Poster Presentation 9th Australasian Virology Society Meeting 2017

Trafficking roles for identified sorting motifs in the cytoplasmic tail of HSV-1 glycoprotein E (#126)

Christopher Denes 1 , Timothy Newsome 2 , Eve Diefenbach 3 , Jing Xue 4 , Anthony L Cunningham 1 , Russell Diefenbach 5
  1. Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia
  2. Molecular Bioscience, School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia
  3. Protein Core Facility, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia
  4. Cell Signalling Unit, Children’s Medical Research Institute, Westmead, New South Wales, Australia
  5. Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia

BACKGROUND

Pulldown analysis identified an interaction between HSV-1 glycoprotein E (gE) cytoplasmic tail (CT) and key components of host intracellular trafficking pathways (the actin WAVE Regulatory Complex (WRC) and the clathrin adaptor protein complex 2 (AP-2)), that may be dependent upon a putative WRC Interacting Receptor Sequence (WIRS) or tyrosine-based endocytic sorting motifs, respectively.

 

AIM

To confirm the requirement of gE motifs for trafficking interactions in the context of HSV-1 egress.

 

METHODS

We have generated a panel of recombinant HSV-1 and plasmid constructs containing mutations in the gE WIRS or endocytic motifs. We are characterising untagged and GFP-tagged gE mutants using GFP-TRAP, proximity ligation assay (PLA) and real-time imaging to establish how these interactions contribute to egress of HSV-1. GFP-TRAP, on-bead digestion and mass spectrometry is also being used to identify a panel of additional gE interactors which may contribute to trafficking.

 

RESULTS

PLA provided evidence for colocalisation between gE and components of the WRC in infected HaCaT and HeLa cells. GST-pulldown using gE(CT) confirmed the interaction of gE with the WRC is WIRS-dependent and demonstrated that tyrosine-to-alanine mutation of the endocytic sorting motifs knocks out AP-2 interaction.

 

CONCLUSIONS

Both interactions likely contribute to trafficking of viral particles during assembly and egress from the host cell. The WIRS motif regulates Arp2/3 complex activation and actin nucleation and recruitment of AP-2 would drive subsequent clathrin-mediated endocytosis. Future research will confirm a role for gE and its interactors in the HSV-1 lifecycle and provide a basis for new drug targets for treatment.