Masahiko Ito

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. IMPORTANCE: Viral 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.

Malignant gliomas are common primary brain tumors. Glioblastoma carries a poor prognosis, with median survival of less than 2 years. Given the urgent need for novel therapies, oncolytic virus (OV) therapy has developed rapidly. However, challenges remain with effective OV delivery to tumor sites and efficient infection of tumor cells. This study investigated the use of stem cells from human exfoliated deciduous teeth (SHED) as a potential vehicle for oncolytic herpes simplex virus-1 therapy (SHED-OV) to enhance treatment efficacy. SHED-OV showed a greater reduction in tumor cell viability in vitro than direct OV infection. Migration assays revealed that SHED-OV actively migrated toward glioma cell-conditioned medium, demonstrating their tumor-homing ability. In vivo experiments demonstrated significantly prolonged survival in both the glioma cell/SHED-OV co-implantation group and the OV-infected glioma cell group compared with controls. In a glioma stem cell model, both SHED-OV and OV alone significantly improved survival compared with controls, with SHED-OV achieving anti-tumor effects comparable to those of direct OV administration and complete tumor regression in a subset of mice. SHED-OV also exhibited tumor-homing ability. These results suggest that SHED could serve as an effective vehicle for OV delivery, potentially improving tumor infection and therapeutic efficacy.

Hepatitis C virus (HCV) exploits lipid droplets (LDs) during the production of infectious particles. Although several host factors, including stress granule (SG) proteins, have been reported to localize to LDs in HCV-infected cells, the mechanism by which these proteins are recruited to LDs has remained unclear. In this study, we show that Ras-GAP SH3 domain-binding protein 1 (G3BP1), an essential SG component, is recruited to LDs with a time lag of several hours or more after the HCV Core protein accumulates on LDs following infection, and that G3BP1 retains its ability to assemble SGs even when localized to LDs during the late phase of infection. Treatment of HCV-infected cells with compounds that disrupt liquid–liquid phase separation (LLPS) abolished the LD localization of G3BP1, as well as another SG protein, T-cell intracellular antigen-1, whereas the LD localization of the viral proteins Core and NS5A was not affected. Finally, we demonstrate that ectopic expression of HCV Core alone is insufficient to recruit G3BP1 to LDs; however, co-expression of Core together with subgenomic HCV RNA, even a replication-defective replicon RNA, leads to the accumulation of G3BP1 around LDs. Collectively, these findings suggest that during the HCV life cycle, the initial localization of Core to LDs, followed by the recruitment of newly synthesized viral RNA to the LD surface, drives LLPS-mediated assembly of biomolecular condensates containing G3BP1 and other host SG proteins around LDs. IMPORTANCE: During the late stages of hepatitis C virus (HCV) infection, various viral and host proteins are known to accumulate around lipid droplets (LDs), where virion assembly is thought to occur. However, it has remained unclear how the expression of a limited subset of viral factors can induce such an extensive cellular reorganization. Here, we identify that HCV infection induces the relocalization of the stress granule (SG)-associated proteins G3BP1 and TIA-1 onto LDs through liquid–liquid phase separation (LLPS). We further demonstrate that the minimal requirement for G3BP1 recruitment to LDs is the coexistence of the HCV Core and the viral RNA. Because G3BP1 plays a central role in the interaction network underlying SG formation and is capable of mobilizing additional SG components, our findings suggest that LLPS-dependent assembly of biomolecular condensates occurs around LDs during HCV infection. This work provides mechanistic insight into HCV particle formation, including viral genome packaging, and offers a conceptual basis for developing future antiviral strategies.

Background: Abnormalities in energy and amino acid metabolism are potentially involved in hepatocellular carcinoma (HCC) development. This study aimed to identify serum metabolites predictive of HCC following sustained virological response (SVR) with hepatitis C virus (HCV) treatment. Methods: Comparative metabolomics was conducted using time-course serum samples from patients who failed interferon-based therapy but subsequently achieved SVR with direct-acting antivirals (DAAs), minimizing inter-individual variability. Predictive biomarkers for post-SVR HCC were extracted from the results and validated by comparing 29 patients who developed post-SVR HCC with 58 age-matched patients who remained HCC-free during follow-up. Results: Metabolite concentrations changed more markedly after treatment in SVR cases than in non-SVR cases. Significant changes in methionine (Met), methionine sulfoxide (MetO), and ornithine (Orn) levels before and after treatment (Pre- and Post-Tx) were found only in the non-HCC group. Regression and survival analyses identified high levels of Pre- and Post-Tx Orn, Pre-Tx Met, and Post-Tx MetO as predictors of post-SVR HCC and enabled risk stratification. The integration of these metabolites with the fibrosis-4 (FIB-4) index and alpha-fetoprotein (AFP) facilitated risk stratification and discriminated between high- and low-risk patients. The Pre-Tx FIB-4/Met model and the Post-Tx AFP/MetO/Orn model identified low- and high-risk groups with 3-year HCC incidence rates of 6.4% and 81.8%, respectively. Conclusions: Serum Met, MetO, and Orn were identified as candidate biomarkers associated with post-SVR HCC development, which remains a concern in the fight against hepatitis C. Combining these metabolites with established clinical markers may improve post-SVR HCC risk stratification.

Persistent hepatitis B surface antigen (HBsAg) production from both covalently closed circular DNA (cccDNA) and integrated HBV DNA (iDNA) remains a major barrier to a functional cure of chronic hepatitis B. Current antiviral therapies, including nucleos(t)ide analogues and pegylated interferon-a, do not effectively eliminate HBsAg derived from both cccDNA and iDNA. Thus, novel strategies targeting HBsAg irrespective of its source are needed. Here, we screened a library of 38 poly(ADP-ribose) polymerase (PARP) inhibitors using Hep38.7-Tet cells in the absence of tetracycline and identified Pamiparib as a compound that reduces extracellular HBsAg levels across multiple HBV models. Pamiparib consistently reduced extracellular HBsAg levels, including iDNA (PLC/PRF/5 cells) and de novo infection models (HepG2-NTCP-Myc-His6 and PXB cells), and retained activity against HBV genotypes A-D. In contrast, Pamiparib did not measurably alter intracellular cccDNA levels, preS1 and preS2/S promoter activities, or intracellular HBV RNA levels, but increased intracellular HBsAg accumulation, supporting an effect on HBsAg release rather than viral transcription. Importantly, Pamiparib also reduced the release of infectious HBV particles, while showing little effect on HBeAg secretion or HBsAg release from cells expressing HBs proteins alone, consistent with selective interference with the multivesicular body-associated egress pathway. In silico and co-immunoprecipitation analyses further suggest that Pamiparib interferes with the interaction between the LHBs and the host trafficking factor g2-Adaptin. Taken together, these findings identify Pamiparib as a host-targeting leading compound that suppresses HBsAg secretion from both cccDNA- and iDNA-derived sources and may support future combination strategies aimed at functional cure of chronic hepatitis B.

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up‐regulation of transforming growth factor β (TGF‐β), especially TGF‐β2, which is mediated by activation of liver‐enriched transcription factor cAMP‐responsive element‐binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV‐infected cells with hepatic stellate cells (HSCs). Compared to TGF‐β1, expression of TGF‐β2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF‐β2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0‐F2. TGF‐β2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF‐β2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core‐NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV‐infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV‐induced fibrogenic response was further demonstrated in the CREBH null‐mutant mouse model. Fibrogenic phenotypes were assessed using co‐cultures of HCV‐infected cells and HSCs. Expressions of fibrogenic factors and TGF‐β1 increasing in the co‐cultures was prevented by TGF‐β2‐ or CREBH silencing. Conclusion: CREBH was identified as a key positive regulator of TGF‐β2 transcription in HCV‐infected cells. TGF‐β2 released from infected cells potentially contributes to cross‐induction of TGF‐β in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430–1443).