title: Quantitative proteomic analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 (GBF1) in dengue infection.
abstract: Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.
authors.affiliation: From the ‡Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109;
authors.forename: Lindsay N
authors.initials: LN
authors.lastname: Carpp
authors.affiliation: ‖Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Rogers
authors.affiliation: §Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Moritz
authors.affiliation: From the ‡Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109; §Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109, ‖Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109. john.aitchison@seattlebiomed.org.
authors.forename: John D
authors.initials: JD
authors.lastname: Aitchison
mesh_terms: D000330:Aedes; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D030562:Databases, Protein; D003715:Dengue / Q000235:genetics / Q000473:pathology / Q000821:virology; D003716:Dengue Virus / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D050958:HSP47 Heat-Shock Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism; D060066:Protein Interaction Maps; D020365:RNA Helicases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D050958:HSP47 Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D020365:RNA Helicases; D034741:RNA, Small Interfering; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C119087:ARFGEF1 protein, human; C120445:GBF1 protein, human; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C582223:NS5 protein, dengue virus; C494660:SERPINH1 protein, human; C488261:ZW10 protein, human; C019995:7-dehydrocholesterol reductase