TI - Discussion . AB - We have demonstrated that Us9 is enriched in detergent-resistant membranes of non-polarized and polarized PC12 cells . This enrichment is cholesterol dependent , and is essential for Us9 -mediated anterograde spread of infection in primary SCG neurons . Us9 is responsible for the axonal sorting of viral capsids [15] , as well as the viral glycoproteins gB , gC and gE [16] . These viral membrane proteins are associated with lipid rafts on the surface of PRV infected swine kidney cells and monocytes , and monospecific antibody -induced patching of one of these proteins led to the copatching of the others [18] . These patches were enriched in the raft marker GM1 , . These findings are consistent with the raft partitioning of these viral glycoproteins in polarized PC12 cells ( Figure 3 ) . In addition , Us9 -dependent targeting of viral membrane proteins requires maturation of cultured SCG neurons ( Tomishima and Enquist , unpublished observations ) . Lipid raft formation during neuronal polarization is likely a key step in this sorting process [4] . Taken together , these data support a role for lipid rafts in the axonal sorting of alpha herpesvirus proteins and structures in the mammalian nervous system . PRV gE , which is in a heterodimeric complex with the viral glycoprotein gI [22] , is also necessary for the efficient axonal sorting of PRV structural components [54] . We propose a model in which Us9 , gE/gI and lipid rafts direct the sorting of vesicles into the axon of infected neurons ( Figure 9 ) . Us9 and gE/gI likely associate with lipid rafts in the trans-Golgi network ( TGN ) , the putative site of viral assembly [20] , [67] , [68] . The presence of Us9 and gE/gI in lipid rafts ( those decorating the surface of cellular vesicles ) would recruit axonal sorting machinery to a small number of viral assembly complexes in the TGN , i.e.vesicles with viral membrane proteins only , those containing mature virus particles or L-particles ( illustrated as "three vesicle populations" in Figure 9 ) . A limited number of vesicles containing virion components would then be targeted to the axon . Though a Us9-gE/gI complex in a lipid raft is required for efficient axonal transport , Us9 is clearly the more critical component , and the presence of gE/gI seems to enhance this process [17] . It is noteworthy that Us9/gE/gI are not required for infectious particle formation in the cell body of SCG neurons [16] , [54] , nor for retrograde transport in the mammalian nervous system [11] , [69] . Furthermore , we have not observed the accumulation of viral capsids or membrane proteins in the cell body of a Us9-null , gE/gI-null or Bartha strain lacking all three genes ( unpublished observations ) . Our findings are consistent with a model in which the virus assembly and axonal sorting compartment within the TGN are identical ( ie both processes use the same material for assembly ) . A small number of assembly complexes would bind a sorting adaptor proteins and go to axons ; the majority of assembly complexes would egress the cell body locally , perhaps at sites where axons contact the infected cell body ( Figure 9 ) . What determines whether a vesicle laden with viral structural proteins is directed to the axon as opposed to being released from the cell body? We propose that PHOSphorylation of Us9 , subsequent to its recruitment into lipid rafts , may be the pivotal step . The PRV Us9 acidic domain region is heavily PHOSphorylated during infection [26] , and is essential for anterograde , transneuronal spread in vivo[26] . PHOSphorylation occurs predominantly on two serine residues within the 10-amino-acid acidic domain , and mutating these serines to alanines dramatically decreases anterograde spread in the rat visual system [26] . This PHOSphorylation event would occur after Us9 enters the raft , as the acidic cluster domain is not required for raft association of Us9 (Figure 4B) . We are currently investigating whether 1 ) the association of Us9 with lipid rafts coincides with its phosphorylaTION inside infected cells and 2 ) the PHOSphorylation state of the Us9-TfR chimera is reduced since it is no longer enriched in lipid rafts . Perhaps not surprisingly , the Us9 acidic domain region is highly conserved among Us9 homologs of other neurotropic herpesviruses , including the human pathogens herpes simplex virus ( HSV ) and varicella zoster virus ( VZV ) , as well as the animal pathogens equine herpesvirus ( EHV ) and bovine herpesvirus ( BHV ) , suggesting an important role for this domain in the anterograde spread of virus in the mammalian nervous system [26] , [70] -[72] . We predict that PHOSphorylation of the Us9 acidic domain ( within the context of a lipid raft ) is necessary for binding an axonal sorting adaptor , which would then mediate anterograde transport inside the axon [16] , [73] . Our model addresses how viral glycoproteins/vesicles are sorted into the axon of infected neurons , but does not suggest a mechanism for their function in cell -to cell spread of infection in cultured epithelial cells [74] -[76] . It may be that these two processes are fundamentally different since 1 ) gE mutants with a small-plaque phenotype on MDBK cells have wild-type anterograde spread kinetics in the rat visual system [77] , 2 ) deletion of PRV Us9 has no effect on cell -to cell spread of infection in epithelial cells , but a dramatic impact on anterograde sorting [11] , and 3 ) gB mutants with a small-plaque phenotype on ST cells [78] have wild-type anterograde neuron-to cell spread kinetics in our trichamber system ( Curanovic and Enquist , unpublished findings ) . It was intriguing to discover that PRV gH was not associated with detergent-resistant membranes in non-polarized and polarized PC12 cells , and was completely solubilized with 1% TX-100 ( as was the non-raft marker transferrin receptor ) . The virus fusion machinery is composed of gB trimers , as well as gH/gL heterodimeric complexes ( reviewed in [79] ) . PRV gH is essential for entry into uninfected cells , cell -to cell spread of infection in tissue culture [80] , and transneuronal spread of infection in mice [81] . Does gH enter the axon in a Us9 or gE -dependent manner despite its apparent exclusion from DRMs? We are currently investigating this question . Cross-linking experiments performed on purified HSV virions found that hetero-oligomers of gB , gC and gD were closely associated with one another in the virion envelope ( within 114A ) . The gH and gL proteins could also be cross-linked within the envelope as one might predict . Interestingly , gL was never cross-linked to gB , leading the authors to suggest that organization of these proteins in the membrane "precludes associations of gH/gL with gB" [82] . One explanation for this finding is that gB is present in a lipid raft microdomain , whereas gH is not ( a small proportion of HSV gH has been shown to be in the DRM fraction of infected COS cells [83] ) . It is also feasible that PRV gH may indeed be raft-associated , but solubilization with cold detergent is too stringent . Triton X-100 and CHAPS are reported to be the most reliable detergents for analyzing raft association [84] . However , some membrane proteins solubilized by Triton X-100 do associate with lipid rafts by antibody copatching [18] , [51] , [85] , [86] . At this time it is unclear if gH has a weak affinity for rafts , or is indeed a "true" non-raft protein as is transferrin receptor . Our findings also highlight the importance of the Us9 TMD domain in raft targeting . Several studies have addressed the importance of the transmembrane segment in partitioning viral and cellular membrane proteins into lipid rafts : influenza virus hemagglutinin [60] , [64] and neuraminidase [59] , [63] , the LMP-1 oncoprotein of Epstein-Barr virus ( EBV ) [87] , and the human immunoreceptor FcgammaRIIA [88] . It is clear from these studies that amino acids within the TMD ( even single amino acids ) have dramatic effects on raft partitioning , sorting , or signaling events . A comprehensive analysis of the Us9 TMD may reveal residues important for protein -lipid/protein protein interactions that are key in promoting axonal sorting of mature virus particles . Several alpha herpesvirus proteins have been shown to associate with DRMs during virus replication . In addition to PRV membrane proteins ([18]) ; ( this study ) the virion host shutoff ( vhs ) protein of HSV-1 was shown to be enriched in organellar membrane fractions which contain virus assembly intermediates [83] . HSV gB is proposed to mobilize lipid rafts during entry , perhaps to mediate cell signaling [81] . The UL11 protein of HSV-2 , a myristoyl and palmitoyl tegument protein , associated with DRMs of infected Caco-2 cells [89] . The UL56 protein of HSV-2 , a tail -anchored type II membrane similar to PRV Us9 , was present in detergent-insoluble lipid rafts ; it is predicted to be involved in vesicular trafficking in HSV-2 infected cells [90] . These findings demonstrate that lipid rafts play an important role in the replication cycle of alpha herpesviruses , and underscore the importance of lipid rafts in virus biology [91] -[94] . Two competing models have been presented for anterograde , axonal transport of alpha herpesviruses in neurons ( reviewed in [95] ) . Viral capsids are either transported down the axon independently from viral membrane proteins ( and assemble prior to egress ) , or they are sorted and transported together as a mature virus particle within a vesicle . Recent studies have shown that efficient capsid transport is dependent on atleast two viral membrane proteins , Us9 and gE . Deleting either of these genes from HSV or PRV results in a marked decrease of viral capsids from entering axons of infected neurons [12] , [15] , [54] , [96] . It is difficult to conceive how viral membrane proteins could impact capsid sorting unless the two were tightly coupled during axonal entry and transport ( ie if capsids entered axons separate from viral membrane proteins , deletion of gE and Us9 would have no effect on capsid sorting ) . These recent findings are consistent with a model where viral capsids and membrane proteins traffic together in axons as mature virus particles ( within a vesicle ) ( Figure 9 ) . In conclusion , we have shown that PRV Us9 is highly enriched in DRMs of non-polarized and polarized PC12 cells , and this enrichment is critical to axonal targeting and subsequently in neuron-to cell spread . This is the first report to implicate lipid rafts in the axonal sorting of alpha herpesvirus structural proteins in mammalian neurons . Our future plans include isolating lipid rafts from polarized PC12 cells infected with wild-type PRV , and identifying the cellular and viral proteins present within these lipid microdomains .