title: Dual Role of Herpes Simplex Virus 1 pUS9 in Virus Anterograde Axonal Transport and Final Assembly in Growth Cones in Distal Axons.
abstract: The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9(-)), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (∼90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD, and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40 to 75%) in varicosities and growth cones infected with strain 17, F, and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with the KOS strain virus (which also lacks pUS9) and still lower (<2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, first in anterograde axonal transport and second in virus assembly in growth cones in distal axons.
  
  
  OBJECTIVE
  HSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal cell body are not impaired in the absence of pUS9. Thus, our findings indicate that pUS9 contributes to the overall HSV-1 anterograde axonal transport, including a major role in virus assembly at the axon terminus, which is not essential in the neuronal cell body. Overall, our data suggest that the process of virus assembly at the growth cones differs from that in the neuronal cell body and that HSV-1 has evolved different mechanisms for virus assembly and exit from different cellular compartments.
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, New South Wales, Australia monica.miranda@sydney.edu.au tony.cunningham@sydney.edu.au.
authors.forename: Monica
authors.initials: M
authors.lastname: Miranda-Saksena
authors.affiliation: Electron Microscope Research Facility, The Westmead Institute for Medical Research, The University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Boadle
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Russell J
authors.identifier: http://orcid.org/0000-0002-8433-4336
authors.initials: RJ
authors.lastname: Diefenbach
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, New South Wales, Australia monica.miranda@sydney.edu.au tony.cunningham@sydney.edu.au.
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Cunningham
mesh_terms: D000818:Animals; D001370:Axonal Transport; D005727:Ganglia, Spinal / Q000166:cytology / Q000821:virology; D020439:Growth Cones / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008074:Lipoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009924:Organ Culture Techniques; D010750:Phosphoproteins / Q000378:metabolism*; D017208:Rats, Wistar; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008074:Lipoproteins; D010750:Phosphoproteins; C109622:US9 protein, Human herpesvirus 1; D014764:Viral Proteins