Phosphoprotein-enriched proteomics identifies heat shock cognate 71 protein as a host factor supporting chikungunya virus replication in macrophages

Abstract The Chikungunya virus (CHIKV) infection induces an acute illness and ongoing inflammatory symptoms, with macrophages believed to play significant yet not fully understood roles. To characterize host cellular responses associated with CHIKV infection, we performed mass spectrometry–based phosphoprotein enrichment-based proteomic analysis of CHIKV-infected RAW264.7 macrophages. Analysis of enriched phosphoproteins identified 2,096 altered proteins compared with mock-infected cells, of which 212 were differentially regulated, including 140 upregulated and 72 downregulated proteins (fold change ≥ 2, p < 0.05). Gene ontology enrichment analysis revealed that differentially regulated proteins were predominantly associated with cellular processes, biological regulation, and metabolic pathways, with molecular functions largely related to binding and catalytic activity, and localization primarily within intracellular compartments, including the cytoplasm and organelles. Notably, stress-response and protein homeostasis pathways were consistently enriched, including the upregulation of heat shock cognate 71 kDa protein (HSC71). Network analysis linked HSC71 to key cytosolic and metabolic regulatory pathways. Pharmacological inhibition of HSC71 resulted in reduced CHIKV replication without significant cytotoxicity. The findings indicate that host protein homeostasis and phosphorylation-dependent pathways may be implicated in CHIKV infection within macrophages. The direct mechanistic role of HSC71 requires further investigation, which could facilitate the future development of antiviral agents.