Biology subjects

Matsuura, Y.

Publications and source records attributed to Matsuura, Y..

At least 19 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↗

bioRxiv2026

Machine learning-based framework for predicting human infection potential of coronavirus associated with tri-amino acid motifs, KIQ and LEP in spike protein

Assessing the human infection potential of emerging coronaviruses remains a critical challenge for global health preparedness. In this study, we developed a machine learning-based framework to predict the human infection potential of coronaviruses and to identify associated sequence motifs using spike (S) protein sequences. A total of 3,904 complete S protein sequences were collected, annotated as human or non-human infection and encoded using trimer-based k-mer features. Model benchmarking was conducted across 27 machine learning algorithms, followed by hyperparameter optimization of the selected model. Robustness and generalizability were evaluated using k-fold cross-validation and independent external validation. Feature interpretability was further assessed using SHAP analysis to identify sequence determinants associated with infection potential. The Random Forest classifier achieved the best performance, with accuracy, sensitivity, and specificity of 97.8%, 99%, and 97.4%, respectively, and demonstrated stable predictive performance across validation datasets. Notably, the KIQ and LEP motifs were strongly associated with human infection coronaviruses and mapped to the HR1 and N-terminal domain regions of the S protein. Overall, this framework provides a practical approach for risk assessment and surveillance of emerging coronaviruses. Author summaryEmerging coronaviruses continue to threaten global public health, but rapidly identifying viruses with the potential to infect humans remains challenging. Traditional experimental approaches are time-consuming and resource-intensive, limiting their use for large-scale surveillance. In this study, we developed a machine learning based workflow to assess the human infection potential of coronaviruses using spike protein sequences. By analyzing sequence patterns across a diverse set of coronaviruses, our framework enables rapid screening of coronaviruses from multiple host species. Unlike previous studies focused on limited coronavirus genera, our approach integrates all four genera and systematically evaluates multiple learning strategies. Importantly, our analysis identifies conserved sequence motifs linked to human infection potential, bridging predictive performance with biological interpretability. Our findings demonstrate computational approaches support early warning systems for identifying high risk coronaviruses, contributing to prioritize viruses for experimental validation, guide surveillance efforts, and strengthen global pandemic preparedness under a One Health perspective.

bioinformatics↗

bioRxiv2025

Generation of a HiBiT-expressing recombinant rat hepacivirus supporting both in vivo and in vitro infection

The lack of immuno-competent animal models of hepatitis C virus (HCV) infection has been an obstacle to vaccine development and research on immune responses. Hepacivirus ratti (Norway rat hepacivirus-1: NRHV1) is a virus closely related to HCV that specifically infects the liver and induces hepatocellular carcinoma in rats, making it a promising surrogate model for HCV. NRHV1 expressing a reporter gene serves as a powerful tool for analyzing the in vivo dynamics and pathogenicity mechanisms of NRHV1. In this study, we developed a reporter NRHV1 capable of infection and replication in both mice and cultured cells and established a platform for generating diverse reporter viruses. A reporter virus containing the HiBiT gene in the coding region of NS5A domain III was constructed using circular polymerase extension reaction (CPER). Infection with this reporter virus led to HiBiT activity in infected cells and the activity was correlated with the amount of intracellular viral RNA. In addition, this reporter virus established persistent infection in NOD-SCID mice and led to the generation of HiBiT activity in the livers of infected mice. Furthermore, reporter virus recovered from infected mice could infect and generate HiBiT activity in cultured cells. These results demonstrate that the reporter virus can infect hepatocytes in vivo and in vitro. This platform provides a versatile tool for in vitro quantitative antiviral screening and in vivo pathogenicity studies. Author summaryHepatitis C virus (HCV) infects millions of people and causes severe liver diseases, yet vaccine development has been hindered by the lack of practical small-animal models with intact immunity. Hepacivirus ratti (NRHV1), a close relative of HCV that naturally infects rats, is a promising HCV surrogate, but tools for monitoring NRHV1 infection dynamics have been limited. Here, we developed the first reporter NRHV1 by inserting a small luminescent HiBiT tag into a tolerable region of the NS5A protein. Using a CPER-based reverse genetics system, we generated a recombinant virus that efficiently infected cultured cells and established persistent infection in mice while retaining liver tropism. HiBiT activity closely reflected viral RNA levels, enabling the quantitative monitoring of infection and sensitive evaluation of antiviral drugs and neutralizing antibodies. This reporter NRHV1 system provides a versatile platform for studying hepacivirus pathogenesis and antiviral immunity and will support future efforts toward HCV vaccine development.

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

Identification of claudin-3 as an entry factor for rat hepacivirus

Approximately 58 million people worldwide are believed to be infected with hepatitis C virus (HCV), a major causative agent of chronic liver diseases. Hepacivirus ratti strain rn-1, which was discovered from Rattus norvegicus (Norway rat) and designated Norway rat hepacivirus 1 (NRHV1), shares similar properties with HCV in terms of genetic homology, target cell tropism, pathogenicity and the immune response. In vivo infection systems for NRHV1 will help overcome the challenges in HCV research for vaccine development. However, the virological characteristics of NRHV1, such as the mechanisms of cell entry, remain largely unexplored, in part owing to a paucity of cell culture systems for NRHV1. Here, we identified the host factors that facilitate NRHV1 entry by profiling the gene expression of two cell lines with different susceptibilities to NRHV1 infection. NRHV1 employs rodent orthologues of HCV entry factors, including scavenger receptor class BI, CD81, and occludin, and utilizes claudin-3 (CLDN3) but not claudin-1. The expression of rat and mouse, but not human, CLDN3 facilitates the entry of NRHV1 into murine cell lines that are insusceptible to NRHV1 infection. The host-specific cell entry of CLDN3 is determined by two amino acid residues, Ile44 and Trp46, in extracellular loop 1. These findings suggest that some hepacivirus species share an HCV-like entry mechanism but show evidence of adaptive evolution of their virus-specific entry factors to host animals.

microbiology↗

bioRxiv2025

The facultative intracellular symbiont Lariskella is neutral for lifetime fitness and spreads through cytoplasmic incompatibility in the leaffooted bug, Leptoglossus zonatus

The maternally-inherited, intracellular bacterium Lariskella (Alphaproteobacteria: Midichloreaceae) has been widely detected in arthropods including true bugs, beetles, a wasp, a moth, and pathogen-vectoring fleas and ticks. Despite its prevalence, its role in the biology of its hosts has been unknown. We set out to determine the role of this symbiont in the leaffooted bug, Leptoglossus zonatus (Hempitera: Coreidae). To examine the effects of Lariskella on bug performance and reproduction as well as in possible interactions with the bugs obligate nutritional symbiont, Caballeronia, bugs were reared in a factorial experiment with both Lariskella and Caballeronia positive and negative treatments. Lifetime survival analysis ([~]120 days) showed significant developmental delays and decrease in survival for bugs that lacked Caballeronia, and Caballeronia-free bugs did not reproduce. However, among the Caballeronia carrying treatments, there were no significant differences in lifetime survival or reproduction in treatments with and without Lariskella, suggesting this symbiont is neutral for overall bug fitness. To test for reproductive manipulation, crossing among Lariskella-positive and negative individuals was performed. When Lariskella-negative females were mated with Lariskella positive males, fewer eggs survived early embryogenesis, consistent with a cytoplasmic incompatibility (CI) phenotype. Wild L. zonatus from California and Arizona showed high but not fixed Lariskella infection rates. Within individuals, Lariskella titer was low during early development (1st-3rd instar), followed by an increase that coincided with development of reproductive tissues. Our results reveal Lariskella to be among a growing number of microbial symbionts that cause CI, a phenotype that increases the relative fitness of females harboring the symbiont. Understanding the mechanism of how Lariskella manipulates reproduction can provide insights into the evolution of reproductive manipulators and may eventually provide tools for management of hosts of Lariskella, including pathogen-vectoring ticks and fleas. Author SummaryArthropods are a highly diverse group of animals with ancient relationships with bacteria. Some maternally-inherited, intracellular bacterial symbionts have evolved strategies to manipulate arthropod host reproduction, ensuring their own spread and persistence. One such strategy is cytoplasmic incompatibility (CI), where symbionts in males prevent viable offspring when these males mate with uninfected females. However, reproduction is rescued in crosses with both mates infected, promoting bacterial transmission within host populations. In the leaffooted bug Leptoglossus zonatus, crossing experiments revealed a significant reduction in egg survival rates when Lariskella-negative females mated with Lariskella-positive males, a typical CI phenotype discovered anew for this symbiont. Additional experiments included measuring performance and fitness of bugs with combinations of Lariskella and Caballeronia (the primary nutritional symbiont of L. zonatus). While Caballeronia-negative treatments caused developmental delays and mortality, the presence of Lariskella had no effect on development or reproduction. Lariskella has been frequently found in pathogen-vectoring arthropods, but its specific role remained unclear. Understanding the role that Lariskella plays in L. zonatus can potentially help us find future pest control measures against blood-sucking pathogen-vectors by exploiting novel CI mechanism to suppress host populations and hence the transmission of human diseases.

ecology↗

bioRxiv2024

BCR, not TCR, repertoire diversity is associated with favorable COVID-19 prognosis

The SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients. In this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages. The study included three unvaccinated patients from each outcome category. Although expanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, they represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased. This study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.

immunology↗

bioRxiv2024

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (PRC1) (J. Virol, 2021, 95, e01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28{gamma}-binding protein. The proteasome activator PA28{gamma} destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28{gamma} knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28{gamma}, while PA28{gamma} knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28{gamma} at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28{gamma}-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28{gamma} mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28{gamma} and the lysine acetyltransferase CBP/p300 and then accelerated PA28{gamma} acetylation and heptamerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28{gamma} to increase the proteasomal targeting of RNF2. IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis and hepatocellular carcinoma. PA28{gamma}, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28{gamma}-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28{gamma} acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

microbiology↗

bioRxiv2024

Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in 8 genes, TP53, NOTCH1, KMT2D, CREBBP, KRAS, PTEN, CARD11, and MYD88. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to congenital mutations, high impact mutations affecting protein translation, and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylctosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection. ImportanceEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

cancer biology↗

bioRxiv2024

Augmented-reality-based exercise increases salivary oxytocin

Exercise is known to have positive effects on psychological well-being, with team sports often associated with superior mental health compared to individual sports. Augmented reality (AR) technology has the potential to convert solitary exercise into multi-person exercise. Given the role of oxytocin in mediating the psychological benefits of exercise and sports, this study aimed to investigate the impact of AR-based multi-person exercise on mood and salivary oxytocin levels. Fourteen participants underwent three distinct regimens: non-exercise (Rest), standard solitary cycling exercise (Ex), and AR-based multi-person cycling exercise (Ex+AR). In both exercise conditions (Ex and Ex+AR), participants engaged in cycling at a self-regulated pace to maintain a Rating of Perceived Exertion of 10. In the Ex+AR condition, participants avatars were projected onto a tablet screen, allowing them to cycle alongside ten other virtual avatars in an AR environment. Mood states (assessed using POMS2) and saliva samples were collected before and immediately after each 10-minute regimen. Subsequently, the levels of salivary oxytocin were measured. Participants exhibited higher cycling speeds during Ex+AR compared to Ex, despite comparable levels of self-reported fatigue between the two groups. Notably, only the Ex+AR condition significantly improved mood states associated with depression-dejection and exhibited a trend toward suppressing anger-hostility in participants. Moreover, the Ex+AR condition led to a significant elevation in salivary oxytocin levels, while the Ex condition showed a trend toward an increase. However, changes in salivary oxytocin did not show a significant correlation with changes in mood states. These findings suggest that Ex+AR enhances mood states and promotes oxytocin release. AR-based multi-person exercise may offer greater psychological benefits compared to standard solitary exercise, although the relationship between oxytocin and mood changes remains inconclusive. HighlightsO_LIExercise, especially team sports, has positive effects on mood state, and oxytocin partially supports these effects. C_LIO_LIAugmented reality (AR) technology can potentially convert standard solitary exercise into multi-person exercise. C_LIO_LIAR-based multi-person exercise (Ex+AR) significantly ameliorated depression-dejection and increased salivary oxytocin levels, but standard solitary exercise did not. C_LIO_LIEx+AR may provide more significant psychological benefits than standard solitary exercise. C_LI

physiology↗

bioRxiv2023

Paleocene origin of a streamlined digestive symbiosis in leaf beetles

Timing the acquisition of a beneficial microbe relative to the evolutionary history of its host can shed light on the adaptive impact of a partnership. Here, we investigated the onset and molecular evolution of an obligate symbiosis between Cassidinae leaf beetles and Candidatus Stammera capleta, a {gamma}-proteobacterium. Residing extracellularly within foregut symbiotic organs, Stammera upgrades the digestive physiology of its host by supplementing plant cell wall-degrading enzymes. We observe that Stammera is a shared symbiont across tortoise and hispine beetles that collectively comprise the Cassidinae subfamily, despite differences in their folivorous habits. In contrast to its transcriptional profile during vertical transmission, Stammera elevates the expression of genes encoding digestive enzymes while in the foregut symbiotic organs, matching the nutritional requirements of its host. Symbiont acquisition during the Paleocene ([~]62 Mya) did not coincide with the origin of Cassidinae beetles, despite the widespread distribution of Stammera across the subfamily. Early-diverging lineages lack the symbiont and the specialized organs that house it. Reconstructing the ancestral state of host-beneficial factors revealed that Stammera encoded three digestive enzymes at the onset of symbiosis, including polygalacturonase - a pectinase that is universally shared. While non-symbiotic cassidines encode polygalacturonase endogenously, their repertoire of plant cell wall-degrading enzymes is more limited compared to symbiotic beetles supplemented with digestive enzymes from Stammera. Highlighting the potential impact of a symbiotic condition and an upgraded metabolic potential, Stammera-harboring beetles exploit a greater variety of plants and are more speciose compared to non-symbiotic members of the Cassidinae.

evolutionary biology↗

bioRxiv2023

Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kinetics in vivo using reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualization in vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualized in vivo infection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.

microbiology↗

bioRxiv2023

Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production.

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens such as Japanese encephalitis virus (JEV), dengue virus (DENV), zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear localization in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-{beta} family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear localization. To understand the importance of core protein nuclear localization, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These data suggested that IPO7-mediated nuclear localization of core proteins plays a role in the release of infectious virus particles of flaviviruses.

microbiology↗

bioRxiv2023

NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA

Chronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA (rcDNA) levels in hepatocellular carcinoma. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA levels while knockdown or knockout of the gene encoding N4BP1 rescued rcDNA levels. N4BP1 possesses the KH-like and RNase domains and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and degraded both the 3.5 kb and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by promoting the degradation of 3.5 kb and 2.1/2.4 kb HBV RNA. ImportanceThere is still a large number of HBV-infected people in the world today because of no curative treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related to HCC progression. This novel factor can potentially become a key to new HBV treatments.

microbiology↗

bioRxiv2023

Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus

Lujo virus (LUJV), which belongs to Mammarenavirus, family arenaviridae, has emerged as pathogen causing severe hemorrhage fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drug and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 M in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections. ImportanceTo investigate antiviral drugs against Lujo virus (LUJV), we screened chemical compound libraries to identify effective antivirals against LUJV entry. CB1 antagonists were identified as robust inhibitors of LUJV entry. The cell fusion activity of LUJV GP was suppressed by CB1 inhibitors. Furthermore, CB1 antagonists reduced the infectivity of authentic LUJV. These findings suggest promising candidates for developing new therapies against LUJV infections.

microbiology↗

bioRxiv2023

Bovine leukemia virus-derived long-noncoding RNA, AS1-S, binds to bovine hnRNPM and alters interaction properties between hnRNPM and host mRNAs.

Viruses utilize several strategies to develop latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein encoding RNA that regulates various cellular functions by interacting with RNA binding proteins, is a key factor for viral latency because of its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine hnRNPM, an RNA-binding protein located in the nucleus, as the binding partner for AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motif 1 and 2, located in the N-terminal region of bovine hnRNPM, are responsible for binding AS1-S. Furthermore, an RNA immunoprecipitation assay showed that introduction of AS1-S increased the number of mRNA that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer", AS1-S may affect proliferation and expansion of BLV-infected cells and contribute to tumor progression. ImportanceBLV infects bovine B cells and causes malignant lymphoma, resulting in severe economic losses in the livestock industry. Due to its low incidence rate and long latent period, the molecular mechanisms underlying the progression to lymphoma remain enigmatic. Several non-coding RNAs, such as miRNA and lncRNA, have recently been discovered in the BLV genome and the relationship between BLV pathogenesis and these non-coding RNAs is attracting attention. However, most of the molecular functions of these transcripts remain un-identified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

microbiology↗

bioRxiv2023

An engineered ACE2 decoy broadly neutralizes Omicron subvariants and shows therapeutic effect in SARS-CoV-2-infected cynomolgus macaques

The Omicron variant continuously evolves under the humoral immune pressure obtained by vaccination and SARS-CoV-2 infection and the resultant Omicron subvariants exhibit further immune evasion and antibody escape. Engineered ACE2 decoy composed of high-affinity ACE2 and IgG1 Fc domain is an alternative modality to neutralize SARS-CoV-2 and we previously reported its broad spectrum and therapeutic potential in rodent models. Here, we show that engineered ACE2 decoy retains the neutralization activity against Omicron subvariants including the currently emerging XBB and BQ.1 which completely evade antibodies in clinical use. The culture of SARS-CoV-2 under suboptimal concentration of neutralizing drugs generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against engineered ACE2 decoy. As the efficient drug delivery to respiratory tract infection of SARS-CoV-2, inhalation of aerosolized decoy treated mice infected with SARS-CoV-2 at a 20-fold lower dose than the intravenous administration. Finally, engineered ACE2 decoy exhibited the therapeutic efficacy for COVID-19 in cynomolgus macaques. Collectively, these results indicate that engineered ACE2 decoy is the promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation can be considered as a non-invasive approach to enhance efficacy in the treatment of COVID-19.

microbiology↗