Site-specific Labeling And Dynamic Tracking Of Baculovirus Infection

Analyzing complex biological systems in real time, in situ and in vivo is the emphasis and difficulty of analytical chemistry. Virus-triggered diseases have severely threatened human health. Thus, a comprehensive understanding of virus infection mechanism is of great importance for preventing, diagnosing and developing drugs to treat virus diseases. Virus fluorescence labeling strategies and single-particle tracking are powerful tools for revealing virus infection mechanism. Owning excellent optical properties, Quantum dots (QDs) is an ideal labeling materials for long-time single-particle tracking. However, viral envelope is the common labeling site in previous reports so far, making it impossible to tracking the infection events after envelope-loss. Thus QD-laleling of viral capsid is ergenly needed. Furthermore, previous researches on virus infection lack systematicness and integrality, and have barely provide dynamic information about virus, thus a tracking method for systematically revealing virus infection mechanism is needed. Moreover, the three key viral components (envelope, capsid and nucleic acids) are all involved in virus infection process. However, only dual-component labeling has been realized so far, making it impossible to acquire the comprehensive and detailed infection mechanism. Baculovirus expression systems can presents one or multiple foreign proteins on viral envelope or capsid, providing the possibilities for simultaneous labeling of viral QD-labeling and multi-component labeling.Aiming at the deficiency in previous virus labeling strategies and their application for comprehensive and systemic study on virus infection mechanism, taking baculovirus that owning typical three components as the model, and combining the advantages of baculovirus expression systems, this work mainly studied the following contents:A strategy for site-specific labeling the capsid of enveloped virus with QDs was established. Taking the advantages of baculovirus expression systems, the capsid proteins VP39 of baculovirus were biotinylated during virus intracellular self-replication process. Thus baculovirus capsids were site-specifically labeled with streptavidin conjugated QDs. Such labeling avoided violent chemical reaction, and thus virus infectivity was maintained. Since VP39 are the maior capsid proteins, such labeling has a high labeling efficiency (about 95%). Such QD-labeling of viral internal capsid lays a good foundation fortracking virus infection events both before and after envelope-loss.A systematic method for tracking virus whole infection process was developed. Based on the baculovirus with QD-labeled capsid and single-particle tracking, the interaction between baculoviruses and the infection-related cellular structures including vesicles, acid endosomes, actins and nuclear pore was monitored respectively, thus five stages of baculovirus infection from the cell membrane attachment and nucleus entry were illustrated. The modes and parameters of movement were also provided. The tracking of virus whole infection process was realized based on this method, provding opportunities for revealing vius whole infection mechanism.A strategy for simultaneous site-specific labeling of viral envelope, capsid and nucleic acids was established. Baculovirus envelope proteins GP64 were biotinylated and capsid proteins VP39 were labeled with GFP based on baculovirus expression systems. Combining the host cells incubation with nucleic acids stains SYTO 82 and SA-QDs, virus multi-component labeling was accomplished during virus self-duplication process, avoiding conventional multiple steps for separately labeling each viral component and the removal of the extra labeling materials. Thus virus infectivity was maintained to the largest extent. Owing to the site-specific modification of viral proteins and the high affinity between SYTO 82 and nucleic acids, each viral component was site-specifically and high-efficiently labeled (the labeling efficiency is about 90%). The information about the intracellular separation and transportation of viral multiple components were obtained based on such labeling, providing opportunities for comprehensively and detailed revealing virus infection mechanism and diagnosing and treating virus diseases.