Investigation of endocytic pathways during entry of RNA viruses reveal novel host proteins as lipid raft dependent endocytosis mediators

Entry of viruses inside host cell after successful attachment is an essential step to ensure its genome replication and progeny production using host cell machinery. Targeting viral entry has been proven an effective therapeutic approach to prevent or treat viral infections. Viruses exploit different operational ligand entry routes to gain entry inside the host cell. Host membrane rafts are crucial for membrane mediated events such as ligand binding and internalization, signaling and pathogen entry. However, those host proteins involved in this phenomenon and molecular mechanism of this mode of endocytosis has not yet been elucidated. In present study, we investigated raft-dependent endocytosis as a major route for host cell entry for three different enveloped viruses viz. SARS-CoV-2, DENV and CHIKV. Subsequently, we performed quantitative global proteomics of SARS-CoV-2 infected Vero cells at the time of virus entry and during peak viral infection and compared proteomic changes with uninfected control. Subsequently, we implemented pathway enrichment of differentially regulated host proteins and identified regulated cellular pathways during different stages of infection. Finally, we investigated the role of selected proteins identified as significantly regulated through proteome analysis along with some of those proteins previously reported to be involved in any mode of endocytosis, in the raft-dependent endocytosis using inhibitor assay and further validated their role in viral entry through loss-of-function assays. Our results confirm that enveloped viruses exploit the raft-dependent endocytosis as a major route for host cell entry. We further report novel host cell proteins that participate as mediators of raft-dependent endocytosis.