Structural, functional and bioinformatic analysis of Variola major virus strain Bangladesh 1975 chemokine binding protein
Variola virus, the causative agent for smallpox, infected humans for thousands of years causing mortality estimated in the hundreds of millions before its eradication in 1979. Its success as a pathogen was due in part to the expression of several immunomodulatory proteins. One of these proteins, G3R, is a chemokine binding protein (CKBP), which binds to host chemokines and interferes with the migration of leukocytes to sites of infection and inflammation. Although CKBPs generally act on behalf of the virus against the host targeted by viral infection, they are now being explored as anti-inflammatory agents to treat human disease. This thesis is focused on the study of G3R to understand its structure, function, and origin. The G3R protein was expressed in a nonadherent mammalian cell line and purified using metal affinity and size exclusion chromatography. Following purification, the protein was crystallised in Space Group P21 and its structure was solved at 2.45 Å. The structure adopted a beta-sandwich fold similar to homologs of G3R expressed by other orthopoxviruses. The beta-sandwich is comprised of two closely packed sheets, I and II, with the surface of beta-sheet II, which binds to chemokines, displaying a negative charge of up to -5 kT/e. The chemokine binding pocket of G3R is larger than that of other similar CKBPs and this increased area could contribute to its broad range of chemokine binding. The affinity of the G3R protein for chemokines was measured using both surface plasmon resonance (SPR) and an indirect ELISA. These analyses revealed the broad extent of G3R’s chemokine binding. The ELISA results showed binding of G3R to mouse and human CC and XC chemokines and to some human and mouse CXC chemokines. No binding of G3R to murine or human CX3C chemokines was observed. In SPR studies, which were generally in agreement with the ELISA findings, G3R showed high affinity towards human and mouse CC and XC chemokines, with dissociation constants in the low nanomolar to picomolar range. G3R also bound to members of the CXC chemokine group, but with dissociation constants in the micromolar to nanomolar range. No binding was observed between G3R and murine CX3CL1; however, low affinity binding was observed with human CX3CL1. In vivo migration assays using a variety of immune cells (monocytes, T cells and macrophages) were then used to study the impact of G3R on chemotaxis. G3R proved to be effective in the inhibition of human THP-1 cell migration towards CC chemokines at a molar ratio of 1:4 (CC:CKBP). In addition, G3R reduced the migration of human Jurkat T cells towards CXC chemokines. Also, G3R inhibited the migration of THP-1 cells through an endothelial monolayer. The migration of THP1 cells towards CX3CL was partially inhibited at higher G3R concentrations producing a mild reduction in cell migration. G3R inhibited migration of inflammatory M1 macrophages at a molar ratio of 1:2. (CC:CKBP). G3R was found to reduce breast and cervical cancer cell invasion/migration towards chemokines present in serum. The relationship and origin of the three variola CKBPs — G3R, A44L and the smallpox virus— encoded chemokine receptor (SECRET) of Crm-B — was studied in the context of other poxviral CKBPs using various bioinformatic tools including: multiple sequence alignment, phylogenetic studies, whole genome alignment, Hidden Markov model (HMM) analysis, HHPred and PSI-BLAST. The results showed that the G3R and A44L proteins have a common ancestor although their amino acid sequence identity is less than 30%. The genes coding for A44L homologs are located closer to the core region of the genome, which is more conserved, while G3R and CrmB homologs are located near the terminal region of the genome. The copy number of vCCI genes is reduced in more evolved poxviruses with a narrower host range in comparison to older poxviruses, such as cowpox virus. Based on the analysis of related poxvirus genomes, these findings could be due to point mutations and deletions, which resulted in an early termination, truncation, and loss of these erstwhile functional genes. Furthermore, this analysis showed that the SECRET domain of CrmB might not have originated from the same ancestor as A44L and G3R or might have diverged earlier from their common ancestor.
Advisor: Krause, Kurt; Brown, Christopher; Wise, Lyn
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Chemokine; Protein; Inflammation; Disease
Research Type: Thesis