RESUMO
Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.
Assuntos
Membrana Celular , Colesterol , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Células Vero , Chlorocebus aethiops , Colesterol/metabolismo , Animais , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Membrana Celular/metabolismo , Membrana Celular/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Humanos , Proteínas de Transporte/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Ligação ProteicaRESUMO
In vivo whole-body imaging, using optical tools based on bioluminescence and fluorescence detection, offers tremendous opportunities to specifically determine the spatiotemporal resolution of cancer cells within the tested animals. This enables the study of many aspects of cancer biology, including cell proliferation, trafficking, and invasions. The antitumor therapeutic properties of various tested compounds (e.g., CD19 CAR-T cells, used for cancer immunotherapy) can be monitored within the same animal at different time points, significantly reducing the number of animals used in the study as indicated in this method.