Dissection Of The Entry Mechanism Of Influenza Virus By Quantum Dot-based Single-virus Tracking

Influenza virus has caused global pandemics for many times and severely threatened public safety and economics in history.Its high variable genome necessitates the long-lasting surveillance and prevention,as well as persistent effort on the development of new anti-flu medicines.Studying the infection mechanism of influenza virus contributes to the understanding of pathogeny of influenza virus,and provides us with the rationale for prevention and treatment of flu,so it is of great social and economic significance.Entry is the first critical step for the infection of influenza A virus.The virus overcomes the physical obstacle of the plasma membrane,reaches the cytoplasm of cell during entry and further hijacks the cellular function to serve its replication.Because of the dynamic,complex interactions between virus and molecules on the surface of cell,it is very difficult to study the entry mechanism,but the single-virus tracking technique is an ideal approach to address this issue.Compared with conventional methods only providing the static information on the average state of the whole population of viruses,real-time,in situ single-virus tracking technique can realize the continuous visualization of the entry behavior of a single virus.Especially,with the aid of quantum dot,a kind of inorganic nanocrystal with outstanding optical properties,single-virus tracking technique possesses huge superiorities in the virological research about the dynamic interactions between multiple viruses and molecules.In this work,utilizing the quantum dot-based single-virus tracking technique,we realized the visualization of the entry of influenza virus by endocytosis,revealed the diffusion behavior of virus during this process and proved the clathrin-mediated endocytosis was the main entry route of influenza virus in MDCK cell.We dissected the different functions of three related cellular proteins:clathrin,dynamin and auxilin by comparing their virus-induced recruitments.We also found the asynchronization between the recruitments of clathrin and dynamin,,and further established the temporal model for the clathrin-dependent endocytic route.Futhermore,we observed a large population of events for recruitments of clathrin,dynamin and auxilin induced by viruses that didn't enter the host-cell eventually.These abortive events of recruitment were short-lived,compared to those induced by productive virus entry,suggesting the existence of a screening process during the maturation of clathrin-coated pits.Only a minority of virus-induced clathrin-coated pits could proceed to the membrane fission and become endocytic vesicles.In addition,we also dissected the clathrin-independent entry route of influenza virus,which possessed many features of marcopinocytosis and was also dependent on dynamin.The functional difference of dynamin in these two routes was revealed,Dynamin promoted membrane fission in both entry routes and participated in the maturation of clathrin-coated pit additionally in the clathrin-dependent route additionally.We further visualized the recruitment of proteins related to the assembly of actin filament and revealed that both entry routes of influenza virus were dependent on the assembly machinery of actin filament.We also discussed the function of the actin assembly during the endocytosis of influenza virus.It was indicated that the assembly of actin filament assisted the membrane fission and propelled the virus-containing vesicle toward the inside of cell.