Proteomics Analysis Of PRV Cell-cell Transmission

Because PRV can infect neurons and transsynaptic spread in the nervous system,it has been widely used as a tracer for mapping neuronal circuits.Besides PRV(pseudorabies virus),some other neurotropic virus,such as HSV-1(herpes simplex virus type 1),RV(rabies virus)and VSV(vesicular stomatitis virus),can also be used in neuronal circuits analysis.How neuronal tracing viruses achieve a highly directional transport from cell to cell,especially how the transsynaptic process in neuron operates,remains mostly a mystery.A better understanding of PRV directional transmission will shed light on how neuronal tracing viruses work and affect the precise interpretation of neuronal circuit data.In addition,this kind of study will also provide new insights into viral-host interactions.Host proteins can be incorporated into PRV virions,but the details and function of these host proteins are still unknown.In the first section,we constructed PRV325,a dual fluorescent PRV which contains a VP26-RFP tag and a g M-GFP tag.Based on FACS(Fluorescence Activated Cell Sorting)strategy,we developed a new FACS-based strategy to purify PRV virions.Extracellular and intracellular PRV virions from BHK21 cells infected with PRV325 were obtained after sorting the corresponding proteomes were analyzed using proteomics analysis.In addition to ～ 30 viral proteins,we also identified a large number host proteins.In six biological replicated proteomics experiments,we identified 146 and 598 host proteins in extracellular and intracellular PRV virions respectively.Through functional analysis,we found that IRSp53 and fascin were critical for the egress process and play a role in PRV direct cell-cell transmission.Moreover,we showed that CDC42 and Rac1,both are known to be involved in cytoskeleton related activities,were also involved in the production of mature PRV extracellular virions.Our results suggest that the formation of the filopodia-like cytoskeleton and the rearrangement of the membrane,which are both associated with IRSp53 and fascin,may be important for the transmission of viruses used in neuronal tracing.In the second section,we attempt to identify host proteins which interact with PRV in synapse.First,synaptosomes were purified from mouse cerebral cortex.Then,purified synaptosomes were incubated in vitro with PRV443,followed by Coimmunoprecipitation using anti-PRV and LC-MS/MS analysis.In all,we identified 134 host proteins which may be interact with PRV in synapse.A correlation analysis of four biological replicates(eight IP-MS samples)proved that the data is reproducible.Preliminary bioinformatics analysis revealed the identified host proteins’ category distribution and network information.This PRV interactomics analysis using synaptosomes can provide valuable insights into the transsynaptic process of PRV in the nervous system which should provide clues for further functional studies.