Abstract
Poxviruses are large DNA viruses that infect a wide range of animals including humans. During the course of evolution, these complex viruses have gained an extensive repertoire of immunomodulatory genes to evade host immune responses in order to enhance their own survival. One of these immunomodulators is a secreted soluble chemokine-binding protein (CBP) which has no known mammalian homologue. Chemokines are a large family of chemotactic proteins that regulate recruitment of leukocytes to sites of infection or inflammation, as well as homeostatic migration of leukocytes through lymphoid organs. Members of the chemokine family are classified as CXC, CC, C and CX3C based on the arrangement of cysteine residues at the N terminus. The majority of the known chemokines fall into either the CXC or the CC classes. In general, CXC chemokines are potent chemoattractants of neutrophils or lymphocytes, whereas CC chemokines are chemoattractants for monocytes. The CBP genes have been identified in Orthopoxviruses and Leporipoxviruses, as well as Parapoxviruses (PPVs) that includes Orf virus (ORFV), and genes encoding putative CBPs have been identified in Pseudocowpox virus (PCPV), Bovine papular stomatitis virus (BPSV) and Parapoxvirus of red deer in New Zealand (PVNZ). Previous studies have demonstrated the structural and functional activity of ORFV-CBP, however, there is little known about the CBPs of the other PPVs. This study describes the characterization of CBPs secreted by two less-studied members of the PPVs; BPSV and PVNZ. BPSV and PVNZ infect cattle and red deer, respectively, and induce superficial pustular skin lesions and lesions of the oral mucosa. Further PVNZ infects the velvet of antlers. In the case of BPSV infections, the immunity is short lived, a phenomenon that could be related to the immunomodulators encodes by the virus.
In chapter two, the molecular properties of BPSV and PVNZ CBPs are described. Mass spectrometry revealed that the BPSV-CBP is a homodimeric polypeptide with a MW of 82.4 kDa, and sequence analysis showed around 40% identity and 70% similarity between the CBPs of BPSV and ORFV at the amino acid level. Moreover, structural modelling based on the crystal structure of the orf virus CBP, predicted a similar beta-sheet sandwich for both proteins, suggesting that like ORFV-CBP, BPSV-CBP may have broad-spectrum chemokine binding properties. PVNZ, unlike other PPVs, express three distinct CBPs (namely 112.0, 112.3 and 112.6). The CBPs of PVNZ112.0 and PVNZ112.3 have 46% identity and cluster together, while PVNZ112.6-CBP has only about 30% identify with the other PPV-CBPs and falls into a separate branch of phylogenetic tree. It seems that multiple PVNZ-CBPs have arisen by a process of duplication and divergence from a common ancestral gene. Although sequence analysis of the PVNZ-CBPs showed a moderate degree of identity (30-40%) with the ORFV-CBP, it predicted a conserved structural core for PVNZ-CBPs, suggesting a certain chemokine-binding activity for these viral proteins.
Chapter three describes a comprehensive chemokine-binding screen of BPSV-CBP and the PVNZ-CBPs by surface plasmon resonance (SPR) assay. The SPR results showed high-affinity binding of the BPSV-CBP to a wide-range of inflammatory murine chemokines within the CXC, CC and XC classes. Murine cells and mice were used in subsequent studies to characterise the in vitro and in vivo functions of BPSV-CBP. The SPR assay also revealed that each PVNZ-CBP has a unique chemokine-binding profile. In general, PVNZ-CBP 112.3 binds to CXC chemokines selectively, while PVNZ-CBP 112.6 has strong binding affinity with CC chemokines. Remarkably, PVNZ-CBP 112.0 had no or weak binding against most of the chemokines tested. The spectrum of the inflammatory chemokines targeted by the CBPs of BPSV and PVNZ suggest that like orf virus, these Parapoxviruses target inflammation as a strategy to subvert immune cell activation and recruitment to infected tissue.
In chapter four, murine neutrophils and monocytes were used in transwell migration assays to investigate the in vitro effects of BPSV-CBP on chemokine-induced chemotaxis. Mature neutrophils were differentiated from a mouse promyelocytic (MPRO) cell line and shown as Ly-6Ghi CD11bhi cells that express the chemokine receptor CXCR2. After optimizing the neutrophil migration in response to CXCL1, CXCL2, and CCL3 chemokines, the BPSV-CBP was shown to cause a significant reduction in cell migration at a molar ratio of chemokine-to-CBP of 1:4, 1:1 and 1:1 respectively. Moreover using monocytes derived from mouse bone marrow cells cultured in the presence of M-CSF, and characterized as CD115+ CD11b+ Gr-1+ cells, BPSV-CBP potently inhibited their migration towards skin-related inflammatory chemokines CC2, CCL3, and CCL5 at the molar ratio of chemokine-to-CBP of 1:1, 1:1, and 1:0.5, respectively.
Finally, a lipopolysaccharide (LPS)-induced murine inflammatory skin model was applied to evaluate the in vivo impact of the BPSV-CBP. Histological examination, in conjunction with a myeloperoxidase (MPO) assay of skin samples, revealed that intradermal injection of the BPSV-CBP reduced the influx of neutrophils (Gr-1+ MHC-II+) into the inflamed sites at 12 h post LPS injection. Moreover, the BPSV-CBP reduced infiltration of MHC-II+ monocyte/dendritic cells into inflamed skin. Overall, this study showed that the BPSV-CBP is an antagonist for a wide range of chemokines, and can inhibit the migration of inflammatory immune cells both in vitro and in vivo.
These findings suggest that the CBP could be important in virulence and pathogenesis of BPSV infections. Importantly, the broad binding properties of the CBPs suggest that they may have therapeutic value as anti-inflammatory drugs.