Directional transneuronal infection by pseudorabies virus is dependent on an acidic internalization motif in the Us9 cytoplasmic tail

The Us9 gene is conserved among most alphaherpesviruses. In pseudorabies vi rus (PRV), the Us9 protein is a 98-amino-acid, type II membrane protein fou nd in the virion envelope. It localizes to the trans-Golgi network (TGN) re gion in infected and transfected cells and is maintained in this compartmen t by endocytosis from the plasma membrane. Viruses with Us9 deleted have no observable defects in tissue culture yet have reduced virulence and restri cted spread to retinorecipient neurons in the rodent brain. In this report, we demonstrate that Us9-promoted transneuronal spread in vivo is dependent on a conserved acidic motif previously shown to be essential for the maint enance of Us9 in the TGN region and recycling from the plasma membrane, Mut ant viruses with the acidic motif deleted have an anterograde spread defect indistinguishable from that of Us9 null viruses. Transneuronal spread, how ever, is not dependent on a dileucine endocytosis motif in the Us9 cytoplas mic tail. Through alanine scanning mutagenesis of the acidic motif, we have identified two conserved tyrosine residues that are essential for Us9-medi ated spread as well as two serine residues, comprising putative consensus c asein kinase II sites, that modulate the rate of PRV transneuronal spread i n vivo.