Abstract
Hepatitis C virus (HCV) entry, translation, replication, and assembly occur with defined kinetics in distinct subcellular compartments. It is unclear how HCV spatially and temporally regulates these events within the host cell to coordinate its infection. We have developed a single molecule RNA detection assay that facilitates the simultaneous visualization of HCV (+) and (−) RNA strands at the single cell level using high-resolution confocal microscopy. We detect (+) strand RNAs as early as 2 hours post-infection and (−) strand RNAs as early as 4 hours post-infection. Single cell levels of (+) and (−) RNA vary considerably with an average (+):(−) RNA ratio of 10 and a range from 1–35. We next developed microscopic assays to identify HCV (+) and (−) RNAs associated with actively translating ribosomes, replication, virion assembly and intracellular virions. (+) RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a shift to replication and then virion assembly. (−) RNAs have a strong colocalization with NS5A, but not NS3, at early time points that correlate with replication compartment formation. At later times, only ~30% of the replication complexes appear to be active at a given time, as defined by (−) strand colocalization with either (+) RNA, NS3, or NS5A. While both (+) and (−) RNAs colocalize with the viral proteins NS3 and NS5A, only the plus strand preferentially colocalizes with the viral envelope E2 protein. These results suggest a defined spatiotemporal regulation of HCV infection with highly varied replication efficiencies at the single cell level. This approach can be applicable to all plus strand RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle.
Highlights
Hepatitis C virus (HCV) belongs to the Flaviviridae family of enveloped, positive-stranded RNA viruses
We used RNA in situ hybridization (ISH) to simultaneously detect HCV (+) and (−) RNAs and analyze the kinetics of HCV infection at the single cell level as well as visualize HCV RNAs associated with actively translating ribosomes, markers of viral replication compartment formation, active RNA replication, nucleocapsid assembly, and intracellular virions
Analysis of HCV (−) RNAs revealed that low levels of transient RNA replication occur early after infection prior to the formation of devoted replication compartments and robust replication
Summary
Hepatitis C virus (HCV) belongs to the Flaviviridae family of enveloped, positive-stranded RNA viruses. Synthesized (+) strand RNAs are thought to be shuttled out of replication compartments to serve as templates for further translation by cellular ribosomes or become encapsidated into assembling virions on the surface of lipid droplets (LDs) [12]. These processes are likely linked, a single viral (+) strand RNA can only be involved in either translation, replication or packaging at a given time, and the switch from one process to another has to be regulated [13]. It is possible that HCV (+) RNA fate is spatially regulated by the distinct subcellular localizations of translation, replication and assembly
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