title: The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons.
abstract: The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1.
  
  
  OBJECTIVE
  Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia russell.diefenbach@sydney.edu.au tony.cunningham@sydney.edu.au.
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, NSW, Australia.
authors.forename: April
authors.initials: A
authors.lastname: Davis
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Monica
authors.initials: M
authors.lastname: Miranda-Saksena
authors.affiliation: Centre for Perinatal Infection Research, The Children's Hospital at Westmead, Westmead, NSW, Australia Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, University of Sydney, Westmead, NSW, Australia.
authors.forename: Marian A
authors.initials: MA
authors.lastname: Fernandez
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Kelly
authors.affiliation: Centre for Perinatal Infection Research, The Children's Hospital at Westmead, Westmead, NSW, Australia Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, University of Sydney, Westmead, NSW, Australia.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Jones
authors.affiliation: Departments of Anatomy and Ophthalmology, University of California San Francisco, San Francisco, California, USA.
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: LaVail
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Jing
authors.initials: J
authors.lastname: Xue
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia.
authors.forename: Joey
authors.initials: J
authors.lastname: Lai
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW, Australia russell.diefenbach@sydney.edu.au tony.cunningham@sydney.edu.au.
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Cunningham
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001370:Axonal Transport; D001665:Binding Sites; D003584:Cytological Techniques; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005260:Female; D005727:Ganglia, Spinal / Q000821:virology; D006561:Herpes Simplex / Q000473:pathology / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D016547:Kinesins / Q000378:metabolism*; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000821:virology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017208:Rats, Wistar; D012720:Severity of Illness Index; D012867:Skin / Q000473:pathology / Q000821:virology; D019562:Viral Load; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D057074:Virus Release
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008074:Lipoproteins; D010750:Phosphoproteins; C109622:US9 protein, Human herpesvirus 1; D014764:Viral Proteins; D016547:Kinesins