Exploring Endosomes-and Autophagosomes-associated Infection Behaviors Of Influenza Virus By Single-virus Tracking

Virus-induced diseases pose severe threat to human lives and development,some of which have raged for centuries,and also result in substantial economic impact worldwide.Moreover,newly emerging virus strains continue to pose severe public health threat worldwide.Therefore,thorough dissecting the infection process of virus and understanding of the infection mechanism are of great significance for preventing and treating virus-induced diseases effectively.However,the infection process of virus in living cells is continuous,complicated,dynamic and multi-step.Due to the deficiencies of conventional methods,it is still the emphasis and difficulty in the virology research to precisely dissect the mechanisms of the interactions between viruses and their host cells in real time and in situ.To solve this issue,our group has established a two-dimensional single-virus tracking(SVT)technique,based on the quantum dot(QD)that possesses unique optical properties.With the aid of the QD-based SVT,we have successfully unraveled several previously unreported infection mechanisms of influenza virus.In this dissertation,we have chosen avian influenza virus H9N2 as a paradigm to develop a set of multi-dimensional SVT methods based on QDs,and applied the SVT method to uncover the infection behavior of influenza virus,which have been summarized as follows:(1)Constructing a platform for three-dimensional(3D)SVT and developing a QD-based 3D SVT technique for studying the underlying mechanisms of the endocytic trafficking of viruses.We globally monitored the transport process of QDs-labeled viruses,Rab5-positive endosomes and Rab7-positive endosomes in three dimensions in living cells.We found that Rab5 mainly takes part in the virus infection process from the cell periphery to the perinuclear region of cells,while few of Rab7-positive endosomes participated in this infection process.Rab7 is mainly involved in the intermittent and confined movements of the virus in the perinuclear region of cells.This part of the work revealed the endosomes-associated infection mechanism of influenza virus.Furthermore,the QD-based 3D SVT is proved to be readily applied for studying dynamic interactions between viruses and cellular structures,and may also lead to detailed insights into the intracellular transport of cargoes.(2)On the basis of QD-based SVT technique,a real-time multichannel imaging method was developed for exploring the involvement of autophagy in the early stages of virus infection.Firstly,we investigated the influence of virus infection on the host cell autophagy,and identified that virus infection triggered the formation of autophagosomes and promote the transport of autophagosomes at the early stages.Using QD-based SVT technique,we realized real-time and in situ dissection of the dynamic interactions betweent viruses and autophagosomes.We found that a small fraction(roughly one-fifth)of viruses exploits the autophagic machinery for transport from the cell periphery to the perinuclear region of cells.By QD-based multi-dimensional SVT technique,we revealed two distinct pathways for intracellular transport of viruses,i.e.either endocytic trafficking or autophagic trafficking.Remarkably,further evidence proved that this autophagic trafficking of viruses in their entry is independent of Rab5,a marker primarily associates with early endosomes.Our findings uncovered the intracellular transport mechanism of virus involved in autophagy for the first time and identified that autophagy serves as a parallel pathway for the intracellular transport of virus.This part of the work contributes to a better understanding of relationship between autophagy and virus entry.Moreover,the QD-based SVT coupled with real-time multichannel imaging is proven to be a promising tool for autophagy-related fundamental research.(3)Based on the study of the involvement of autophagy in the intracellular transport of viruses,we investigated Rab7-associated infection behaviors of viruses during the autophagic trafficking process by QD-based multi-dimensional SVT technique.Firstly,we found that the viruses sequestered into autophagosmes are allowed to release their genome,when Rab7 is actively involved in this process.Then,we tracked the Rab7-associated transport process of autophagosomes in real time.By analyzing the transport behavior,we elucidated the mechanism of interactions between autophagosomes and Rab7.Meanwhile,using QD-based multi-dimensional SVT technique,we revealed the role for Rab7 in autophagic trafficking of virus.Collectively,we systematically elucidated the infection behaviors of virus involved in autophagy.These results indicate the possibility of targeting the autophagic pathway during virus entry for antiviral therapy,and open a new window for developing more efficient antiviral strategies.