title: Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important cofactors that promote L-protein stability and RNA synthesis.
abstract: The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication.
  
  
  OBJECTIVE
  Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Diane C
authors.initials: DC
authors.lastname: Munday
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Weining
authors.initials: W
authors.lastname: Wu
authors.affiliation: Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Nikki
authors.initials: N
authors.lastname: Smith
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, Jouy-en-Josas, France.
authors.forename: Jenna
authors.initials: J
authors.lastname: Fix
authors.affiliation: Boston University, Boston, Massachusetts, USA.
authors.forename: Sarah Louise
authors.initials: SL
authors.lastname: Noton
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, Jouy-en-Josas, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Galloux
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Olivier
authors.initials: O
authors.lastname: Touzelet
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Armstrong
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Jenna M
authors.initials: JM
authors.lastname: Dawson
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Waleed
authors.initials: W
authors.lastname: Aljabr
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Easton
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, Jouy-en-Josas, France.
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Rameix-Welti
authors.affiliation: University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Andressa Peres
authors.initials: AP
authors.lastname: de Oliveira
authors.affiliation: Faculty of Applied Sciences, University of Campinas, Limeira, Brazil.
authors.forename: Fernando M
authors.initials: FM
authors.lastname: Simabuco
authors.affiliation: University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Armando M
authors.initials: AM
authors.lastname: Ventura
authors.affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Hughes
authors.affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: John N
authors.initials: JN
authors.lastname: Barr
authors.affiliation: Boston University, Boston, Massachusetts, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Fearns
authors.affiliation: Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Digard
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, Jouy-en-Josas, France jean-francois.eleouet@jouy.Inra.fr julian.hiscox@liverpool.ac.uk.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom jean-francois.eleouet@jouy.Inra.fr julian.hiscox@liverpool.ac.uk.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Hiscox
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D055550:Protein Stability; D018113:Respiratory Syncytial Virus, Human / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases