title: Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions.
abstract: BACKGROUND
  Herpes simplex virus type 1 (HSV1), a member of the alphaherpesvirinae, can cause recurrent facial lesions and encephalitis. Two membrane envelopment processes, one at the inner nuclear membrane and a second at cytoplasmic membranes are crucial for a productive viral infection. Depending on the subfamily, herpesviruses encode more than 11 different transmembrane proteins including members of the tail-anchored protein family. HSV1 encodes three tail-anchored proteins pUL34, pUL56 and pUS9 characterized by a single hydrophobic region positioned at their C-terminal end that needs to be released from the ribosome prior to posttranslational membrane insertion. Asna1/TRC40 is an ATPase that targets tail-anchored proteins to the endoplasmic reticulum in a receptor-dependent manner. Cell biological data point to a critical and general role of Asna1/TRC40 in tail-anchored protein biogenesis. With this study, we aimed to determine the importance of the tail-anchored insertion machinery for HSV1 infection.
  
  
  METHODS
  To determine protein-protein interactions, the yeast-two hybrid system was applied. Asna1/TRC40 was depleted using RNA interference. Transient transfection and virus infection experiments followed by indirect immunofluorescence analysis were applied to analyse the localization of viral proteins as well as the impact of Asna1/TRC40 depletion on virus infection.
  
  
  RESULTS
  All HSV1 tail-anchored proteins specifically bound to Asna1/TRC40 but independently localized to their target membranes. While non-essential for cell viability, Asna1/TRC40 is required for efficient HSV1 replication. We show that early events of the replication cycle like virion entry and overall viral gene expression were unaffected by depletion of Asna1/TRC40. Furthermore, equal amounts of infectious virions were formed and remained cell-associated. This indicated that both nuclear egress of capsids that requires the essential tail-anchored protein pUL34, and secondary envelopment to form infectious virions were successfully completed. Despite large part of the virus life cycle proceeding normally, viral propagation was more than 10 fold reduced. We show that depletion of Asna1/TRC40 specifically affected a step late in infection during release of infectious virions to the extracellular milieu.
  
  
  CONCLUSIONS
  Asna1/TRC40 is required at a late step of herpesviral infection for efficient release of mature virions to the extracellular milieu. This study reveals novel tools to decipher exocytosis of newly formed virions as well as hitherto unknown cellular targets for antiviral therapy.
authors.affiliation: Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, 80336, München, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Ott
authors.affiliation: Institute for Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstrasse 12, 70569, Stuttgart, Germany.
authors.forename: Débora
authors.initials: D
authors.lastname: Marques
authors.affiliation: Institute for Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstrasse 12, 70569, Stuttgart, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Funk
authors.affiliation: Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, 80336, München, Germany. Susanne.Bailer@igvp.uni-stuttgart.de.|Institute for Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Nobelstrasse 12, 70569, Stuttgart, Germany. Susanne.Bailer@igvp.uni-stuttgart.de.
authors.forename: Susanne M
authors.initials: SM
authors.lastname: Bailer
mesh_terms: D000818:Animals; D053501:Arsenite Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D055785:Gene Knockdown Techniques; D018259:Herpesvirus 1, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D008856:Microscopy, Fluorescence; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D057074:Virus Release
chemical_list: C489667:GET3 protein, human; D053501:Arsenite Transporting ATPases
keywords: Asna1/TRC40; Exocytosis; HSV1; Herpesvirus; Integral membrane proteins; Nuclear egress; Tail-anchored protein biogenesis; pUL34; pUL56; pUS9