Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

PLoS Pathogens

Tomomi Ando¹  Hideki Aizaki¹  Toshiki Watanabe¹  Tetsuro Suzuki¹  Takaji Wakita²  Hiromi Imamura²  Ryosuke Suzuki³ 

[1] Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan;Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan;The Hakubi Center and Graduate School of Biostudies, Kyoto University, Kyoto, Japan

关键词: Viral replication;    Cytoplasm;    Fluorescence resonance energy transfer;    Hepatitis C virus;    DNA replication;    RNA synthesis;    Fluorescence microscopy;    Mitochondria;   

DOI  :  10.1371/journal.ppat.1002561

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY