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.
Highlights
Adenosine 59-triphosphate (ATP) is the major energy currency of cells and is involved in a variety of cellular processes, including the virus life cycle, in which ATP-dependent reactions essential for virus multiplication are catalyzed by viral-encoded enzymes or complexes consisting of viral and host-cell proteins [1]
ATeam, which was established in 2009, is a genetically-encoded Forster resonance energy transfer (FRET)-based indicator for ATP that is composed of a small bacterial protein that binds ATP sandwiched between two fluorescent proteins
By applying ATeam to the subgenomic replicon system, we have developed a method to monitor ATP at putative subcellular sites of RNA replication of the hepatitis C virus (HCV), a major human pathogen associated with liver disease, in living cells
Summary
Adenosine 59-triphosphate (ATP) is the major energy currency of cells and is involved in a variety of cellular processes, including the virus life cycle, in which ATP-dependent reactions essential for virus multiplication are catalyzed by viral-encoded enzymes or complexes consisting of viral and host-cell proteins [1]. The lack of a real-time monitoring system for ATP has hindered studies aimed at elucidating the mechanisms by which cellular processes are controlled through ATP. A method for measuring ATP levels in individual living cells has recently been developed using a genetically-encoded FRET-based indicator for ATP, called ATeam, which employs the epsilon subunit of a bacterial F0F1ATPase [2]. ATeam has enabled researchers to examine the subcellular compartmentation of ATP as well as time-dependent changes in cellular ATP levels under various physiological conditions. The ATeambased method has been used to demonstrate that ATP levels within the mitochondrial matrix are lower than those in the cytoplasm and the nucleus [2]
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