Alphaherpes viruses, including pseudorabies virus (PRV) and herpes simplex virus (HSV)-1 and -2, infect the nervous system and establish latent infections in ganglia of the peripheral nervous system (PNS) in their natural hosts. Spread of infection to new hosts requires reactivation of the latent infection and subsequent transport of progeny virions in axons to distant egress sites in the periphery 
. Anterograde transport away from the neuronal soma in axons is essential for spread to innervated epithelial layers, or more rarely into the central nervous system (CNS) 
. Given the polarized nature of mature neurons, i.e. distinct somatodendritic and axonal compartments 
, an active sorting mechanism has evolved to permit progeny virions to enter axons following replication. For PRV, anterograde spread of infection in vivo
along chains of synaptically connected neurons is dependent on the small type II membrane protein Us9 
. The role of Us9 in axonal sorting and transport has been established in vitro
at the cellular level, with studies reporting Us9-dependent axonal sorting and anterograde transport of viral glycoproteins, tegument, and virions 
Enveloped viral particles transport within the lumen of vesicles, likely derived from the trans-Golgi network (TGN) 
. Us9 incorporation into these transport vesicles is necessary for and directly promotes sorting into axons and anterograde transport 
. Co-immunoprecipitation experiments have demonstrated a functional interaction between Us9 and the neuron-specific kinesin-3 motor protein Kif1-A 
. In uninfected neurons, Kif1-A facilitates the anterograde transport of pre-synaptic and dense-core vesicles 
and is likely repurposed by Us9 during infection to modulate the axonal sorting and transport of virions.
The subcellular localization of Us9 is critical for protein functionality. Us9 is enriched in lipid raft membrane microdomains, localization to which is essential for Us9-mediated anterograde transport of virions 
. In polarized neurons, Us9 is sorted into specific vesicular compartments of the Golgi and endosomal networks, and certain non-functional Us9 mutants have aberrant membrane localization patterns 
Though no crystal structure has been established for Us9, one critical domain is a 10-amino acid cluster of negatively charged acidic residues (46–55) 
(). Us9 mutants with the acidic cluster deleted fail to undergo anterograde spread in vivo
. Two tyrosine (Y49 and Y50) residues within the cluster, known not to be phosphorylated or influence subcellular localization, are essential for productive anterograde spread in vivo
and are required for Us9-Kif1A binding 
Schematic of the GFP-Us9 protein and critical residues.
Compilation of PRV strains expressing mutant Us9 variants employed in this study as well as in previous work.
Two serine residues (S51 and S53) within the acidic cluster are phosphorylated, as determined through radiolabelling assays 
, and are part of casein kinase-2 (CK2) consensus sequences. Phosphorylation of S51 and S53 is essential for anterograde spread in vivo
though it is unclear what role phosphorylation plays in the functional biochemistry of Us9 or Us9-directed anterograde transport. These serine residues may influence subcellular localization and/or facilitate interactions with binding partners. Furthermore, homologous, highly conserved serine residues exist in the Us9 protein of other related alphaherpes viruses, including HSV-1, -2 
and bovine herpes virus (BHV)-1, -5 
(). It is also known that the varicella zoster virus Us9 homolog is phosphorylated by CK2 in vitro
. Three other minor serine phosphorylation sites previously undetected by radiolabelling (S38, S46, and S59) have also recently been detected in the PRV Us9 peptide through mass spectrometry 
; however, these residues are not conserved with Us9 homologs from other alphaherpes viruses. Understanding the relevance of serine phosphorylation to Us9 mediated axonal sorting and transport will expand and refine our model of the molecular mechanisms that facilitate alphaherpes virus anterograde spread.
In this study, we characterized serine residues 51 and 53 of the PRV Us9 protein in vitro at the cellular and biochemical level. We visualized the distribution of phosphorylated Us9 in axons using immunofluorescence with a phospho-specific antibody and then analyzed the partitioning of phosphorylated and total Us9 protein species across different membrane microdomains. For live cell imaging and for quantification of anterograde spread, we isolated PRV recombinants expressing GFP-tagged, mutant Us9 variants with alanine substituted for one or both serine residues. We then performed infections of chambered neuronal cultures to quantify the spread defect associated with loss of phosphorylation. Finally, we assessed Us9-Kif1A interactions in the serine mutant background through co-immunoprecipitation experiments. This study of the role of Us9 phosphorylation in anterograde sorting, transport, and spread expands our understanding of the Us9 functional domains.