title: Role of Us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus.
abstract: Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
authors.forename: Radomir
authors.initials: R
authors.lastname: Kratchmarov
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Taylor
authors.forename: Lynn W
authors.initials: LW
authors.lastname: Enquist
mesh_terms: D000818:Animals; D001370:Axonal Transport; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D047908:Intracellular Signaling Peptides and Proteins; D016547:Kinesins / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism* / Q000821:virology*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C490575:Kif1a protein, rat; D008074:Lipoproteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C491630:US9 protein, Suid herpesvirus 1; D014764:Viral Proteins; D016547:Kinesins