Conjugation of β-Glucan with the Hydrazone and Disulfide Linkers Markedly Improves the Immunogenicity of Zika Virus E Protein

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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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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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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Two Genetic Differences between Closely Related Zika Virus Strains Determine Pathogenic Outcome in Mice

Journal of Virology ◽

10.1128/jvi.00618-20 ◽

2020 ◽

Vol 94 (20) ◽

Cited By ~ 3

Author(s):

Derek L. Carbaugh ◽

Shuntai Zhou ◽

Wes Sanders ◽

Nathaniel J. Moorman ◽

Ronald Swanstrom ◽

...

Keyword(s):

Amino Acid ◽

Mouse Models ◽

Zika Virus ◽

Clinical Manifestations ◽

Amino Acid Sequences ◽

Viral Envelope ◽

Laboratory Research ◽

Balanced Polymorphism ◽

E Protein ◽

Virus Strains

ABSTRACT Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in Ifnar1−/− mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1−/− Ifngr1−/− double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3′ quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1−/− mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1−/− and Ifnar1−/− Ifngr1−/− DKO mice). IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only Ifnar1−/− mice but not Ifnar1−/− Ifngr1−/− DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.

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Deep mutational scanning comprehensively maps how Zika envelope protein mutations affect viral growth and antibody escape

10.1101/725556 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marion Sourisseau ◽

Daniel J.P. Lawrence ◽

Megan C. Schwarz ◽

Carina H. Storrs ◽

Ethan C. Veit ◽

...

Keyword(s):

Amino Acid ◽

Zika Virus ◽

Natural Infection ◽

Physical Structure ◽

Functional Constraints ◽

Viral Growth ◽

Viral Neutralization ◽

Future Evolution ◽

E Protein ◽

Amino Acid Mutations

AbstractFunctional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here we take an alternative approach to map functional constraints on Zika virus’s envelope (E) protein by using deep mutational scanning to measure how all amino-acid mutations to the protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function, but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein, and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution.ImportanceZika virus has recently been shown to be associated with severe birth defects. The virus’s E protein mediates its ability to infect cells, and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino-acid mutations to E protein affected the virus’s ability to grow in cells in the lab and escape from several antibodies. The resulting maps relate changes in the E protein’s sequence to changes in viral function, and therefore provide a valuable complement to existing maps of the physical structure of the protein.

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High-throughput fitness profiling of Zika virus E protein reveals different roles for N-linked glycosylation during infection of mammalian and mosquito cells

10.1101/199935 ◽

2017 ◽

Author(s):

Danyang Gong ◽

Tian-hao Zhang ◽

Dawei Zhao ◽

Yushen Du ◽

Travis J. Chapa ◽

...

Keyword(s):

Zika Virus ◽

Cell Receptor ◽

Human Cells ◽

Nucleotide Position ◽

List Type ◽

Zikv Infection ◽

E Protein ◽

Mosquito Cells ◽

Viral Mutant ◽

Replication Fitness

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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A Novel Mechanism for Zika Virus Host-Cell Binding

Viruses ◽

10.3390/v11121101 ◽

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.

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A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Vaccines ◽

10.3390/vaccines8040603 ◽

2020 ◽

Vol 8 (4) ◽

pp. 603

Author(s):

Beatrice Sarah Berneck ◽

Alexandra Rockstroh ◽

Jasmin Fertey ◽

Thomas Grunwald ◽

Sebastian Ulbert

Keyword(s):

Zika Virus ◽

Insect Cell ◽

Neutralizing Antibodies ◽

Structural Similarity ◽

Cross Reactivity ◽

Virus Envelope ◽

E Protein ◽

Neutralizing Capacity ◽

Fusion Loop

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

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Zika Virus-Like Particle (VLP) vaccine displaying Envelope (E) protein CD loop antigen elicits protective and specific immune response in a murine model

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2020.05.161 ◽

2020 ◽

Vol 529 (3) ◽

pp. 805-811 ◽

Cited By ~ 1

Author(s):

Velasco Cimica ◽

Stephanie Williams ◽

Debra Adams-Fish ◽

Conor McMahon ◽

Aarthi Narayanan ◽

...

Keyword(s):

Immune Response ◽

Murine Model ◽

Zika Virus ◽

Specific Immune Response ◽

E Protein ◽

Virus Like Particle

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Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene

Journal of General Virology ◽

10.1099/jgv.0.001588 ◽

2021 ◽

Vol 102 (4) ◽

Author(s):

Jung Ah Choi ◽

Kunyu Wu ◽

Gyoung Nyoun Kim ◽

Nasrin Saeedian ◽

Seung Han Seon ◽

...

Keyword(s):

Immune Responses ◽

Vesicular Stomatitis Virus ◽

Zika Virus ◽

Protein Gene ◽

Transmembrane Domain ◽

Genetically Modified ◽

Lethal Dose ◽

Effective Vaccine ◽

E Protein ◽

Vesicular Stomatitis

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar −/−) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar -/- mice from a lethal dose of the ZIKV challenge.

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