N-linked glycosylation of GETV envelope proteins affect viral infection and pathogenicity

Description

Getah virus (GETV) is an emerging mosquito-borne pathogen with a broad host range, posing a significant threat to livestock and public health. Although putative N-linked glycosylation sites on its envelope proteins have been reported, their precise functional roles remain uncharacterized. In this study, tunicamycin inhibition assays demonstrated that GETV replication depends on the host glycosylation machinery. Enzymatic dissection revealed that the envelope proteins E1 and E2 carry complex-type N-linked glycans, which are critical for infectivity. By constructing envelope protein N-linked glycosylation site mutants, it was revealed that the E1-N141 site influences viral entry, whereas the E2-N262 site was critical for both viral adsorption and invasion. Mutations at all envelope protein N-linked glycosylation sites impaired the infectivity of progeny virions, resulting in delayed replication kinetics and significantly reduced viral titers. In a suckling mouse model, all mutants showed attenuated pathogenicity, with the E2-N200 mutation conferring the most substantial reduction in virulence. Notably, despite reduced virulence, some mutants (E1-N141A and E2-N200A/N262A) elicited neutralizing antibody responses stronger than those induced by wild-type virus and provided complete protection against subsequent challenge with wild-type GETV. These findings highlight the critical role of envelope protein glycosylation in GETV infectivity and pathogenesis, providing a molecular basis for rational vaccine design.

Steps to reproduce

Materials and methods Cell lines, viruses and antibodies Baby Hamster Kidney (BHK‐21) and Swine Testis (ST) cells were purchased from China Center for Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 10% fetal bovine serum (Lonsera, China). The cells were incubated at 37°C in an atmosphere containing 5% carbon dioxide. For infected cells, a maintenance medium with 2% FBS was used. GETV HuN1 strain (GenBank number: MF741771.1) were kept at our laboratory. Mouse anti-GETV Cap protein, E1 protein and E2 protein monoclonal antibodies were produced in our laboratory. Alexa Fluor 488‐conjugated goat anti‐mouse were purchased from AmyJet Scientific (China). Mouse anti-GAPDH antibody and HRP-conjugated Goat Anti-Mouse were purchased from Proteintech (China). Cytotoxicity assay The different concentrations of tunicamycin (MCE, USA) were added to the cell culture medium and the cells were incubated for 24 hours. The cytotoxicity was detected using the Cell Counting Kit-8 (NCM, China) following the manufacturer’s instructions. An equal volume of DMSO was used as the control. Prediction and analysis of GETV membrane protein structure and glycosylation sites The N-linked glycation sites of GETV membrane proteins (E1, E2 and E3) were predicted using the online NetNGlyc server (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/). The structure of membrane protein was predicted using the online SWISS MODEL server (https://swissmodel.expasy.org). The membrane protein sequences of the 10 prevalent genotypes were obtained from GenBank of NCBI. The distribution of the N-glycosylation site in membrane proteins of the 10 prevalent genotypes was analyzed using the online Sequence Alignment (https://www.genome.jp/tools-bin/clustalw, https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

Institutions

• Yangzhou University

  Jiangsu, Yangzhou

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