Temporal dissection of hepatitis C virus and orthoflavivirus infections by continuous luminescence monitoring.

Virus infection is a dynamic process consisting of rapidly evolving viral and host events, yet conventional endpoint assays provide only fragmented snapshots of these processes. Here, we established a unified temporal framework for dissecting virus-host interactions by integrating continuous bioluminescence monitoring with molecular and functional analyses. Real-time monitoring of hepatitis C virus (HCV) single-round infectious particles (SRIPs) revealed a biphasic replication profile consisting of an initial transient phase, followed by replication-dependent amplification. This transition was independently validated by similar HCV RNA kinetics observed after infection with HCV SRIPs or fully infectious HCV. Extension of this approach to dengue, Zika, and yellow fever virus SRIPs demonstrated that infection kinetics are determined by virus-specific replication programs and are modulated by the combination of viral replicons/capsids and prM/E proteins. This temporal framework enabled the identification of virus-host interactions that are difficult to resolve by conventional endpoint analyses. Early-phase cytoplasmic proteomics, combined with functional screening, identified the dynein light chain component DYNLRB2 as a previously unrecognized negative regulator of early HCV infection. Continuous monitoring also distinguished phase-specific antiviral activities, revealing distinct kinetic inhibition profiles for daclatasvir, acetylsalicylic acid, and interferon-α. Furthermore, delayed activation of the ER stress sensor ATF6 was first detected 40-47 h after HCV infection, and transcriptomic analysis at reporter-defined time points characterized the accompanying transition in host gene expression. Together, our study establishes continuous temporal monitoring as a general strategy for resolving dynamic virus-host interactions and for uncovering molecular events that remain obscured by conventional endpoint analyses.IMPORTANCEViral infection is a dynamic process in which entry, genome amplification, host-factor requirements, antiviral effects, and cellular responses occur on different timescales. Conventional endpoint assays can identify changes in these processes but provide limited information about when they emerge or transition from one stage to another. Here, we show that continuous monitoring, when combined with direct molecular and functional measurements, can place these otherwise separate observations within a common temporal framework. Using HCV and related orthoflavivirus SRIPs, we resolved early and replication-associated phases, identified host factors with stage-dependent effects, and distinguished profiles of antiviral inhibition. We further showed that ATF6-mediated ER stress activation emerged later than early HCV infection and viral RNA amplification. Thus, continuous temporal monitoring provides a general strategy for resolving dynamic virus-host interactions by defining the temporal relationships among viral processes, pharmacological effects, and host responses that remain obscured by conventional endpoint analyses.