Bovine herpesvirus 4 Immediate Early II gene is essential and can be duplicated

Abstract

Bovine herpesvirus 4 (BoHV-4) is a virus with a worldwide distribution in cattle population that has been isolated from a lot of different tissues and samples from animal with various clinical manifestations, ranging from conjunctivitis, ocular discharge and genital diseases as post-partum metritis or abortion, but even in apparently healthy animal. Even if a clear correlation between BoHV-4 and any pathologies has never been demonstrated; BoHV-4 is most consistently associated with metritis, a very common postpartum disease of the uterus, in which the role of BoHV-4 as a secondary pathogen is well documented. BoHV-4 has a well known tropism for endometrial stromal and epithelial cells, in which causes non-apoptotic cell death and de novo virus production associated with an increased prostaglandin-endoperoxide synthase 2 protein, cyclo-ossigenase 2 (COX2) and prostaglandin E2 production and secretion from stromal cell. BoHV-4 activation and replication in the bovine endometrium is associated with the early transactivation of the BoHV-4 IE2 gene promoter from endometrial cells. BoHV-4 IE2 gene promoter transactivation and viral replication were associated also with extracellular stimuli belonging to the intrauterine microenvironment such as E. coli LPS and PGE. A model that fits well endometrial BoHV-4 disease is described in literature, involving a vicious circle comprising of bacterial endometritis leading to secretion of PGE, then PGE and LPS stimulating virus replication, which causes further endometrial tissue damage and inflammation. The existence of a virus patho-biotype causing uterine disease appears possible; virus strain adaptation to an organ, tissue or cell type is infact a very important issue for the study of pathology, even because the function of most of the viral genes remains still unknown. In this study a BoHV-4 strain was isolated from the uterus of a persistently infected cow affected with non-responsive post-partum metritis and designated BoHV-4-U. After the characterization this uterine strain was cloned as a bacterial artificial chromosome (BAC) to easily manipulate and study the viral genome. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system, that allows to study single or multiple gene disruptions, opening the way to the clarification of the interactions between BoHV-4 infection and the host endometrial cells. After the generation of the BAC-BoHV-4-U the first gene we decided to investigate was the ORF50/Rta gene, that has been shown to be an essential gene for DNA replication and latency reactivation in many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in co-transfection assays, there is no direct proof of its real indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. Through different strategies the ORF50 gene was interrupted generating before some mutants, replication defective BoHV-4-V.test /IE2 mutants, as a control viruses, and then some BoHV-4-U/IE2 mutants. The BoHV-4-V.test/IE2 mutants was efficiently rescued, with respect to the production of infectious virus and DNA replication, upon the expression of the BoHV-4 ORF50/Rta protein in trans by different complementing cell lines; the BoHV-4-U/IE2 mutants surprisingly were all able to growth, even in non-complementing cell lines. The generation of a suitable probe led to discover that the IE2 gene is duplicated in the genome of BoHV-4-U. These data demonstrated that in BoHV-4, too, ORF50/Rta is the master replication switch gene and, that can be duplicated in some strains, as in our uterine strain. The deletion of the second gene copy of BoHV-4-U Rta unable the virus the capacity to replicate, and this inability was completely complemented by the in trans expression of ORF50/Rta. The generation of this completely replication un-competent virus maybe will provide a new powerful tool for recombinant vaccine production, even in respect to the rescue of the virus through the expression, under a strictly inducible promoter, of the Rta protein.