Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes massive epidemics of a debilitating musculoskeletal inflammatory disease. There are currently no approved CHIKV-specific vaccines or antiviral agents. CHIKV initiates infection after the E2 glycoprotein binds to glycosaminoglycans (GAGs) on the surface of host cells and promotes internalization by clathrin-dependent uptake into the endocytic pathway. Both attenuated and virulent CHIKV strains bind GAGs, and GAG-binding efficiency influences virulence, including in a mouse model of CHIKV disease. However, the molecular basis of CHIKV-GAG interactions is unknown, which precludes a complete understanding of the function of GAGs in CHIKV pathogenesis. Moreover, the host factors that promote CHIKV replication, the precise cellular targets for CHIKV infection in the host, and links between host CHIKV replication factors and disease pathogenesis also remain unclear. The proposed research combines the expertise of the laboratories of Terence Dermody, Michael Diamond, and Thomas Morrison in virus-receptor interactions, RNA virus replication, viral immunology, and mouse models of viral disease to enhance knowledge of CHIKV replication and pathogenesis. Three integrated and interactive specific aims are proposed.
In Specific Aim 1, the mechanisms and pathological significance of CHIKV binding to GAGs will be determined. Specific GAG subtypes bound by CHIKV will be defined using glycan-array screening and genetically altered cell lines with defects in GAG biosynthesis, and sequences in E2 required for binding to GAGs will be defined using structure-guided mutagenesis. The function of GAG binding in acute and chronic CHIKV disease will be determined using mutant CHIKV strains with reduced or abolished GAG-binding capacity and GAG-knockout mice.
In Specific Aim 2, the function in CHIKV infection of COPI coatomer subunits and regulatory factors, which were recently identified in a small-interfering RNA screen, will be elucidated. Cells with diminished COPI transport activity will be infected with CHIKV and tested for formation of viral replication compartments, synthesis of viral RNA, and assembly and release of viral progeny. The function of COPI coatomer ARCN1 and regulatory factor GBF1 in CHIKV pathogenesis will be determined using newly established gene-targeted mice.
In Specific Aim 3, cell types in the mammalian host that contribute to CHIKV pathogenesis and persistence will be identified. CHIKV strains engineered to contain tissue-specific microRNA seed sequences will be tested for acute and chronic disease in mice and elaboration of chemokines and cytokines, infiltration of musculoskeletal tissues with inflammatory leukocytes, and development of humoral immune responses. Overall, studies in this collaborative proposal will enhance our understanding of mechanisms by which CHIKV binds to GAGs, determine the function of COPI transport in CHIKV infection and pathogenesis, and define specific cells in the host targeted by CHIKV to produce disease. Knowledge gained from the proposed research may illuminate new targets for anti-CHIKV therapeutics.
The proposed research will elucidate mechanisms by which chikungunya virus, an emerging arthropod-borne virus of considerable threat to human health, infects specific cell types in the host to cause disease. In the future, this information could be used to develop new therapeutics that inhibit replication of chikungunya virus and possibly other pathogenic alphaviruses.
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