Visualization Of The Interaction Between Viruses And Cells By Using Fluorescent Nanolabel-Based Real-Time Tracking Technology

Virus-host cell interaction has long been a major research field in virology. This was exampled by the Hepatitis C virus (HCV). Cell models such as subgenomic replicon and full-length genomic replicon were established to study the interaction of HCV with its host cells. As a result, two important HCV therapeutic drug targets NS3 Serine protease and NS5B RNA-dependant RNA polymerase were identified.Multiple strategies and methods have been applied to the investigation of virus-host cell interaction.The MβCD and nocodazole together with the fluorescent label technology were used to explore the mechanisms of SV40 virus interaction with Vero cell. Moreover, a variety of novel chemical luminous dyes have recently been used for labeling virus capsid or virus-like particles (VLPs) to elucidate the interactions between virus and its host cell.In this study, we tried to use the VLPs of two non-enveloped viruses, rabbit hemorrhagic disease virus (RHDV) and Human Bocavirus (HBoV) and one wild type porcine parvovirus (PPV) labeled with novel chemical dyes, such as QDs or "light switch" to infect their host cells for following the process of the virus infection. Furthermore, we anticipate exploring the mechanisms of interaction between viruses and cellular proteins, and finally establishing a general approach to define the virus-host cell interaction. This work primarily covered following three parts:1.Expression and purification of RHDV's capsid protein VP601)The VP60 protein gene was inserted into the prokaryotic expression vector pMAL-c2X to generate the recombinant vector pMAL-VP60 and transformed into DH5 a. The recombinant fusion protein was purified by the Amylose resin and verified by SDS-PAGE and Western Blot. The ability of purified VP60 to self assemble into VLPs in vitro was also tested.2)The Bac-to-Bac baculovirus expression system was used to express VP60-VLPs protein in High-Five cells (Hi-5). The recombinant baculovirus expressing VP60 was produced and propagated in Sf9 cells. The VP60-VLPs protein was expressed in Hi-5 cells following the recombinant baculovirus infection. The VP60-VLPs was then purified by both sucrose gradient (60-10%) centrifugation and CsC1 gradient centrifugation. And finally, the purified capsids were examined under the TEM.2.Expression and purification of HBoV's capsid protein VP21)The VP2 protein gene of HBoV was inserted into the prokaryotic expression vector PET-28(a) to generate the recombinant vector PET-28(a)-VP2, which was then transformed into BL21 to express VP2 protein. The purification of protein and the preparation of its antiserum will be done as described by the manual of the manufacturer.2) The capsid gene VP2 of HBoV was inserted into Bacmid and the VP2 protein was expressed in Hi-5 cells to get the VP2-VLPs, and all the procedures are same as above.3) Some useful data through the infection experiments of the Sf9 and RK13 cells by using the Quantum Dots (QDs) to label the VP60-VLPs were collected and analysed.