Effect of cell growth on hepatitis C virus (HCV) replication and a mechanism of cell confluence-based inhibition of HCV RNA and protein expression.
An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.
Nelson
Heather B
HB
Bio Unit I, Chieftan Way, Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.
Tang
Hengli
H
D002452:Cell Count; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015739:Nocodazole / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D049452:Green Fluorescent Proteins; D015739:Nocodazole; D009711:Nucleotides; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase