Expression, purification, and characterization of bovine leukemia virus-like particles produced in Drosophila S2 cells

Material complementario: https://www.frontiersin.org/articles/10.3389/fviro.2021.756559/full#supplementary-materialBovine 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 amajor 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.CSIC I+D 2014ANII: ALI_1_2016_2_129851; POS_NAC_2015_1_109471PEDECIBA-FOCEM: COF 03/11CAP: BFPD_2020_1#2814383

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease

Rotavirus Nonstructural Protein NSP5 Interacts with Major Core Protein VP2

Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA

OH1 from Orf virus: a new tyrosine phosphatase that displays distinct structural features and triple substrate specificity

Manuscript Number: JMB-D-17-00326R2Epub ahead of printViral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homologue in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual specificity phosphatase, and reveals its ability to dephosphorylate phosphatidylinositol 3,5 bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation

Isolation and characterization of canine parvovirus type 2c circulating in Uruguay Isolamento e caracterização da cepa tipo 2c do parvovirus canino circulante no Uruguai

This research reports the first CPV-2c isolation in cell culture (canine fibroma cell line A-72) in Uruguay. The isolates were obtained from 13 rectal swabs of Uruguayan dogs with parvovirosis. Samples were submitted to PCR with two sets of primers, restriction fragment length polymorphism (RFLP), partial sequencing of the gene encoding for VP2 capsid protein and phylogenetic characterization. The strain isolated was confirmed as CPV-2c. These results contribute to a better knowledge of CPV strains circulating in Uruguay and promote an evaluation of the efficacy of heterologous vaccines used to protect against the circulating strains.<br>Este trabalho relata o primeiro isolamento do CPV-2c em cultura de células (linhagem celular de fibroma canino A-72), no Uruguai. Os isolados foram obtidos a partir de 13 suabes retais de cães uruguaios com parvovirose. As amostras foram submetidas à reação em cadeia da polimerase (PCR) com dois pares de primers, polimorfismo de comprimento de fragmentos de restrição (RFLP), sequenciamento parcial do gene que codifica a proteína capsidial VP2 e caracterização filogenética. A cepa isolada foi confirmada como CPV-2c. Os resultados contribuem para um melhor conhecimento das cepas do CPV circulantes no Uruguai e incitam uma maior investigação sobre a eficácia das vacinas produzidas com cepas heterólogas utilizadas atualmente para proteger contra cepas circulantes