Biology subjects

Hirano, J.

Publications and source records attributed to Hirano, J..

8 recordsLinked to original sources

bioRxiv2026

A versatile and stable reporter platform for human rhinovirus and other enteroviruses

Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.

microbiology↗

bioRxiv2026

RNA virus discovery in Australian camelids reveals divergent picornaviruses and the convergent evolution of upstream ORFs

Invasive species can impact viral ecology, evolution and emergence by acquiring and disseminating viruses absent from native hosts. However, the extent to which invasive species harbour previously unrecognized RNA viruses and transmit these to native species is uncertain. We performed metatranscriptomic sequencing of invasive camelids in Australia and identified several previously undescribed vertebrate-associated RNA viruses, including an astrovirus closely related to avian-associated viruses suggesting a recent host jump. We also identified highly divergent picornaviruses that differed sufficiently from recognized taxa in genome organization and polyprotein phylogeny to establish a new genus. Notably, one virus encoded a putative upstream ORF (uORF) in the 5' genomic region. Across the Picornaviridae, putative uORF gain and loss appear to have occurred multiple times independently. In addition, although most of these uORF-encoded proteins exhibited little to no amino acid sequence homology, a subset showed overlapping ranges of secondary structure composition and intrinsic disorder, and when heterologously expressed, these proteins were translated and triggered reproducible transcriptional responses in a cell line. While no single pathway was uniformly affected across all uORFs, distinct uORFs from divergent lineages consistently perturbed overlapping sets of cellular pathways, supporting broadly analogous functional effects despite a lack of sequence homology. These findings demonstrate that uORFs represent a recurrent and selectable functional module within RNA virus genomes, suggest that the upstream genomic position itself constitutes a "hotspot" for the repeated acquisition of a functional module, and provide experimental evidence that their functional properties have converged across evolutionarily independent lineages. Author SummaryRNA viruses evolve under strong genomic constraints, forcing them to repeatedly adopt similar solutions to common challenges posed by their host environment. Invasive species can impact viral ecology, evolution and emergence by acquiring and disseminating non-native viruses. By characterizing RNA viruses infecting invasive camelids in Australia, we discovered previously unrecognized RNA viruses and recurrent patterns of genome evolution. Notably, we identified a small additional gene located at the 5' genomic region, known as an upstream open reading frame (uORF). uORFs were independently acquired and lost across multiple viral lineages, revealing that the same genomic region can be repeatedly exploited to acquire new functions. Although uORF-encoded proteins share little or no amino acid sequence homology, some of the encoded proteins showed comparable secondary structure composition and induce overlapping host cellular responses when expressed in cells. Hence, RNA viruses that have followed different evolutionary paths can converge on similar functional strategies.

microbiology↗

bioRxiv2025

Establishment of a green fluorescent protein (GFP)-based reporter for picornaviral 3C proteases

Picornaviruses represent a diverse group of plus-stranded RNA viruses, many of which have been linked to severe diseases in both humans and animals. The viral 3C protease is essential for the maturation of viral proteins and the propagation of picornaviruses and, owing to its cleavage activity against multiple host proteins, is associated with the pathogenesis of picornaviruses. The picornaviral 3C protease is an ideal drug target for inhibiting viral propagation and mitigating pathogenesis; however, methodology to evaluate and compare the activity of phylogenetically diverse proteases remains lacking. To address this, herein, we propose a novel green fluorescent protein (GFP)-based reporter optimized to visualize the enzymatic activity of picornaviral 3C proteases in cells by using the conformational change of a GFP variant induced by the 3C protease, generating fluorescence emission linked to the enzymatic activity. Upon treatment of picornaviruses with a known 3C protease inhibitor, the fluorescence decreased in a dose-dependent manner, demonstrating that the signal depended on the activity of the 3C protease. The reporter system for the 3C protease can be applied to major pathogenic human picornaviruses, such as those in the genera Enterovirus, Rhinovirus, Cosavirus, Salivirus, and Kobuvirus. Furthermore, the fluorescent signal from the reporter was confirmed in various animal- derived picornaviruses, such as those from bats, rodents, and primates. Therefore, the reporter could be widely used to analyze the activity of several 3C proteases from currently prevalent picornaviruses and those that may emerge in the future. To demonstrate the flexibility of the reporter in comparing phylogenetically different proteases, the enzymatic activity of the 3C protease derived from clinical strains of enterovirus A71 (EV-A71) was tested and compared. The results showed that the amino acid residues of the 3C protease affect its activity by utilizing the reporter system. Additionally, clinical EV- A71 strains had different effects on the activity of 3C protease against host proteins. Our findings will aid in elucidating the molecular characteristics of 3C proteases among picornaviruses and developing therapeutics to mitigate the pathogenesis of these viruses.

cell biology↗

bioRxiv2025

Evaluation of anti-noroviral compounds on human norovirus replication using 2D monolayers of human intestinal organoids

We evaluated the anti-noroviral effects of compounds including molnupiravir, on human norovirus (HuNoV) replication using 2D monolayers of human intestinal organoids (HIOs), which have been widely used in HuNoV infection studies. We found that these compounds exhibited lower antiviral activity in 2D HIO monolayers compared with that in apical-out 3D HIOs, a recently developed culture system for HuNoV. This suggests that the anti-noroviral efficacy of these compounds may vary depending on the assay system used.

microbiology↗

bioRxiv2023

Reconstructing visual illusory experiences from human brain activity

Visual illusions provide significant insights into the brains interpretation of the world given sensory inputs. However, the precise manner in which brain activity translates into illusory experiences remains largely unknown. Here we leverage a brain decoding technique combined with deep neural network (DNN) representations to reconstruct illusory percepts as images from brain activity. The reconstruction model was trained on natural images to establish a link between brain activity and perceptual features and then tested on two types of illusions: illusory lines and neon color spreading. Reconstructions revealed lines and colors consistent with illusory experiences, which varied across the source visual cortical areas. This framework offers a way to materialize subjective experiences, shedding new light on the brains internal representations of the world.

neuroscience↗

bioRxiv2021

Single chain models illustrate the 3D RNA folding shape during translation

The three-dimensional conformation of RNA is important in the function and fate of the molecule. The common conformation of mRNA is formed based on the closed-loop structure and internal base pairings with the activity of the ribosome movements. However, recent reports suggest that the closed-loop structure might not be formed in many mRNAs. This implies that mRNA can be considered as a single polymer in the cell. We developed TRIP; Three-dimensional RNA Illustration Program, to model the three-dimensional RNA folding shape based on single-chain models. We identified the angle restriction of each bead component from previously reported single-molecule FISH experimental data. This simulation method was able to recapitulate the mRNA conformation change of the translation activity and three-dimensional positional interaction between organelle and its localized mRNAs. Within the analyzed cases base-pairing interactions only have minor effects on the three-dimensional mRNA conformation, and instead single-chain polymer characteristics have a more significant impact on the conformation. This method will be used to predict the aggregation mechanism of mRNA and long noncoding RNA in specific cellular conditions such as nucleolus and phase-separated granules.

biophysics↗

bioRxiv2021

Dasabuvir inhibits human norovirus infection in human intestinal enteroids

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, with a human intestinal enteroid (HIE) culture system where HuNoVs are able to replicate reproducibly, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity. Dasabuvir, which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human A rotavirus (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation.

microbiology↗

bioRxiv2018

USP15 participates in HCV propagation through the regulation of viral RNA translation and lipid droplet formation

Hepatitis C virus (HCV) utilizes cellular factors for an efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of de-ubiquitinating enzymes (DUBs) or overexpression of non-specific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up an RNAi screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR122 cells) but not in a non-hepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo. We also found that USP15-deficient Huh7 cells showed reductions in the sizes and numbers of lipid droplets (LDs), and addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and formation of LDs.

microbiology↗