scholarly journals Participation of Extracellular Vesicles from Zika-Virus-Infected Mosquito Cells in the Modification of Naïve Cells’ Behavior by Mediating Cell-to-Cell Transmission of Viral Elements

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 123 ◽  
Author(s):  
Pedro Pablo Martínez-Rojas ◽  
Elizabeth Quiroz-García ◽  
Verónica Monroy-Martínez ◽  
Lourdes Teresa Agredano-Moreno ◽  
Luis Felipe Jiménez-García ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Cell Damage ◽  
Infection Process ◽  
Host Cells ◽  
Infected Mosquito ◽  
Zikv Infection ◽  
Necrosis Factor Alpha ◽  
Inflammatory State

To date, no safe vaccine or antivirals for Zika virus (ZIKV) infection have been found. The pathogenesis of severe Zika, where host and viral factors participate, remains unclear. For the control of Zika, it is important to understand how ZIKV interacts with different host cells. Knowledge of the targeted cellular pathways which allow ZIKV to productively replicate and/or establish prolonged viral persistence contributes to novel vaccines and therapies. Monocytes and endothelial vascular cells are the main ZIKV targets. During the infection process, cells are capable of releasing extracellular vesicles (EVs). EVs are mediators of intercellular communication. We found that mosquito EVs released from ZIKV-infected (C6/36) cells carry viral RNA and ZIKV-E protein and are able to infect and activate naïve mosquito and mammalian cells. ZIKV C6/36 EVs promote the differentiation of naïve monocytes and induce a pro-inflammatory state with tumor necrosis factor-alpha (TNF-α) mRNA expression. ZIKV C6/36 EVs participate in endothelial vascular cell damage by inducing coagulation (TF) and inflammation (PAR-1) receptors at the endothelial surface of the cell membranes and promote a pro-inflammatory state with increased endothelial permeability. These data suggest that ZIKV C6/36 EVs may contribute to the pathogenesis of ZIKV infection in human hosts.

The epidermal growth factor receptor is a relevant host factor in the early stages of Zika virus life cycle in vitro

2021 ◽  
Author(s):  
Catarina Sabino ◽  
Daniela Bender ◽  
Marie-Luise Herrlein ◽  
Eberhard Hildt
Keyword(s):  
Life Cycle ◽  
Zika Virus ◽  
A549 Cells ◽  
Growth Factor Receptor ◽  
Viral Life Cycle ◽  
Infection Process ◽  
Zikv Infection ◽  
Early Stages ◽  
Egfr Expression

Zika virus (ZIKV) is a flavivirus well-known for the epidemic in the Americas in 2015-2016, where microcephaly in newborns and other neurological complications were connected to ZIKV infection. Many aspects of the viral life cycle, including binding and entry into the host cell, are still enigmatic. Based on the observation that CHO cells lack the expression of EGFR and are not permissive for various ZIKV strains, the relevance of EGFR for the viral life cycle was analyzed. Infection of A549 cells by ZIKV leads to a rapid internalization of EGFR that colocalizes with the endosomal marker EEA1. Moreover, the infection by different ZIKV strains is associated with an activation of EGFR and subsequent activation of the MAPK/ERK signaling cascade. However, treatment of the cells with MβCD, which on the one hand leads to an activation of EGFR but on the other hand prevents EGFR internalization, impairs ZIKV infection. Specific inhibition of EGFR or of the RAS-RAF-MEK-ERK signal transduction cascade hinders ZIKV infection by inhibition of ZIKV entry. In accordance to this, knockout of EGFR expression impedes ZIKV entry. In case of an already established infection, inhibition of EGFR or of downstream signaling does not affect viral replication. Taken together, these data demonstrate the relevance of EGFR in the early stages of ZIKV infection and identify EGFR as a target for antiviral strategies. Importance These data deepen the knowledge about the ZIKV infection process and demonstrate the relevance of EGFR for ZIKV entry. In light of the fact that a variety of specific and efficient inhibitors of EGFR and of EGFR-dependent signaling were developed and licensed, repurposing of these substances could be a helpful tool to prevent the spreading of ZIKV infection in an epidemic outbreak.

scholarly journals TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 519
Author(s):  
Yuchen Wang ◽  
Ryuta Uraki ◽  
Jesse Hwang ◽  
Erol Fikrig
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Host Factors ◽  
Ns1 Protein ◽  
Zikv Infection ◽  
Binding Partner ◽  
Promising Therapeutic Target ◽  
Critical Components ◽  
Congenital Microcephaly

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain–Barré syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target.

scholarly journals Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 45
Author(s):  
Benedita K. L. Feron ◽  
Joachim J. Bugert ◽  
Simon C. W. Richardson
Keyword(s):  
Cell Viability ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Sirna Delivery ◽  
Lethal Factor ◽  
Firefly Luciferase ◽  
Delivery Systems ◽  
Zikv Infection ◽  
Huh7 Cells

Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (p = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (p = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant Bacillus anthracis PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of B. anthracis lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (p = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (p = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the PierceTM Firefly Luciferase Glow Assay Kit by Thermo ScientificTM. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models.

scholarly journals Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

2010 ◽  
Vol 78 (4) ◽  
pp. 1601-1609 ◽  
Author(s):  
Débora L. Oliveira ◽  
Célio G. Freire-de-Lima ◽  
Joshua D. Nosanchuk ◽  
Arturo Casadevall ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Extracellular Vesicles ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Interleukin 10 ◽  
Biologically Active ◽  
Host Cells ◽  
Phagocytic Cells ◽  
Necrosis Factor Alpha

ABSTRACT Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.

scholarly journals Adaptation to host cell environment during experimental evolution of Zika virus

2021 ◽  
Author(s):  
Vincent Grass ◽  
Emilie Hardy ◽  
Kassian Kobert ◽  
Soheil Rastgou Talemi ◽  
Elodie Décembre ◽  
...  
Keyword(s):  
Host Cell ◽  
Zika Virus ◽  
Immune Escape ◽  
Viral Evolution ◽  
Time Lag ◽  
Host Cells ◽  
Host Responses ◽  
Type I ◽  
Zikv Infection ◽  
Cellular Environment

Abstract Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV populations pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated S455L, a substitution in ZIKV envelope (E) protein, recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Taken together, our results uncovered ZIKV adaptations to the cellular environment leading to accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into Zika virus adaptation to host cells and immune escape mechanisms.

scholarly journals ADP-Ribosyltransferase PARP11 Suppresses Zika Virus in Synergy with PARP12

2021 ◽  
Author(s):  
Lili Li ◽  
Yueyue Shi ◽  
Sirui Li ◽  
Junxiao Liu ◽  
Shulong Zu ◽  
...  
Keyword(s):  
Zika Virus ◽  
Neurological Complications ◽  
Host Cells ◽  
Interferon Response ◽  
Type I ◽  
Zikv Infection ◽  
Interferon Stimulated Genes ◽  
Ns3 Protein ◽  
Global Threat ◽  
Adp Ribosyltransferase

Abstract Zika virus (ZIKV) infection and ZIKV epidemic have been continuously spreading silently throughout the world and its associated microcephaly and other serious congenital neurological complications poses a significant global threat to public health. ZIKV infection stimulates type I interferon response in host cells which suppresses viral replication by inducing the expression of interferon-stimulated genes (ISGs). Here, we identified ADP-ribosyltransferase PARP11 as an anti-ZIKV ISG and found that PARP11 suppressed ZIKV independently on itself PARP enzyme activity. Furthermore, PARP11 interacted with PARP12 and promoted PARP12-mediating ZIKV NS1 and NS3 protein degradation. Homo family PARP11 and PARP12 cooperated with each other on ZIKV suppression and the anti-ZIKV function of PARP11 mostly dependent on the existence of PARP12. Our findings have broadened the understanding of the anti-viral function of PARP11, and more importantly suggest a potential therapeutics target against ZIKV infection.

scholarly journals One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

2019 ◽  
Author(s):  
Ricardo Vieira Araujo ◽  
Fabiana Feitosa-Suntheimer ◽  
Alexander S. Gold ◽  
Berlin Londono-Renteria ◽  
Tonya Michelle Colpitts
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Infected Mosquito ◽  
Zikv Infection ◽  
Rna Detection ◽  
High Background ◽  
Qrt Pcr ◽  
One Step ◽  
Nonspecific Amplification ◽  
Lower Background

Abstract Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is qRT-PCR, yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito RNA. In this work we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing for confident detection.Methods: Our new oligo design to detect ZIKV RNA included in silicoanalysis of both viral and mosquito (Ae. aegyptiand Ae. albopictus)genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedesgenomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes.Standard curves constructed with in vitrotranscribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegyptiimmune response to virus infection.Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegyptipresented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

scholarly journals Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Shu Liu ◽  
André Hossinger ◽  
Julia P. Hofmann ◽  
Philip Denner ◽  
Ina M. Vorberg
Keyword(s):  
Extracellular Vesicles ◽  
Mammalian Cells ◽  
Information Transfer ◽  
Quaternary Structure ◽  
Horizontal Transmission ◽  
Biologically Active ◽  
Host Cells ◽  
Transmissible Spongiform Encephalopathies ◽  
Yeast Prion ◽  
Long Distance

ABSTRACT Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast ( Saccharomyces cerevisiae ) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. IMPORTANCE Prions are proteinaceous infectious particles that propagate by templating their quaternary structure onto nascent proteins of the same kind. Prions in yeast act as heritable epigenetic elements that can alter the phenotype when transmitted to daughter cells or during mating. Prion activity is conferred by so-called prion domains often enriched in glutamine and asparagine residues. Interestingly, many mammalian proteins also contain domains with compositional similarity to yeast prion domains. We have recently provided a proof-of-principle demonstration that a yeast prion domain also retains its prion activity in mammalian cells. We demonstrate here that cytosolic prions composed of a yeast prion domain are also packaged into extracellular vesicles that transmit the prion phenotype to bystander cells. Thus, proteins with prion-like domains can behave as proteinaceous information molecules that exploit the cellular vesicle trafficking machinery for intercellular long-distance dissemination.

scholarly journals ADP-ribosyltransferase PARP11 suppresses Zika virus in synergy with PARP12

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lili Li ◽  
Yueyue Shi ◽  
Sirui Li ◽  
Junxiao Liu ◽  
Shulong Zu ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cell Lines ◽  
Zika Virus ◽  
Hek293t Cell ◽  
Host Cells ◽  
Interferon Response ◽  
Type I ◽  
Zikv Infection ◽  
Ns3 Protein ◽  
Adp Ribosyltransferase

Abstract Background Zika virus (ZIKV) infection and ZIKV epidemic have been continuously spreading silently throughout the world and its associated microcephaly and other serious congenital neurological complications poses a significant global threat to public health. Type I interferon response to ZIKV infection in host cells suppresses viral replication by inducing the expression of interferon-stimulated genes (ISGs). Methods The study aims to demonstrate the anti-ZIKV mechanism of PARP11. PARP11 knock out and overexpressing A549 cell lines were constructed to evaluate the anti-ZIKV function of PARP11. PARP11−/−, PARP12−/− and PARP11−/−PARP12−/− HEK293T cell lines were constructed to explain the synergistic effect of PARP11 and PARP12 on NS1 and NS3 protein degradation. Western blotting, immunofluorescence and immunoprecipitation assay were performed to illustrate the interaction between PARP11 and PARP12. Results Both mRNA and protein levels of PARP11 were induced in WT but not IFNAR1−/− cells in response to IFNα or IFNβ stimulation and ZIKV infection. ZIKV replication was suppressed in cells expressed PARP11 but was enhanced in PARP11−/− cells. PARP11 suppressed ZIKV independently on itself PARP enzyme activity. PARP11 interacted with PARP12 and promoted PARP12-mediated ZIKV NS1 and NS3 protein degradation. Conclusion We identified ADP-ribosyltransferase PARP11 as an anti-ZIKV ISG and found that it cooperated with PARP12 to enhance ZIKV NS1 and NS3 protein degradation. Our findings have broadened the understanding of the anti-viral function of ADP-ribosyltransferase family members, and provided potential therapeutic targets against viral ZIKV infection.

scholarly journals Heat shock protein 70 (Hsp70) is involved in the Zika virus cellular infection process

2017 ◽  
Author(s):  
Sujit Pujhari ◽  
Vanessa M. Macias ◽  
Ruth H. Nissly ◽  
Masashi Nomura ◽  
Suresh V. Kuchipudi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Zika Virus ◽  
Heat Shock Protein 70 ◽  
Membrane Surface ◽  
Infection Process ◽  
Western Hemisphere ◽  
Hsp70 Expression ◽  
Zikv Infection ◽  
Hsp70 Protein

AbstractZika virus (ZIKV) is a historically neglected flavivirus that has recently caused epidemics in the western hemisphere. ZIKV has been associated with severe symptoms including infant microcephaly and Guillain Barré syndrome, stimulating interest in understanding factors governing ZIKV infection. Heat shock protein 70 (Hsp70) has been shown to be an infection factor for multiple viruses. We investigated the role of Hsp70 in the ZIKV infection process. We localized Hsp70 protein to the Vero cell membrane surface by confocal microscopy and demonstrated that, inside the cell, there is significant co-localization between Hsp70 and ZIKV E protein. Inducing and suppressing Hsp70 expression increased and decreased ZIKV production, respectively. Antibody blocking cell surface-localized Hsp70 decreased ZIKV cell infection rates and production of infectious virus particles, as did competition with recombinant Hsp70 protein. Our data suggest that Hsp70 is an important factor in the ZIKV infection process. Understanding the interactions between Hsp70 and ZIKV may lead to novel therapeutics for ZIKV infection, particularly for pregnant women and fetuses.

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