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Marek’s disease virus (MDV) is a cell-associated and highly oncogenic alpha-herpesvirus that infects chickens. The MDV genome consists of a unique long (UL) and a unique short region (US), each flanked by inverted repeat regions (RL, RS).
In the RL, a number of unique genes are located that are involved in MDV pathogenesis and tumorigenesis. To facilitate generation of recombinant viruses harboring mutations in the MDV RL region, I deleted most of the internal repeat long (IRL) in pRB-1B (vΔIRL) leaving short sequence ends of the region intact to allow restoration of the sequence via homologous recombination during MDV replication.
I used vΔIRL as a tool to modify the viral interleukin-8 (vIL-8), a CXC chemokine expressed during both the lytic and latent stages of MDV infection to investigate its role in MDV pathogenesis. Previously, a virus with a deletion of the entire vIL-8 open reading frame (ORF) was shown to be severely impaired in disease progression and tumor development in infected chickens. Marek’s disease (MD) and tumor incidence was reduced to 4-10% in chickens infected with vIL-8 deletion viruses compared to more than 90% upon infection with parental virus. However, it remained unclear whether this phenotype was caused by the lack of secreted vIL-8 or vIL-8 splice variants that fuse exons II and III of vIL-8 to several upstream open reading frames, including the viral oncoprotein Meq, RLORF4 and RLORF5a.
To specifically examine the role of secreted vIL-8 in MDV pathogenesis, I constructed a recombinant virus in which the vIL-8 start codon located in exon I was mutated (vΔMetvIL- 8). This mutant lacked expression of vIL-8 without affecting Meq-vIL-8 splice variants. Loss of secreted vIL-8 resulted in a highly reduced disease and tumor incidence in chickens infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals via the natural route, infection and lymphomagenesis in contact animals was severely impaired. To determine the target cells of the vIL-8 chemokine, I generated purified recombinant vIL-8 and could demonstrate that it efficiently binds to and induces chemotaxis of B cells, the main target for lytic MDV replication. Furthermore, I could show that vIL-8 also interacts with CD4+CD25+ T cells, a putative target for MDV transformation.
Our data provide evidence that vIL-8 attracts B cells and CD4+CD25+ T cells the targets for both lytic and latent infection.
Chemokines usually contain a number of conserved motifs that are important for chemokine function. vIL-8 contains a DKR motif at the N-terminus. In other CXC chemokines, the motif in this position is responsible for the specific binding of the chemokine to its receptor on target cells. To address the role of the DKR motif in the binding specificity, I mutated the DKR motif to ELR which is present in the closely related chemokine interleukin-8 (IL-8). Mutating the DKR motif to ELR did not alter the binding properties of vIL-8 suggesting that DKR is not important for specificity of vIL-8 binding.
Previously, a role for C-terminal domains of CXC cytokines in angiogenesis during tumor formation has been suggested. To determine whether the vIL-8 C-terminus can influence tumor formation, I generated a C-terminal deletion mutant (vΔCT-vIL-8). In vitro, this virus replicated comparable to parental and revertant virus.
In vivo, we only observed only a slight reduction in disease and tumor incidence in chickens infected with vΔCT-vIL-8, suggesting that the C-terminus plays a minor role in the tumorigenesis and pathogenesis and neither affects vIL-8 nor vIL-8 splice variant function.
Despite a plethora of studies addressing the establishment of MDV infection and pathogenesis, the exact mechanisms are still not well understood. To develop a tool that facilitates detection of and discrimination between lytic and latently infected cells in vivo, we generated a markervirus containing fluorescently labeled proteins indicating the state of the infection of the infected cells. For this purpose, we fused the red fluorescent protein (RFP) to the C-terminus of UL47, a protein expressed only during lytic replication, and the green fluorescent protein (GFP) to the C-terminus of Meq, a protein expressed during latency and in transformed cells (vUL47-RFP_Meq-GFP). In vitro, vUL47-RFP_Meq-GFP replicated comparable to parental virus and expression of the fluorescent proteins could be observed. Intriguingly, vUL47-RFP_Meq-GFP did not induce disease, suggesting that fusion of GFP to the C-terminus of Meq affects its function in transformation and tumorigenesis.