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

Abstract

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.

Characterization and application of recombinant Bovine Leukemia Virus Env protein

“…We think a similar scenario could occur with sBLV-Env Fm, with gp51-gp30 complexes retaining all antigenic epitopes and constraining the presence of different Env conformers that could divert the activation of the immune system towards the recognition of non-neutralizing epitopes . In this regard our group has recently demonstrated that a similar BLV-EnvFm protein containing the transmembrane (TM) and cytosolic domains is able to form VLPs when co-expressed with Gag protein 42 . In the present work, this construct was modified by replacing the TM domain with a C-terminal Streptag to obtain a soluble protein easy to purify and quantify.…”

“…Recently, Olivero et al have demonstrated that Drosophila S2 cells co-transfected with the env and gag sequences were able to form virus-like particles (VLPs) displaying a BLV Env which retains structural motifs on its surface 42 . Stably transfected Drosophila melanogaster S2 cells have appeared as a major platform for recombinant protein expression 43 , 44 .…”

Soluble SARS‐CoV‐2 RBD and human ACE2 peptidase domain produced in DrosophilaS2 cells show functions evoking virus–cell interface

“…Sample preparation from SDS‐PAGE bands for MALDI‐TOF/TOF was done as before (Rossello et al, 2017), while for proteins in solution, tryptic peptides were obtained and desalted using C18 OMIX tips (Agilent). For MALDI‐TOF/TOF, elution was performed on a plate, and for LC–MS/MS, eluates were vacuum‐dried and resuspended in 0.1% formic acid as previously described (Olivero‐Deibe et al, 2021).…”

“…Tryptic peptides were subjected to LC–MS/MS as before (Olivero‐Deibe et al, 2021) using a gradient from 0% to 35% of mobile phase B over 90 min, and mass analysis was performed in a data‐dependent mode using a top 12 method (Rivera et al, 2020). Database searches were performed using a D. melanogaster target‐decoy database containing RBD sequence from Uniprot (June 2020) using PatternLab V (Santos et al, 2022).…”

Kinetics of Bovine leukemia virus aspartic protease reveals its dimerization and conformational change

“…The main substrate of PR is Gag, a precursor polyprotein relevant for capsid self-assembling. Gag is processed by PR, releasing matrix (MA), capsid (CA) and nucleocapsid (NC) proteins, which undergo dramatic conformational rearrangments which confer infectivity to virions [19]. Other longer versions of Gag are also expressed and processed by PR.…”