The Ablation of Envelope Protein Glycosylation Enhances the Neurovirulence of ZIKV and Cell Apoptosis in Newborn Mice.
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ABSTRACT:
SUBMITTER: Guo Y
PROVIDER: S-EPMC8302398 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zika virus (ZIKV) can infect a wide range of tissues including the developmental brain of human fetus. Whether specific viral genetic variants are linked to neuropathology is incompletely understood. To address this, we have intracranially serially passaged a clinical ZIKV isolate (SW01) in neonatal mice and discovered variants that exhibit markedly increased virulence and neurotropism. Deep sequencing analysis combining with molecular virology studies revealed that a single 67D (Aspartic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism in vivo. Notably, virus clones with D67N mutation had higher viral production and caused more severe cytopathic effect (CPE) in human neural astrocytes U251 cells in vitro, indicating its potential neurological toxicity to human brain. These findings revealed that a single mutation D67N on ZIKV envelope may lead to severe neuro lesion that may help to explain the neurovirulence of ZIKV and suggest monitoring the occurrence of this mutation during nature infection may be important.
| S-EPMC8922429 | biostudies-literature
Project description:Recent outbreaks of Zika virus (ZIKV) infection have highlighted the need for a better understanding of ZIKV-specific immune responses. The ZIKV envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface and it is the main target of the protective immune response. EZIKV protein contains the central domain (EDI), a dimerization domain containing the fusion peptide (EDII), and a domain that binds to the cell surface receptor (EDIII). In this study, we performed a systematic comparison of the specific immune response induced by different EZIKV recombinant proteins (EZIKV, EDI/IIZIKV or EDIIIZIKV) in two mice strains. Immunization induced high titers of E-specific antibodies which recognized ZIKV-infected cells and neutralized the virus. Furthermore, immunization with EZIKV, EDI/IIZIKV and EDIIIZIKV proteins induced specific IFNγ-producing cells and polyfunctional CD4+ and CD8+ T cells. Finally, we identified 4 peptides present in the envelope protein (E1-20, E51-70, E351-370 and E361-380), capable of inducing a cellular immune response to the H-2Kd and H-2Kb haplotypes. In summary, our work provides a detailed assessment of the immune responses induced after immunization with different regions of the ZIKV envelope protein.
| S-EPMC9492693 | biostudies-literature
Project description: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.
| S-EPMC6613755 | biostudies-literature
Project description:Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts.
| S-EPMC5708593 | biostudies-literature
Project description:Infection with Zika virus (ZIKV) came first to public attention after it was found to be associated with congenital microcephaly during the outbreak in Brazil (2015-2016). Diagnosis of ZIKV suffers from extensive cross-reactivity with other Flaviviruses, which are circulating in many ZIKV epidemic areas. Due to the fatal outcome of ZIKV infection during pregnancy, detailed knowledge about neutralizing and non-neutralizing epitopes is crucial for the development of robust detection systems of protective antibodies. Therefore, additional information about ZIKV immunogenicity and antibody response is required. In this project, we report the production, purification and characterization of six different polyclonal antibodies against ZIKV envelope (E) protein. The produced antibodies bind to isolated ZIKV E protein as well as to the surface of ZIKV particles, interestingly without being potently neutralizing. Surface plasmon resonance measurement showed that these antibodies bind with high affinity to ZIKV E protein. Epitope mapping revealed that the epitopes are distributed among the three ZIKV E domains with seven binding sites. These identified binding sites overlap only partially with the previously described epitopes recognized by neutralizing antibodies, which is in accordance with their lack of potent neutralizing activity. Additionally, these antibodies showed neither cross-reactivity nor potent neutralizing activity against West Nile virus, a related flavivirus. The gained set of data helps to extend our understanding about the distribution of neutralizing and non-/weak-neutralizing epitopes in ZIKV E protein, and provides a rationale for ZIKV vaccine design and development of robust detection assays for neutralizing antibodies.
| S-EPMC6723789 | biostudies-literature
Project description:We previously reported that overexpression of an O-type glycosyltransferase, GALNT6 (polypeptide N-acetylgalactosaminyltransferase 6) played critical roles in mammary carcinogenesis. To further investigate the biological function of GALNT6, we screened a substrate protein(s) of GALNT6 using a VVA (Vicia villosa agglutinin) lectin (specific to GalNAc-Ser/Thr) pull-down method followed by mass spectrometry analysis. Here we report GRP78 (glucose-regulated protein 78, also known as HSPA5, heat shock 70 kDa protein 5), which is highly expressed in cancer cells and indicated to play important roles in various cellular processes including ER (endoplasmic reticulum) stress and autophagy, as a novel substrate of GALNT6. We found that GALNT6-induced O-glycosylation is critical for the stability of GRP78, its subcellular localization in ER, and its anti-apoptotic function. Furthermore, we demonstrated that overexpression of GRP78 could be important for Golgi-to-ER relocation of GALNT6. Collectively, our study revealed biological significances of O-glycosylation of GRP78 protein, which might play significant roles in the survival of cancer cells, and thus provided a new insight in cancer cell death and useful information for development of anti-cancer treatment targeting the GALNT6-GRP78 pathway.
| S-EPMC6197318 | biostudies-literature
Project description:BackgroundChikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications.MethodsIn the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors.ResultsCHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice.ConclusionsThese findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.
| S-EPMC10792792 | biostudies-literature
Project description:UnlabelledThe gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation.ImportanceDevelopment of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.
| S-EPMC4524065 | biostudies-literature
Project description:Duck Tembusu virus (DTMUV), an emergent flavivirus, causes domestic waterfowls to suffer from severe egg-drop syndrome and fatal encephalitis, greatly threatens duck production globally. Like other mosquito-borne flaviviruses, the envelope (E) protein of all DTMUV strains was N-glycosylated at the amino acid position 154. Thus far, the biological roles of DTMUV E glycosylation have remained largely unexplored. Herein, we demonstrated the key roles of E glycosylation in the replication and pathogenicity of DTMUV in ducks by characterizing the reverse-genetics-derived DTMUV wild-type MC strain and MC bearing mutations (N154Q and N154I) that abolish the E glycosylation. Our data showed that the disruption of E glycosylation could substantially impair virus attachment, entry, and infectivity in DEFs and C6/36 cells. Notably, ducks inoculated intracerebrally with the wild-type virus exhibited severe disease onset. In contrast, those inoculated with mutant viruses were mildly affected as manifested by minimal weight loss, no mortality, lower viral loads in the various tissues, and reduced brain lesions. Attenuated phenotypes of the mutant viruses might be partly associated with lower inflammatory cytokines expression in the brains of infected ducks. Our study offers the first evidence that E glycosylation is vital for DTMUV replication, pathogenicity, and neurovirulence in vivo.
| S-EPMC8437475 | biostudies-literature
Project description:A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.
| S-EPMC514991 | biostudies-literature