title: Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.
abstract: Adenosine 5'-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Ando
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Suzuki
authors.forename: Hideki
authors.initials: H
authors.lastname: Aizaki
authors.forename: Toshiki
authors.initials: T
authors.lastname: Watanabe
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003593:Cytoplasm / Q000821:virology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D008113:Liver Neoplasms; D012367:RNA, Viral / Q000096:biosynthesis*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D012367:RNA, Viral; D000255:Adenosine Triphosphate