Fachbereich Veterinärmedizin



    The Mareks disease virus MDV chemokine vIL8 binds the cCXCR5 receptor and is a functional orthologue of cCXCL13 (2016)

    Alzuheir, Ibrahim Mahmoud (WE 5)
    Berlin: Mensch und Buch Verlag, 2016 — XVI, 98 Seiten
    ISBN: 978-3-86387-741-5
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000102851
    Institut für Virologie

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    +49 30 838 51833

    Abstract / Zusammenfassung

    Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes Marek’s disease (MD) in chickens. Infection of susceptible birds with virulent strain results in economic loss due to a high mortality, visceral lymphomas, paralysis and immunosuppression. The MDV genome encodes a CXC chemokine (vIL-8) that was named after the first identified chicken interlukin-8 (cCXCL8) based on the sequence similarity. However, recent functional data suggest that vIL-8 is not a cCXCL8 homologue. Identification of the cellular orthologue and receptor of vIL-8 are needed to understand its role in MDV pathogenesis. In the first part of this thesis, the evolutionary relationship was analyzed and vIL-8 functions characterized in vitro. Phylogenic and amino acid analysis of vIL-8 and all so far known human and chicken CXC chemokines revealed that vIL-8 lacks the conserved ELR (Glutamic acid- Leucine- Arginine) motif of CXC chemokines. The analysis also demonstrated vIL-8 has the highest sequence homology to cCXCL13L1 also known as B lymphocyte chemoattractant (BLC), which also lacks the ELR motif. Unlike the typical four exons structure of CXC chemokine, vIL-8 and CXCL13 have only three exons. In addition, we identified cCXCR5 as the receptor of vIL-8, which is the exclusive receptor of cCXCL13 and is present on chicken B and certain T cell subsets.
    To address if cCXCL13 can complement vIL-8 in MDV pathogenesis and tumor formation, I used the bacterial artificial chromosome (BAC) system harboring the sequence of the very virulent MDV (vvMDV) RB-1B strain to generate recombinant viruses that express the cellular cCXCL13s in the absence of vIL-8. First, I inserted cCXCL13L1 downstream of vIL-8, but this locus did not allow secretion of cCXCL13. Next, I inserted the three variants of cCXCL13 (L1, L2 and L3) under the control of the immediate early cytomegalovirus promoter (pCMV) in the unique long region (at the C-terminus of UL35 and UL45), these recombinant mutants were unable to replicate in vitro. We then target the non-essential mini-F locus to insert CXCL13 driven by pCMV; however, the recombinant virus of CXCL13L1 was unable to replicate in vitro. Finally, insertion of cCXCL13 in mini-F driven by Thymidine kinase promoter (TK) allows reconstitution of recombinant viruses that secreted cCXCL13 and replicated comparable to wild type virus in vitro. We conduct in vivo experiment by infect one day chicks with the recombinant viruses and keep some as contacts. The recombinant viruses were replicate slightly lower that wild type in the infected animals, but unable to tarnsmitte and replicate in contact birds. In addition, the mutants couldn’t complement the loss of vIL-8 in MDV pathogenicity.
    To explain the lack of complementation between vIL-8 and its cellular orthologue, I tagged vIL-8 with a polyhistidine-tag (6xHis-tag). This tagging enables comparable quantification of vIL-8 expression driven by its original promoter with TK-CXCL13 encoded in the mini-F. The results demonstrate that the expression level of vIL-8 is much higher than cCXCL13s driven by the TK promoter in mini-F, indicated that expression levels and kinetics are crucial for chemokine function.