Functional characterization of bovine herpesvirus 4 ORF45

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to Rhadinovirus genus, with no strict clear association with disease, even if increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle are reported. BoHV-4 potential as a gene delivery vector for immuno-prophylaxis and gene therapy has been already well documented, thanks to its favorable molecular and biological characteristics, such as little or no pathogenicity, absence of oncogenicity; capability to accommodate large amounts of foreign genetic material and the possibility to be manipulated using infectious BoHV-4-derived bacterial artificial chromosome (BAC) genomes. Molecular studies on its ORFs and gene products are on-going, to better clarify the interaction mechanisms between the viral particles and the host cells and also to deeper understand its application as a viral vector. Genome and genes structure are well conserved in Gammaherpesvirus, and ORF45 gene and its product, orf45, is one of those. BoHV-4 Open Reading Frame 45 (ORF45) codifies for a protein (orf45) of unknown function. Although preserved the homologues of ORF45 differ greatly in the length of the protein product and most likely perform different biological activities in various viruses. In addition to differing in the length of the protein, the overall homology of the sequences between homologues is very low. In fact, only a few brief and discrete regions can be aligned with each other. Between these regions, the carboxy-terminal (C-terminal) end shows the highest homology, implying the possibility that this region has a very important biological role. Similarly, another highly preserved region is the one where the nuclear location sequence (NLS) is present. For example, in Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) ORF45 presents the longest protein product among the various gammaherpesviruses, with its 407aa, while in BoHV-4 is only 241aa and in Murine Gammaherpesvirus-68 (MHV-68) is 353aa. In recent years the research is focusing a lot on ORF45 and its protein product, in fact in literature there are numerous studies describing different biological activities attributed to the protein product of the ORF45 gene, in particular its belonging to the early-expression protein, expressed at the stage of infection crucial for viral avoidance from the host’s immune surveillance. In particular, ORF45 is a gene not yet investigated in BoHV-4 and we thought to deepen its biological and molecular characteristics. First we wanted to confirm that also in BoHV-4 the protein product of ORF45 is a nuclear protein and for this we generated a construct with the expression cassette for ORF45 fused with GFP, the green fluorescent protein, placed under the transcriptional control of the heterologous promoter of Human Cytomegalovirus (CMV); following transient transfection in HEK293 of the construct p-CMV-ORF45/EFGP we observed through the acquisition of the images by high resolution confocal microscope that the protein of our interest, that is, ORF45 fused with GFP, was abundantly present in the nuclear compartment of the cells compared to our control given by the transfection of a mock construct expressing only the gene for GFP that unlike was more localized in the cytoplasmic compartment. These data confirm that reported in the literature where ORF45 is defined as a nuclear protein. Moreover, we went deeper into the various biological characteristics and activities of ORF45 in BoHV-4, such as being an essential protein for virus replication and its localization in the viral integument area. To demonstrate our thinkings we have generated and characterized a BoHV-4 mutant ORF45-null exploiting the BAC homologous recombination proces. To generate a recombinant virus, a BoHV-4 genome clone was isolated from the milk cell fraction from a healthy cow and cloned as BAC and propagated within the bacterial strain E.coli, SW102. Furthermore, in the first phase of this process called TARGETING we replaced ORF45 in its entirety within the BoHV-4 genome, with a selectable expression cassette for kanamycin resistance and the Galaktokinase gene, This allowed us to discriminate positive clones (that is, where homologous recombination has been successfully carried out and therefore the ORF has been replaced with our DNA of interest) with negative ones, by positive selection on solid plates with kanamycin. We then carried out a second growth screening in liquid medium, positive for kanamycin and chloramphenicol. After extracting the BAC DNA, we performed an enzymatic digestion analysis with the restriction enzyme HindIII. The resultant recombinant pBAC-BoHV-4-AΔORF45-KanaGalK genome of BoHV-4 was electroporated into BEK bovine permissive cells and BEKcre cells. We could observe that it was not completely able to replenish vital and infectious viral particles (IRVP) and replicate, confirming that ORF45 is crucial for BoHV-4 replication. To give further confirmation of the fundamental importance of ORF45 for BoHV-4 replication, also by homologous recombination we generated a revertant clone pBAC-BoHV-4-ΔORF45-Revertant, where the expression cassette of ORF45, driven by a CMV heterologous promoter, it was positioned in the opposite direction to natural ORF45 in the BoHV-4 genome. Also, in this case after extracting the DNA of the BAC we carried out an analysis through enzymatic digestion with the restriction enzyme HindIII and through Southern blotting using a specific probe for ORF45 we confirmed the successful insertion of o the revertant DNA sequence. In this case, after electroporation in permissive BEK cells, , the reconstitution of the vital and infectious viral particles was successfully achieved, confirming the data. We then electroporated the DNA of the recombinant clone also in the BEKcre cells, thanks to the enzyme cre-recombinase the BAC floxed cassette is excised and therefore the green fluorescence appears no longer visible, since the GFP gene, being present in the BAC cassette, in turn inserted between two sites LoxP, will be excised along with the BAC cassette. We then evaluated and compared the growth kinetics of the recombinant virus pBAC-BoHV- 4-ΔORF45-Revertant with a parental virus and a slight decrease in growth kinetics of the recombinant virus is observed. Since ORF45 was also tagged with an HA epitope (hemoagglutinin), we were able to demonstrate that the product of the ORF45 gene is associated with the virion particles by the western blotting technique, confirming that the protein belongs to the tegument of the virus. Moreover, the impact of BoHV-4 ORF45 on cellular transcriptome was investigated; many cellular transcriptional pathways were found to be alterated, mainly those involving p90 ribosomal S6 kinase (RSK) and signal-regulated kinase (ERK) complex (RSK/ERK). This work demonstrates that BoHV-4 replicating cycle is dependent on ORF45 gene product and provides direct evidence that ORF45 gene product is necessary for BoHV-4 lytic replication and thus highlighting the authentic character of BoHV-4 ORF45 and paving the way to further investigations.