N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle

Asparagine (N)-linked protein glycosylation plays an important role in protein synthesis and modification. Two Zika virus (ZIKV) structural proteins, the pre-membrane (prM) and envelope (E) protein are N-glycosylated. The prM protein of all ZIKV strains contains a single N-linked glycosylation site, while not all strains contain an N-linked site in the E protein. Our aim was to examine the impact of prM and E N-linked glycosylation on ZIKV infectivity and cell trafficking. Using a ZIKV infectious clone, we found that when the N-glycan sites were removed, the prM- and the prM/E-double mutants did not produce an infectious virus in the supernatant. Further, by using ZIKV prME constructs, we found that N-glycosylation was necessary for effective secretion of ZIKV virions. The absence of the N-glycan on prM or E caused protein aggregation in the rough endoplasmatic reticulum (ER) compartment. The aggregation was more pronounced for the prM-mutation, and the mutant virus lost the ER-Golgi intermediate compartment (ERGIC) localization. In addition, lack of the N-glycan on prM induced nuclear translocation of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker. To conclude, we show that the prM N-glycan is essential for the ZIKV infectious cycle, and plays an important role in viral protein trafficking, protein folding, and virion assembly.

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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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N-Linked Glycosylation of West Nile Virus Envelope Proteins Influences Particle Assembly and Infectivity

Journal of Virology ◽

10.1128/jvi.79.21.13262-13274.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13262-13274 ◽

Cited By ~ 163

Author(s):

Sheri L. Hanna ◽

Theodore C. Pierson ◽

Melissa D. Sanchez ◽

Asim A. Ahmed ◽

Mariam M. Murtadha ◽

...

Keyword(s):

West Nile Virus ◽

Envelope Proteins ◽

Glycosylation Site ◽

Protein Glycosylation ◽

E Proteins ◽

Virus Envelope ◽

West Nile ◽

Particle Assembly ◽

E Protein ◽

The Impact

ABSTRACT West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.

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Envelope Protein Glycosylation Mediates Zika Virus Pathogenesis

Journal of Virology ◽

10.1128/jvi.00113-19 ◽

2019 ◽

Vol 93 (12) ◽

Cited By ~ 29

Author(s):

Derek L. Carbaugh ◽

Ralph S. Baric ◽

Helen M. Lazear

Keyword(s):

Zika Virus ◽

Envelope Protein ◽

Glycosylation Site ◽

Viral Envelope ◽

Protein Glycosylation ◽

Guillain Barre Syndrome ◽

Viral Loads ◽

E Protein ◽

Guillain Barré Syndrome ◽

Guillain Barré

ABSTRACT Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations (congenital Zika syndrome) and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. All contemporary ZIKV isolates encode an N-linked glycosylation site in the envelope (E) protein (N154), but this glycosylation site is absent in many historical ZIKV isolates. Here, we investigated the role of E protein glycosylation in ZIKV pathogenesis using two contemporary Asian-lineage strains (H/PF/2013 and PRVABC59) and the historical African-lineage strain (MR766). We found that glycosylated viruses were highly pathogenic in Ifnar1−/− mice. In contrast, nonglycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously but remaining neurovirulent when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, we found that glycosylation facilitated infection of cells expressing the lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) or DC-SIGN-related (DC-SIGNR), suggesting that inefficient infection of lectin-expressing leukocytes could contribute to the attenuation of nonglycosylated ZIKV in mice. IMPORTANCE It is unclear why the ability of Zika virus (ZIKV) to cause serious disease, including Guillain-Barré syndrome and birth defects, was not recognized until recent outbreaks. One contributing factor could be genetic differences between contemporary ZIKV strains and historical ZIKV strains. All isolates from recent outbreaks encode a viral envelope protein that is glycosylated, whereas many historical ZIKV strains lack this glycosylation. We generated nonglycosylated ZIKV mutants from contemporary and historical strains and evaluated their virulence in mice. We found that nonglycosylated viruses were attenuated and produced lower viral loads in serum and brains. Our studies suggest that envelope protein glycosylation contributes to ZIKV pathogenesis, possibly by facilitating attachment to and infection of lectin-expressing leukocytes.

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Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Journal of Virology ◽

10.1128/jvi.01348-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 49

Author(s):

Arun S. Annamalai ◽

Aryamav Pattnaik ◽

Bikash R. Sahoo ◽

Ezhumalai Muthukrishnan ◽

Sathish Kumar Natarajan ◽

...

Keyword(s):

Mouse Model ◽

Zika Virus ◽

Glycosylation Site ◽

Human Diseases ◽

Guillain Barre Syndrome ◽

Sequence Motif ◽

Genetic Changes ◽

E Protein ◽

Guillain Barré Syndrome ◽

Guillain Barré

ABSTRACT Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion. IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

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Determinants of attenuation in the envelope protein of the flavivirus Alfuy

Journal of General Virology ◽

10.1099/vir.0.034793-0 ◽

2011 ◽

Vol 92 (10) ◽

pp. 2286-2296 ◽

Cited By ~ 14

Author(s):

Natalie A. Prow ◽

Fiona J. May ◽

Daniel J. Westlake ◽

Robert J. Hurrelbrink ◽

Rebecca M. Biron ◽

...

Keyword(s):

Infectious Clone ◽

Glycosylation Site ◽

Protein Glycosylation ◽

Hinge Region ◽

Wild Type ◽

E Proteins ◽

Time To Death ◽

Naturally Occurring ◽

High Doses ◽

Delayed Time

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM–E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273–277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD50. Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

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Computationally Designed Peptides for Zika Virus Detection: An Incremental Construction Approach

Biomolecules ◽

10.3390/biom9090498 ◽

2019 ◽

Vol 9 (9) ◽

pp. 498 ◽

Cited By ~ 1

Author(s):

Marcello Mascini ◽

Emre Dikici ◽

Marta Robles Mañueco ◽

Julio A. Perez-Erviti ◽

Sapna K. Deo ◽

...

Keyword(s):

Zika Virus ◽

Dynamic Range ◽

Acute Infection ◽

Detection Limits ◽

Analytical Signal ◽

Cross Reactivity ◽

Glycosylation Site ◽

Protein Glycosylation ◽

Enzyme Linked Immunosorbent Assay ◽

Intact Virus

Herein, and in contrast to current production of anti-Zika virus antibodies, we propose a semi-combinatorial virtual strategy to select short peptides as biomimetic antibodies/binding agents for the detection of intact Zika virus (ZIKV) particles. The virtual approach was based on generating different docking cycles of tetra, penta, hexa, and heptapeptide libraries by maximizing the discrimination between the amino acid motif in the ZIKV and dengue virus (DENV) envelope protein glycosylation site. Eight peptides, two for each length (tetra, penta, hexa, and heptapeptide) were then synthesized and tested vs. intact ZIKV particles by using a direct enzyme linked immunosorbent assay (ELISA). As a reference, we employed a well-established anti-ZIKV antibody, the antibody 4G2. Three peptide-based assays had good detection limits with dynamic range starting from 105 copies/mL of intact ZIKV particles; this was one order magnitude lower than the other peptides or antibodies. These three peptides showed slight cross-reactivity against the three serotypes of DENV (DENV-1, -2, and -3) at a concentration of 106 copies/mL of intact virus particles, but the discrimination between the DENV and ZIKV was lost when the coating concentration was increased to 107 copies/mL of the virus. The sensitivity of the peptides was tested in the presence of two biological matrices, serum and urine diluted 1:10 and 1:1, respectively. The detection limits decreased about one order of magnitude for ZIKV detection in serum or urine, albeit still having for two of the three peptides tested a distinct analytical signal starting from 106 copies/mL, the concentration of ZIKV in acute infection.

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The Ablation of Envelope Protein Glycosylation Enhances the Neurovirulence of ZIKV and Cell Apoptosis in Newborn Mice

Journal of Immunology Research ◽

10.1155/2021/5317662 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Yanqing Guo ◽

Linlin Bao ◽

Yanfeng Xu ◽

Fengdi Li ◽

Qi Lv ◽

...

Keyword(s):

Cell Apoptosis ◽

Serial Passage ◽

Causal Link ◽

Protein Glycosylation ◽

Fourth Generation ◽

Supporting Evidence ◽

Newborn Mouse ◽

Newborn Mice ◽

E Protein ◽

The Impact

Zika virus (ZIKV) has attracted the wide global attention due to its causal link to microcephaly. In this study, two amino acid (aa) mutation (E143K and R3394K) were identified at the fourth generation (named ZKC2P4) during the serial passage of ZIKV-Asian lineage ZKC2/2016 strain in the newborn mouse brain, while another seven aa deletions in envelope (E) protein were detected in ZKC2P6. ZKC2P6 is a novel nonglycosylated E protein Asian ZIKV we first identified and provides the first direct supporting evidence that glycosylation motif could be lost during the passage in neonatal mice. To study the impact of E protein glycosylation ablation, we compared the pathogenicity of ZKC2P6 with that of ZKC2P4. The results showed that the loss of E protein glycosylation accelerated the disease progression, as evidenced by an earlier weight loss and death, a thinner cerebral cortex, and more serious tissue lesions and inflammation/necrosis. Furthermore, ZKC2P6 exhibited a greater ability to replicate and caused severer cell apoptosis than that of ZKC2P4. Therefore, the ablation of E glycosylation generally enhances the neurovirulence of ZIKV and cell apoptosis in newborn mice.

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N-glycosylation of Viral E Protein Is the Determinant for Vector Midgut Invasion by Flaviviruses

mBio ◽

10.1128/mbio.00046-18 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 16

Author(s):

Dan Wen ◽

Suhua Li ◽

Fangfang Dong ◽

Yanan Zhang ◽

Yongfang Lin ◽

...

Keyword(s):

Immune System ◽

Zika Virus ◽

Critical Role ◽

Blood Feeding ◽

Oral Route ◽

Glycosylation Site ◽

Immune Defense ◽

Host Interactions ◽

Mosquito Midgut ◽

E Protein

ABSTRACT Transmission of flaviviruses by hematophagous insects such as mosquitoes requires acquisition of the virus during blood feeding on the host, with midgut as the primary infection site. Here, we report that N-glycosylation of the E protein, which is conserved among most flaviviruses, is critical for the Zika virus (ZIKV) to invade the vector midgut by inhibiting the reactive oxygen species (ROS) pathway of the mosquito immune system. Our data further show that removal of the ZIKV E glycosylation site prevents mosquito infection by flaviviruses via the oral route, whereas there is no effect on infection by intrathoracic microinjection, which bypasses the midgut. Interestingly, the defect in infection of the mosquito midgut by the mutant virus through blood feeding is rescued by reduction of the ROS level by application of vitamin C, a well-known antioxidant. Therefore, our data demonstrate that ZIKV utilizes the glycosylation on the envelope to antagonize the vector immune defense during infection. IMPORTANCE Most flaviviruses, including Zika virus (ZIKV), are transmitted between hosts by arthropod vectors, such as mosquitoes, which acquire the virus during a blood meal. Here, by mutagenesis, we found a major role of the N-glycosylation of flavivirus E protein in its transmission circle, facilitating its survival against the vector immune system during invasion of the mosquito midgut while blood feeding on the host. In spite of the extensive studies of the involvement of N-glycan modification of flavivirus E protein in virus-host interactions, we discovered its critical role in virus-vector interaction and the evolution of flavivirus. Given the deleterious effects of ZIKV on human health, this study might have a significant impact on development of novel transmission-blocking strategies.

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Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development

F1000Research ◽

10.12688/f1000research.16454.2 ◽

2019 ◽

Vol 7 ◽

pp. 1624

Author(s):

Iman Almansour ◽

Rahaf Alfares ◽

Halah Aljofi

Keyword(s):

B Cell ◽

Zika Virus ◽

Large Scale ◽

Human Mabs ◽

Bioinformatics Analyses ◽

Multiple Sequence ◽

B Cell Epitopes ◽

E Protein ◽

The Impact

Background:Cases of the re-emergence of Zika virus in 2015 were associated with severe neurologic complications, including Gillien-Barre syndrome in adults and congenital Zika syndrome in newborns. The major structural determinant of immunity to the Zika virus is the E protein. Although B-cell epitopes of Zika E protein were recently identified, data regarding epitope variations among Zika strains in pre-epidemic and epidemic periods are lacking.Methods:Here, we conducted systematic bioinformatics analyses of Zika strains isolated between 1968 and 2017. Multiple sequence alignment of E protein as well as B-cell epitopes annotations were performed. In addition, homology-based approach was utilized to construct three-dimensional structures of monomeric E glycoproteins to annotate epitope variations. Lastly, prediction of ofN-glycosylation patterns and prediction of protein stability upon mutations were also investigated.Results:Our analyses indicates that epitopes recognized by human mAbs ZIKV-117, ZIKV-15, and ZIKV-19 were highly conserved, suggesting as attractive targets for the development of vaccines and immunotherapeutics directed against diverse Zika strains. In addition, the epitope recognized by ZIKV-E-2A10G6 mAb derived from immunized mice was mostly conserved across Zika strains.Conclusions:Our data provide new insights regarding antigenic similarities between Zika strains circulating worldwide. These data are essential for understanding the impact of evolution on antigenic cross-reactivity between Zika lineages and strains. Furtherin-vitroanalyses are needed to determine how mutationsat predefined epitopes could impact the development of vaccines that can effectively neutralize Zika viruses.

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Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development

F1000Research ◽

10.12688/f1000research.16454.1 ◽

2018 ◽

Vol 7 ◽

pp. 1624 ◽

Cited By ~ 2

Author(s):

Iman Almansour ◽

Rahaf Alfares ◽

Halah Aljofi

Keyword(s):

B Cell ◽

Zika Virus ◽

Large Scale ◽

Human Mabs ◽

Bioinformatics Analyses ◽

Multiple Sequence ◽

B Cell Epitopes ◽

E Protein ◽

The Impact

Background:Cases of the re-emergence of Zika virus in 2015 were associated with severe neurologic complications, including Gillien-Barre syndrome in adults and congenital Zika syndrome in newborns. The major structural determinant of immunity to the Zika virus is the E protein. Although B-cell epitopes of Zika E protein were recently identified, data regarding epitope variations among Zika strains in pre-epidemic and epidemic periods are lacking.Methods:Here, we conducted systematic bioinformatics analyses of Zika strains isolated between 1968 and 2017. Multiple sequence alignment of E protein as well as B-cell epitopes annotations were performed. In addition, homology-based approach was utilized to construct three-dimensional structures of monomeric E glycoproteins to annotate epitope variations. Lastly, ofN-glycosylation patterns and prediction of protein stability upon mutations were also investigated.Results:Our analyses indicates that epitopes recognized by human mAbs ZIKV-117, ZIKV-15, and ZIKV-119 were highly conserved, suggesting as attractive targets for the development of vaccines and immunotherapeutics directed against diverse Zika strains. In addition, the epitope recognized by ZIKV-E-2A10G6 mAb derived from immunized mice was highly conserved across Zika strains.Conclusions:Our data provide new insights regarding antigenic similarities between Zika strains circulating worldwide. These data are essential for understanding the impact of evolution on antigenic cross-reactivity between Zika lineages and strains. Furtherin-vitroanalyses are needed to determine how mutations could impact the development of vaccines that can effectively neutralize Zika viruses.

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