scholarly journals Title: Mechanisms of Host Cell Binding and Neurotropism of Zika Virus

2018 ◽  
Author(s):  
C.A. Rieder ◽  
J. Rieder ◽  
S. Sannajust ◽  
D. Goode ◽  
R. Geguchadze ◽  
...  
Keyword(s):  
Zika Virus ◽  
Annexin V ◽  
Vero Cells ◽  
Western Hemisphere ◽  
Host Cells ◽  
Binding Mechanism ◽  
E Protein ◽  
Clinical Presentations ◽  
Cell Adsorption ◽  
Protein Cell

AbstractZika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two distinct binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue ASN154 and viral phosphatidylserine (PS). Short (20-mer) peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts, expecting interactions to be representative of ZIKV E protein/cell interactions, and bound MDCK or Vero cells and primary neurons significantly above a scrambled PRVABC59 control peptide. Peptides also significantly inhibited Vero cell adsorption by ZIKV strains MR_766 and PRVABC59, indicating that we have identified a binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains.Pretreatment of ZIKV MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59, but not MR_766, suggesting that Western hemisphere strains are additionally utilizing PS-mediated entry to infect host cells. Taken together, these data indicate that we have identified an ancestral binding mechanism of ZIKV, and a secondary binding mechanism utilized by Western Hemisphere strains.

scholarly journals A Novel Mechanism for Zika Virus Host-Cell Binding

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1101
Author(s):  
Courtney A. Rieder ◽  
Jonathan Rieder ◽  
Sebastién Sannajust ◽  
Diana Goode ◽  
Ramaz Geguchadze ◽  
...  
Keyword(s):  
Zika Virus ◽  
Neuronal Cells ◽  
Annexin V ◽  
Vero Cells ◽  
Western Hemisphere ◽  
Host Cells ◽  
E Protein ◽  
Clinical Presentations ◽  
Secondary Mechanism ◽  
Protein Cell

Zika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two putative binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue asparagine 154 (ASN154) and viral phosphatidylserine (PS). Synthetic peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts to model ZIKV E protein/cell interactions and bound MDCK or Vero cells and primary neurons significantly. Peptides significantly inhibited Vero cell infectivity by ZIKV strains MR_766 and PRVABC59, indicating that this region represents a putative binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains. Pretreatment of ZIKV strains MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59 but not MR_766, suggesting that Western hemisphere strains may additionally be capable of utilizing PS-mediated entry to infect host cells. These data indicate that the region surrounding E protein ASN154 is capable of binding fibroblasts and primary neuronal cells and that PS-mediated entry may be a secondary mechanism for infectivity utilized by Western Hemisphere strains.

scholarly journals The Envelope Residues E152/156/158 of Zika Virus Influence the Early Stages of Virus Infection in Human Cells

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1444 ◽  
Author(s):  
Sandra Bos ◽  
Wildriss Viranaicken ◽  
Etienne Frumence ◽  
Ge Li ◽  
Philippe Desprès ◽  
...  
Keyword(s):  
Viral Infection ◽  
Zika Virus ◽  
Molecular Mechanisms ◽  
Pacific Islands ◽  
Human Cells ◽  
Host Cells ◽  
Zikv Infection ◽  
Viral Attachment ◽  
Human Host ◽  
E Protein

Emerging infections of mosquito-borne Zika virus (ZIKV) pose an increasing threat to human health, as documented over the recent years in South Pacific islands and the Americas in recent years. To better understand molecular mechanisms underlying the increase in human cases with severe pathologies, we recently demonstrated the functional roles of structural proteins capsid (C), pre-membrane (prM), and envelop (E) of ZIKV epidemic strains with the initiation of viral infection in human cells. Specifically, we found that the C-prM region contributes to permissiveness of human host cells to ZIKV infection and ZIKV-induced cytopathic effects, whereas the E protein is associated with viral attachment and early infection. In the present study, we further characterize ZIKV E proteins by investigating the roles of residues isoleucine 152 (Ile152), threonine 156 (Thr156), and histidine 158 (His158) (i.e., the E-152/156/158 residues), which surround a unique N-glycosylation site (E-154), in permissiveness of human host cells to epidemic ZIKV infection. For comparison purpose, we generated mutant molecular clones of epidemic BeH819015 (BR15) and historical MR766-NIID (MR766) strains that carry each other’s E-152/156/158 residues, respectively. We observed that the BR15 mutant containing the E-152/156/158 residues from MR766 was less infectious in A549-Dual™ cells than parental virus. In contrast, the MR766 mutant containing E-152/156/158 residues from BR15 displayed increased infectivity. The observed differences in infectivity were, however, not correlated with changes in viral binding onto host-cells or cellular responses to viral infection. Instead, the E-152/156/158 residues from BR15 were associated with an increased efficiency of viral membrane fusion inside infected cells due to conformational changes of E protein that enhance exposure of the fusion loop. Our data highlight an important contribution of E-152/156/158 residues to the early steps of ZIKV infection in human cells.

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

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.

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

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
S. Saif Hasan ◽  
Andrew Miller ◽  
Gopal Sapparapu ◽  
Estefania Fernandez ◽  
Thomas Klose ◽  
...  
Keyword(s):  
Zika Virus ◽  
Human Antibody ◽  
E Protein

scholarly journals An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 547 ◽  
Author(s):  
Silvia Márquez-Jurado ◽  
Aitor Nogales ◽  
Ginés Ávila-Pérez ◽  
Francisco Iborra ◽  
Luis Martínez-Sobrido ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Zika Virus ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Vero Cells ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

scholarly journals Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Jonathan L. Miller ◽  
Darin J. Weed ◽  
Becky H. Lee ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Cell Lines ◽  
Pseudorabies Virus ◽  
Vero Cells ◽  
Low Ph ◽  
Host Cells ◽  
Multiple Model ◽  
Trade Restrictions ◽  
African Green Monkey Kidney ◽  
Entry Pathway ◽  
Model Cell

ABSTRACTThe alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low-pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines, including porcine kidney (PK15) cells and African green monkey kidney (Vero) cells, was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membranes reduced entry into PK15 and Vero cells by >80%. Together, these findings suggest that endocytosis followed by fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.IMPORTANCEPRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurological, respiratory, and reproductive disorders, often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low-pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

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

2017 ◽  
Vol 91 (15) ◽  
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.

scholarly journals DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS

2016 ◽  
Vol 58 (0) ◽  
Author(s):  
Laura Masami SUMITA ◽  
Jaqueline Polizeli RODRIGUES ◽  
Noely Evangelista FERREIRA ◽  
Alvina Clara FELIX ◽  
Nathalia Caroline Santiago SOUZA ◽  
...  
Keyword(s):  
Zika Virus ◽  
Vero Cells ◽  
Preliminary Results

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

2017 ◽  
Vol 91 (14) ◽  
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.

scholarly journals Zika Virus Update: More on an Emerging Arboviral Disease in the Western Hemisphere

2016 ◽  
Vol 11 (2) ◽  
pp. 163-167 ◽  
Author(s):  
Kelly G. Vest
Keyword(s):  
Public Health ◽  
Zika Virus ◽  
Primary Care Providers ◽  
Western Hemisphere ◽  
Disease Spread ◽  
Care Providers ◽  
Neurologic Diseases ◽  
The Public ◽  
The World ◽  
Arboviral Disease

AbstractZika virus has captivated the world with its quick spread throughout the Western Hemisphere. Increased emphasis has been placed on the infection of pregnant women and subsequent adverse and severe effects in the developing fetus and newborn. This article supplements a previous article and provides updated information on new and evolving evidence that strengthens the association between Zika virus and unique congenital and neurologic diseases, updates what is known about the epidemiology of the disease, and provides new and updated material for primary care providers as they counsel patients who may be exposed or infected. With the extent of disease spread, it is expected that Zika virus will become endemic to the Western Hemisphere and will change the public health parameters and approach in this area of the world. (Disaster Med Public Health Preparedness. 2017;11:163–167)

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