Single N -glycosylation site of bovine leukemia virus SU is involved in conformation and viral escape

Rizzo, Giorgia; Forti, Katia; Serroni, Anna; Cagiola, Monica; Baglivo, Sara; Scoccia, Eleonora; Giuseppe, Antonio De

doi:10.1016/j.vetmic.2016.10.024

Cited by 8 publications

(14 citation statements)

References 19 publications

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“…Regarding the glycosylation of EnvFm, we identified two paucimannosidic residues at asparagine 203, in agreement with previous results obtained with the recombinant soluble ectodomain of BLV-Env expressed in S2 Drosophila cells in our laboratory (63) and in accordance with the type of glycosylation described in insect cells (70,71). The presence of an N-glycosylation site at this position, which has been demonstrated to be essential for BLV in vitro infection (72), remains as an important feature in our attempt to produce noninfectious particles showing epitopes mimicking those found in native virions. N-glycosylation of Env protein is essential for BLV infectivity and could be responsible for antigenic determinants (72,73).…”

Section: Discussionsupporting

confidence: 91%

Expression, Purification, and Characterization of Bovine Leukemia Virus-Like Particles Produced in Drosophila S2 Cells

et al. 2021

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Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects cattle worldwide. In Uruguay, it is estimated that more than 70% of dairy cattle are infected, causing serious economic losses due to decreased milk production, increased calving interval, and livestock losses due to lymphosarcoma. Several attempts to develop vaccine candidates that activate protective immune responses against BLV were performed, but up to date, there is no vaccine that ensures efficient protection and/or decreased viral transmission. The development and application of new vaccines that effectively control BLV infection represent a major challenge for countries with a high prevalence of infection. In this study, we generated two Drosophila melanogaster S2 stable cell lines capable of producing BLV virus-like particles (BLV-VLPs). One of them, BLV-VLP1, expressed both Gag and Env wild-type (Envwt) full-length proteins, whereas BLV-VLP2 contain Gag together with a mutant form of Env non-susceptible to proteolytic maturation by cellular furin type enzymes (EnvFm). We showed that Envwt is properly cleaved by cellular furin, whereas EnvFm is produced as a full-length gp72 precursor, which undergoes some partial cleavage. We observed that said mutation does not drastically affect its expression or its entry into the secretory pathway of S2 insect cells. In addition, it is expressed on the membrane and retains significant structural motifs when expressed in S2 insect cells. Morphology and size of purified BLV-VLPs were analyzed by transmission electron microscopy and dynamic light scattering, showing numerous non-aggregated and approximately spherical particles of variable diameter (70–200 nm) as previously reported for retroviral VLPs produced using different expression systems. Furthermore, we identified two N-glycosylation patterns rich in mannose in EnvFm protein displayed on VLP2. Our results suggest that the VLPs produced in Drosophila S2 cells could be a potential immunogen to be used in the development of BLV vaccines that might contribute, in conjunction with other control strategies, to reduce the transmission of the virus.

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Section: Discussionsupporting

confidence: 91%

Expression, Purification, and Characterization of Bovine Leukemia Virus-Like Particles Produced in Drosophila S2 Cells

et al. 2021

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“…gp51 contains 9 N-glycosylation sites, most of them towards the C-terminus. Although the most appropriate expression system for expressing a glycoprotein would have been a eukaryotic one, previous studies by Rizzo et al ., published in 2016 [ 39 ] have shown that gp51 interaction with its receptor was mediated by specific domains but sugars did not play any role in such interaction, as syncytia formation with maintenance of BLV particle infectivity remained when N-glycosylation sites of gp51 were mutated. A prokaryotic system was thus used here, taking its advantages into account, in terms of cost and ease of use.…”

Section: Resultsmentioning

confidence: 99%

In silico and in vitro analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51

et al. 2018

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The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes’ 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain’s functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.

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“…Envelope glycosylation is believed to be critical in viral immune evasion [39, 40]. Recently, de Brogniez Alix et al confirmed that N-linked glycosylation of the BLV SU protein could affect its pathogenicity and found that N129 was a probable glycosylation site [41], which was beneficial for viral escape from neutralization [42]. In the variation analysis of our isolates, the N129 glycosylation site in the ND2 region was not affected by the NAS to NAT substitution.…”

Section: Discussionmentioning

confidence: 99%

Genotyping bovine leukemia virus in dairy cattle of Heilongjiang, northeastern China

Wang

Zhou

et al. 2019

BMC Vet Res

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Background Bovine leukemia virus (BLV) causes enzootic bovine leukosis in cattle and leads to heavy economic losses in the husbandry industry. Heilongjiang Province, China, is rich in dairy cattle. However, its current BLV epidemiology and genotypes have still not been evaluated and confirmed. In this report, we investigated the BLV epidemiology in dairy cattle in the major regions of Heilongjiang Province via the nested PCR assay. Results A total of 730 blood samples were collected from nine different farms in six regions of Heilongjiang. The results showed that the infection rate of these regions ranged from null to 31%. With a clustering analysis of 60 published BLV env sequences, genotypes 1 and 6 were confirmed to be circulating in Heilongjiang. Importantly, a new genotype, 11, and a new subgenotype, 6E, were also identified in the Harbin and Daqing regions, respectively. An epitope analysis showed that a cluster of T-X-D-X-R-XXXX-A sequences in genotype 11 gp51 neutralizing domain 2 was unique among all currently known BLV isolates and was therefore a defining feature of this new genotype. Conclusions BLV epidemics and genotypes were initially investigated in dairy cattle of Heilongjiang. A relatively high infection rate was found in some regions of this province. A new genotype, G11, with a highly specific motif, was identified and thus added as a new member to the current BLV genotype family. This report provides an initial reference for future investigations and subsequent control of BLV transmission and spread in this region. Electronic supplementary material The online version of this article (10.1186/s12917-019-1863-3) contains supplementary material, which is available to authorized users.

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Single N -glycosylation site of bovine leukemia virus SU is involved in conformation and viral escape

Cited by 8 publications

References 19 publications

Expression, Purification, and Characterization of Bovine Leukemia Virus-Like Particles Produced in Drosophila S2 Cells

Expression, Purification, and Characterization of Bovine Leukemia Virus-Like Particles Produced in Drosophila S2 Cells

In silico and in vitro analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51

Genotyping bovine leukemia virus in dairy cattle of Heilongjiang, northeastern China

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