Evaluation of the anti-apoptotic activity of bovine alphaherpesvirus type 5 US3 protein kinase in insect cells using a recombinant baculovirus

Abstract

Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in cattle in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1, and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus-based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic p35 gene was constructed using the Bac-to-Bac™ system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the recombinant US3 protein in insect cells comparing with two other AcMNPV recombinants, one containing a functional copy of the AcMNPVanti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV.

Drosophila X virus‐like particles as delivery carriers for improved oral insecticidal efficacy of scorpion Androctonus australis peptide against the invasive fruit fly, Drosophila suzukii

“…Around 5−7 d later, the morphology of transfected cells was analyzed under the light microscope. Once the obvious cytopathic effects (CPE) appeared (Silva et al, 2020), the cells were subjected to centrifugation at 1000 × g for 10 min for the collection of supernatants which were stored at −80 °C as virus stocks. Cell pellets were used for the detection of the fusion protein by western blot.…”