Date of Award
Doctor of Philosophy
Pamela Small, Mark Sangster, Elias Fernandez
CMV is the leading cause of both non-hereditary mental retardation and hearing loss and CMV infection following transplantation carries a serious risk for complications. The development of a CMV vaccine or better therapeutic treatment is desired but to develop these a more complete understanding of CMV pathogenesis is necessary. Sequence comparisons between attenuated and virulent strains of HCMV map major differences to a 15kb region (ULb’) containing the chemokine homolog, vCXCL-1. The vCXCL-1 protein of the Toledo (Tol) strain was previously shown to function in vitro as a CXC chemokine. Murine CMV (MCMV) also encodes a viral chemokine, MCK2. Prior research has shown that infection of mice with a recombinant MCMV, RM4511, which lacks a functional MCK2, resulted in decreased inflammation at the site of infection and decreased dissemination to the salivary gland. Analysis of the role of vCXCL-1 in the context of HCMV infection is limited by the species specificity of the CMVs. One possible model system for analyzing the function of this chemokine is the chimpanzee model of CMV infection. Chimpanzee CMV (CCMV) has at least one gene with similarity to the vCXCL-1 gene of the Toledo strain of HCMV. The hypothesis of this study was that vCXCL-1CCMV is a functional CXC chemokine that contributes to viral dissemination. To address this we initially compared the functional and biochemical characteristics in vitro of vCXCL-1 from Toledo HCMV (vCXCL-1Tol) and CCMV (vCXCL-1ccmv) by analyzing receptor binding, activation, chemotaxis, signaling, and anti apoptotic suppression in neutrophils. The CCMV chemokine had a ~70-fold lower affinity for human CXCR2 compared to (vCXCL-1Tol. Nevertheless, the homologs functioned very similarly and we only observed differences in integrin induction and the ability of the two chemokines to reduce neutrophil apoptosis. We evaluated the in vivo function of vCXCL-1ccmv using MCMV infection in mice. Due to the strict species specificity of CMV we used MCMV RM4511 to produce recombinant MCMVs expressing vCXCL-1ccmv or a control host chemokine, mCXCL1, under the control of the HCMV IE promoter. RMvCXCL1ccmv and RMmCXCL1 grew with similar kinetics to RM4511 in cell culture and expressed vCXCL-1ccmv and mCXCL1, respectively. Primary dissemination of RMvCXCL1ccmv and RMmCXCL1 was similar to RM4511 but neither recombinant was recovered from the salivary gland at any time point. Maintenance of this phenotype in SCID mice showed the recombinant viruses were not cleared from the salivary gland by the adaptive immune response. RMvCXCL1ccmv and RMmCXCL1 were also not recovered in neutrophil-depleted mice, although RM4511 titers were reduced in these mice indicating neutrophils may play a role in dissemination of MCMV to the salivary gland. This study is the first to characterize the CCMV viral chemokine, vCXCL-1, and show it is functionally similar to vCXCL-1Tol This provides evidence that the study of vCXCL-1ccmv may contribute to a better understanding of its HCMV homolog. Furthermore, this research is the first to evaluate the role of a CMV CXC chemokine in vivo Future work aimed at evaluating the role of other viral and host chemokines in vivo using the mouse model will likely advance our understanding of viral chemokines and their role in virus-host interactions.
Miller-Kittrell, Mindy L., "The Role of Cytomegalovirus Viral Chemokines in Neutrophil Activation and Viral Dissemination. " PhD diss., University of Tennessee, 2008.