Genome-wide analysis of Marek's disease virus proteins and their role in modulating the innate immune response in chickens

Abstract

Marek’s disease virus (MDV), the causative agent of Marek’s disease in chicken, is an important oncogenic avian pathogen which leads to world-wide economic losses in the poultry industry. It targets the chicken's immune system by initially causing a lytic infection in B-lymphocytes in lymphoid organs (spleen, bursa of Fabricius and thymus), followed by a latent infection of T-lymphocytes, which may lead to tumour formation. Despite the presence of well-established vaccination programs against MDV, it is still a major concern for the poultry industry due to the emergence of more virulent strains. As MDV is also considered an excellent model for herpesvirusinduced oncogenicity and immunosuppression, a better understanding of its pathogenesis, including the functional roles of individual MDV proteins, is of both biomedical as well as economical importance. All open reading frames (ORFs) of the CVI988 vaccine strain and the RB1B virulent strain were PCR-amplified from BAC DNA and cloned into the pDONR 207 entry vector by recombinatorial cloning (Gateway® system). Subsequently, all ORFs were subcloned into the yeast-two-hybrid (Y2H) vectors pGBKT7–DEST (bait) and pGADT7-DEST (prey), as well as other expression vectors. The Y2H bait and prey vector clone collections were transformed into the yeast strains AH109 and Y187, respectively. More than 140 ORFs, or ORF fragments, were analysed against each other in a comprehensive Y2H assay. Of > 20.000 interactions tested, 435 positive interactions between 115 ORFs were observed. Several of these interactions have previously been reported in other species of herpesvirus indicating that they may be conserved within the family. A subset of the positive interactions were confirmed using co-immunoprecipitation and LUMIER pull-down assays as a second independent assay. In the second part of the project all MDV proteins were tested for their ability to inhibit the chicken interferon-alpha (chIFN-α)-induced immune response. In functional luciferase reporter assay with a chicken Mx1 promoter containing an interferon stimulated responsive element (ISRE), four MDV-encoded chIFN-α inhibitors were identified, including UL12, UL26, UL50, and Meq, the main MDV oncoprotein. Both isoforms Meq and L-Meq derived from the oncogenic and the non-oncogenic vaccine strain, respectively, similarly inhibited the interferon response in a dose-dependent way, and Meq deletion mutants revealed that the Cterminal, proline-rich transactivating domain is not required for this inhibitory effect. In transient transfection experiments, Meq induced a dose-dependent proteasomal degradation of the chicken interferon regulatory factor 7 (chIRF7), which is required for chIFN-α- induced activation of ISRE. Over-expression of chIRF7 lead to a dosedependent degradation of Meq and its accumulation in the cytoplasm, suggesting that proteasomal degradation of both Meq and chIRF-7 is linked. Consistent with these findings, MDV deletion mutant lacking both copies of the Meq gene was more sensitive to chIFN-α treatment compared to wild-type virus.