infection with Zika virus (ZIKV) leads to the production by the host of antibodies that target the viral surface envelope (E) protein. A subset of these antibodies can inhibit virus infection, thus making E a suitable candidate for the development of vaccine against the virus. However, the anti-ZIKV E antibodies can cross-react with the E protein of the related dengue virus on account of the high level of similarity exhibited by the two viral proteins. Such a scenario may lead to severe dengue disease. Therefore, the design of a ZIKV vaccine requires particular care. Here, we tested two candidate vaccines containing a recombinant form of the ZIKV E protein that is forced in a covalently stable dimeric conformation (cvD). They were generated with an explicit aim to reduce the exposure of the cross-reactive epitopes. One vaccine is composed of a soluble form of the E protein (sE-cvD), the other is a more complex virus-like particle (VLP-cvD). We used the two candidate vaccines to immunize mice and later infected them with ZIKV. The animals produced a high level of inhibitory antibodies and were protected from the infection. The VLP-cvD was the most effective, and we believe it represents a promising ZIKV vaccine candidate.

scholarly journals Human Serum With High Neutralizing Antibody Titers Against Both Zika and Dengue Virus Shows Delayed In Vitro Antibody-Dependent Enhancement of Dengue Virus Infection

2018 ◽  
Vol 5 (7) ◽  
Author(s):  
William G Valiant ◽  
Tahaniyat Lalani ◽  
Heather C Yun ◽  
Anjali Kunz ◽  
Timothy H Burgess ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Dengue Infection ◽  
Neutralizing Antibody ◽  
Naive Subject ◽  
Dengue Virus Infection ◽  
Antibody Titers

Abstract Zika virus infection in a dengue virus–naïve subject was associated with the induction of high levels of cross-reactive binding antibodies. These responses were, however, largely non-neutralizing and displayed a capacity to enhance dengue infection in vitro at significantly low dilution (1:10). In contrast, a subject who had high levels of neutralizing antibodies against both dengue and Zika viruses enhanced infection at a dilution of 1:10 000. These results suggest that high levels of dengue cross-neutralizing antibodies could potentially prevent the enhancement of dengue infection in Zika virus–convalescent individuals.

scholarly journals Zika virus-like particles bearing covalent dimer of envelope protein protect mice from lethal challenge

2020 ◽  
Author(s):  
Giuditta De Lorenzo ◽  
Rapeepat Tandavanitj ◽  
Jennifer Doig ◽  
Chayanee Setthapramote ◽  
Monica Poggianella ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Wild Type ◽  
Lethal Challenge ◽  
E Protein ◽  
Virus Like Particle ◽  
High Level

AbstractZika virus (ZIKV) envelope (E) protein is the major target of neutralizing antibodies in infected host, and thus represents a candidate of interest for vaccine design. However, a major concern in the development of vaccines against ZIKV and the related dengue virus is the induction of cross-reactive poorly neutralizing antibodies that can cause antibody-dependent enhancement (ADE) of infection. This risk necessitates particular care in vaccine design. Specifically, the engineered immunogens should have their cross-reactive epitopes masked, and they should be optimized for eliciting virus-specific strongly neutralizing antibodies upon vaccination. Here, we developed ZIKV subunit- and virus-like particle (VLP)-based vaccines displaying E in its wild type form, or E locked in a covalently linked dimeric (cvD) conformation to enhance the exposure of E dimers to the immune system. Compared with their wild-type derivatives, cvD immunogens elicited antibody with higher capacity of neutralizing virus infection of cultured cells. More importantly, these immunogens protected animals from lethal challenge with both the African and Asian lineages of ZIKV, impairing virus dissemination to brain and sexual organs. Moreover, the locked conformation of E reduced the exposure of epitopes recognized by cross-reactive antibodies and therefore showed a lower potential to induce ADE in vitro. Our data demonstrated a higher efficacy of the VLPs in comparison with the soluble dimer and support VLP-cvD as a promising ZIKV vaccine.Author SummaryInfection with Zika virus (ZIKV) leads to the production by host of antibodies that target the viral surface envelope (E) protein. A subset of these antibodies can inhibit virus infection, thus making E as a suitable candidate for the development of vaccine against the virus. However, the anti-ZIKV E antibodies can cross-react with the E protein of the related dengue virus on account of the high level of similarity exhibited by the two viral proteins. Such a scenario may lead to severe dengue disease. Therefore, the design of a ZIKV vaccine requires particular care. Here, we tested two candidate vaccines containing a recombinant form of the ZIKV E protein that is forced in a covalently stable dimeric conformation (cvD). They were generated with an explicit aim to reduce the exposure of the cross-reactive epitopes. One vaccine is composed of a soluble form of the E protein (sE-cvD), the other is a more complex virus-like particle (VLP-cvD). We used the two candidate vaccines to immunize mice and later infected with ZIKV. The animals produced high level of inhibitory antibodies and were protected from the infection. The VLP-cvD was the most effective and we believe it represents a promising ZIKV vaccine candidate.

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