| Marek’s disease virus（MDV）is considered a unique member of the Alphaherpesvirinae subfamily that induces rapid onset of T cell lymphoma in chickens,inhibit the host immunity,and constitute a major threat to the healthy development of the global chicken industry.Compared with other conserved UL56 gene homologues of herpesviruses,little is known about the roles of MDV UL56 gene,while recent studies of mammalian herpesvirus p UL56 proteins have revealed their involvement in promoting ubiquitination of the Nedd4（Neural precursor cell expressed developmentally down-regulated protein4）-like E3 ubiquitin ligases for proteasomal degradation and in modulating host immune responses.To better obtain anti-MDV p UL56 polyclonal antibody,we conducted in silico analysis of the antigenic properties for MDV p UL56 and a polypeptide(28IEEEPVLSDIDEQSE42)was synthesized as the immunogen to make p UL56-specific polyclonal antibody and analyzed of antibody specificity by IFA and Western blotting.The results showed that the anti-MDV p UL56 antibody after subjected to antigen-affinity purification specifically recognizes the p UL56protein expressed by MDV-infected and plasmid-transfected cells.To determine the expression kinetics of UL56 gene products,chicken embryo fibroblasts were infected with very virulent GX20NNM1 or attenuated814 strain and analyzed by quantitative PCR and Western blotting.The results showed that during the time course of infection,the levels of UL56 m RNA transcripts increased consistently.At the translational level,the p UL56 protein encoded by UL56 gene was expressed in the size of 32 k Da,which emerged as early as 12 h post-infection（hpi）but otherwise began to wane at 72 hpi thereafter.With the treatment of viral DNA synthesis inhibitors,the p UL56expression was significantly reduced,featuring the virus dynamics of a late（γ）-gene product.By confocal imaging,p UL56 was found to reside in the Golgi compartment.Both the L-domain motifs and the C-terminal tail-anchored transmembrane were essential for its intracellular localization.Noticeably,p UL56 co-localized with a truncated mutant of the chicken Nedd4-like family protein harboring only the LDI-1-WW domains;However,co-immunoprecipitation assay established no direct interaction between them,and the ectopic expression of p UL56 did not alter the abundance of endogenous Nedd4-like protein.In order to gain a comprehensive understanding of the interactions between the viral protein and host cell,the chicken DF-1 cell line was used for transfection with a plasmid expressing MDV p UL56 that was tagged with red fluorescent protein.Then cells were sorted for transient p UL56 expression by flow cytometry,and only the positive cell populations were enriched for high-throughput sequencing followed by profiling m RNA transcriptome.The sequencing results showed that 914 differentially expressed genes（DEGs）were identified in the DF-1 cells expressing p UL56,which included 462 up-regulated and 452 down-regulated genes,respectively.According to the combined enrichment analyses with GO,KEGG and Reactome networks,the enriched significant DEGs were mapped to the signaling pathways associated with innate immune responses and cytokine production.In more detail,certain genes in terms of type I interferon signaling and inflammatory cytokines were selected for verification by RT-q PCR.The gene m RNA expression levels,such as IFI6,IFIH1,CD83,HSP90AA1,IL-1βand IL-8L1 were elevated,except for the decreased expression of IL-12β.These results were in agreement with those from high-throughput sequencing.Overall,the present study provides a caveat that the p UL56 homologues of different herpesviruses with structural similarities might vary in expression patterns and probably in functional consequences.The present study suggested that p UL56,as a MDV non-structural protein,might play an extensive role in regulation of host cellular processes.For this reason,further investigation should be encouraged to focus on the potential association between UL56 gene and MDV pathogenesis in the context of engineered viral mutants. |
