High-Throughput Fitness Profiling of Zika Virus E Protein Reveals Different Roles for Glycosylation during Infection of Mammalian and Mosquito Cells

AbstractZika virus (ZIKV) infection causes Guillain-Barré syndrome and severe birth defects. ZIKV envelope (E) protein is the major viral protein involved in cell receptor binding and entry and therefore considered one of the major determinants in ZIKV pathogenesis. Here, we report a gene-wide mapping of functional residues of ZIKV E protein using a mutant library with changes covering every nucleotide position. By comparing the replication fitness of every viral mutant between mosquito and human cells, we identified that mutations affecting N-linked glycosylation at N154 position display the most divergence. Through characterizing individual mutants, we show that, while ablation of N-linked glycosylation selectively benefits ZIKV infection of mosquito cells by enhancing cell entry, it either had little impact on ZIKV infection on certain human cells or decreased infection through entry factor DC-SIGN. In conclusion, we define the roles of individual residues of ZIKV envelope protein, which contribute to ZIKV replication fitness in human and mosquito cells.HighlightsGene-wide mapping of functional residues of E protein in human and mosquito cells.Mutations affecting N-linked glycosylation display the most dramatic difference.N-linked glycosylation decreases ZIKV entry into mosquito cells.N-linked glycosylation is important for DC-SIGN mediated infection of human cells.

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Faculty Opinions recommendation of Differential Metabolic Reprogramming by Zika Virus Promotes Cell Death in Human versus Mosquito Cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735240309.793562464 ◽

2019 ◽

Author(s):

Eng Eong Ooi ◽

Kuan Rong Chan

Keyword(s):

Cell Death ◽

Zika Virus ◽

Metabolic Reprogramming ◽

Mosquito Cells

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Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection

Nature Communications ◽

10.1038/s41467-021-22966-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Rommel J. Gestuveo ◽

Jamie Royle ◽

Claire L. Donald ◽

Douglas J. Lamont ◽

Edward C. Hutchinson ◽

...

Keyword(s):

Aedes Aegypti ◽

Protein Interactions ◽

Zika Virus ◽

Label Free ◽

Protein Protein Interactions ◽

Zikv Infection ◽

Ubiquitin Protein Ligase ◽

Mosquito Cells ◽

Endoplasmic Reticulum Protein ◽

Arboviral Diseases

AbstractThe escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.

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Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

Scientific Reports ◽

10.1038/s41598-021-84682-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexander Pralow ◽

Alexander Nikolay ◽

Arnaud Leon ◽

Yvonne Genzel ◽

Erdmann Rapp ◽

...

Keyword(s):

Zika Virus ◽

Animal Cell ◽

Gradient Centrifugation ◽

Viral Envelope ◽

Protein Glycosylation ◽

High Yield ◽

Structural Protein ◽

Site Specific ◽

E Protein ◽

The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

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Discovery of an imidazonaphthyridine and a riminophenazine as potent anti-Zika virus agents through a replicon-based high-throughput screening

Virus Research ◽

10.1016/j.virusres.2021.198388 ◽

2021 ◽

Vol 299 ◽

pp. 198388

Author(s):

Rafaela Sachetto Fernandes ◽

Andre Schutzer de Godoy ◽

Igor Andrade Santos ◽

Gabriela Dias Noske ◽

Ketllyn Irene Zagato de Oliveira ◽

...

Keyword(s):

High Throughput ◽

Zika Virus ◽

High Throughput Screening

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A human antibody against Zika virus crosslinks the E protein to prevent infection

Nature Communications ◽

10.1038/ncomms14722 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 74

Author(s):

S. Saif Hasan ◽

Andrew Miller ◽

Gopal Sapparapu ◽

Estefania Fernandez ◽

Thomas Klose ◽

...

Keyword(s):

Zika Virus ◽

Human Antibody ◽

E Protein

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Gut-associated bacteria invade the midgut epithelium of Aedes aegypti and stimulate innate immunity and suppress Zika virus infection in cells

10.1101/866897 ◽

2019 ◽

Cited By ~ 1

Author(s):

Shivanand Hegde ◽

Denis Voronin ◽

Aitor Casas-Sanchez ◽

Miguel A. Saldaña ◽

Eva Heinz ◽

...

Keyword(s):

Zika Virus ◽

Control Strategies ◽

Fluorescent Microscopy ◽

Cellular Level ◽

Intracellular Bacteria ◽

Bacterial Symbionts ◽

Mosquito Cells ◽

Transmission Electron ◽

Bacterial Replication

AbstractMicrobiota within mosquitoes influence nutrition, immunity, fecundity, and the capacity to transmit pathogens. Despite their importance, we have a limited understanding of host-microbiota interactions, especially at the cellular level. It is evident bacterial symbionts that are localized within the midgut also infect other organs within the mosquito; however, the route these symbionts take to colonize other tissues is unknown. Here, utilizing the gentamicin protection assay, we showed that the bacterial symbionts Cedecea and Serratia have the capacity to invade and reside intracellularly within mosquito cells. Symbiotic bacteria were found within a vacuole and bacterial replication was observed in mosquito cell by transmission electron microscopy, indicating bacteria were adapted to the intracellular milieu. Using gene silencing, we determined that bacteria exploited host factors, including actin and integrin receptors, to actively invade mosquito cells. As microbiota can affect pathogens within mosquitoes, we examined the influence of intracellular symbionts on Zika virus (ZIKV) infection. Mosquito cells harbouring intracellular bacteria had significantly less ZIKV compared to uninfected cells or cells exposed to non-invasive bacteria. Intracellular bacteria were observed to substantially upregulate the Toll and IMD innate immune pathways, providing a possible mechanism mediating these anti-viral effects. Examining mono-axenically infected mosquitoes using transmission electron and fluorescent microscopy revealed that bacteria occupied an intracellular niche in vivo. Our results provided evidence that bacteria that associate with the midgut of mosquitoes have intracellular lifestyles which likely have implications for mosquito biology and pathogen infection. This study expands our understanding of host-microbiota interactions in mosquitoes, which is important as symbiont microbes are being exploited for vector control strategies.

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Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion

Journal of Virology ◽

10.1128/jvi.00698-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 23

Author(s):

Benjamin O. Fulton ◽

David Sachs ◽

Megan C. Schwarz ◽

Peter Palese ◽

Matthew J. Evans

Keyword(s):

Zika Virus ◽

Viral Evolution ◽

Rna Replication ◽

Transposon Mutagenesis ◽

Ns1 Protein ◽

Genetic Constraints ◽

E Protein ◽

A Genome ◽

Nucleotide Insertions ◽

Fusion Loop

ABSTRACT The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.

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Variable Inhibition of Zika Virus Replication by Different Wolbachia Strains in Mosquito Cell Cultures

Journal of Virology ◽

10.1128/jvi.00339-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 24

Author(s):

Michaela J. Schultz ◽

Sharon Isern ◽

Scott F. Michael ◽

Ronald B. Corley ◽

John H. Connor ◽

...

Keyword(s):

Virus Replication ◽

Zika Virus ◽

Western Hemisphere ◽

Mosquito Cell ◽

Mosquito Vector ◽

Viral Inhibition ◽

Virus Growth ◽

Content Type ◽

Bacterial Endosymbiont ◽

Mosquito Cells

ABSTRACT Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread. IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia. Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.

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