Study Of Interaction Between Pseudorabies Virus Tegument Protein PUL21 And Host Cytoplasmic Dynein

Pseudorabies virus(PRV)is the causative agent of Aujeszky's disease.It brings enormous harm to sustainable pig production in the world.PRV is characterized by its neuroinvasiveness,neurovirulence and latent infection in neurons.After entering host cells by membrane fusion,the capsid-tegument complex of PRV proceeds to the nucleus by retrograde transport along the microtubule.The aim of retrograde transport is to deliver the viral genome to the nucleus for DNA replication.Hence,it is vital to virus life cycle.Cytoplasmic dynein is a category of motor proteins for retrograde transport on microtubules in host cells.Cytoplasmic dynein is involved in retrograde transport of PRV but the detailed mechanism is still undefined.In this study,the potential interaction partner protein of PRV inner tegument protein pUL21 from cytoplasmic dynein was screened by bimolecular fluorescence complementation(BiFC).It turned out that the carboxyl terminus of tegument protein pUL21 could interact with cytoplasmic dynein light chain Roadblock-1.Further studies were accomplished.Here are the details: 1 pUL21 is an inner tegument protein of PRVMature PRV extracellular virions were pelleted and purified.Residual proteins on capsids were analyzed by Western blot after detergent and KCl treatment of virions.pUL21 was tightly combined with the capsid after detergent and KCl treatment as another innter tegument protein VP1/2.In a word,pUL21 is an inner tegument protein of PRV.2 PRV tegument protein pUL21 interacts with cytoplasmic dynein light chain Roadblock-111 genes of host cell cytoplasmic dynein were cloned.The interaction between PRV tegument protein pUL21 and cytoplasmic dynein light chain Roadblock-1 was found by BiFC and verified by co-immunoprecipitation(Co-IP).The subcellular location of pUL21 and Roadblock-1 were studied by immunofluorescence assay(IFA).pUL21 and Roadblock-1 co-localized in the perinuclear region in cytoplasm.The interaction domain was mapped by truncation and co-IP.pUL21 interacts with Roadblock-1 with its caboxyl terminus.3 Functions of the carboxyl terminus of pUL21According to the interaction domain mapped by truncation and co-IP,PRV UL21 C?300 and PRV UL21 C?300 R were constructed using PRV bacterial artificial chromosome(BAC).Although the replication and titer are not affected,the transmission ability of PRV UL21 C?300 is weaker than that of PRV WT in epithelial cells.Deletion of the carboxyl terminus of pUL21 delays PRV retrograde axonal transport in explanted neurons.Deletion of the carboxyl terminus of pUL21 reduces the neuroinvasiveness and neurovirulence of PRV in mice.