Biology subjects

Yoshimura, K.

Publications and source records attributed to Yoshimura, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of loop regions as motifs determining cellular and organ chirality in Myosin 1C

Left-right (LR) asymmetry occurs throughout the animal kingdom, from microscopic to macroscopic scales; however, how microscale LR asymmetry (chirality) is integrated into higher-order structural LR asymmetry remains unclear. In Drosophila, the actin motor proteins Myosin1D and Myosin1C impose opposite chirality states on cell shape and intracellular F-actin flow, thereby directing dextral and sinistral morphogenesis, respectively. However, the protein motifs that confer their distinct activities in specifying chiral states have remained unknown. Here, we show that loop motifs within the myosin head domain that form actin-binding sites determine enantiomeric chirality. By swapping these loop motifs between Myosin1D and Myosin1C, we demonstrate that Myosin1D acquires sinistral activity when carrying Myosin1C loops. AlphaFold3 and molecular dynamics analyses reveal differences in loop structure and actin-binding energetics that may account for the contrasting chiral activities of Myosin1D and Myosin1C. Our findings identify specific protein motifs that dictate the handedness of actin flow and organ asymmetry, providing a mechanistic link between molecular interactions and macroscopic left-right patterning.

cell biology↗

bioRxiv2025

RNA switch model for localization and translation of the myelin basic protein mRNA

Oligodendrocytes myelinate the central nervous system by extending cellular projections that ensheath axons and elongate to form lipid-rich myelin. Classic studies visualizing RNA dynamics showed that myelin basic protein (MBP), one of the most abundant myelin proteins, is locally synthesized at the myelin sheath through the transport and local translation of Mbp mRNA. Mbp transport requires its 1.5-kb 3 untranslated region (3 UTR) and prior work identified candidate sub-sequences that may act as cis-acting transport stimulating RNA elements, including one with putative secondary structure. Here, a high-throughput reporter assay, dimethyl sulfate (DMS)-based RNA structure probing, and microscopy in primary rat oligodendrocytes identify a structured 127-nt region that we name the Mbp localization signal (MLS) as both necessary and sufficient for RNA enrichment to oligodendrocyte projections. Lysate pulldown experiments further identify hnRNP-F - a known constituent of the Mbp RNA granule that can suppress mRNA translation - as associated with the MLS; paradoxically, binding of this protein should compete with the ordered MLS RNA structure. These results suggest a model in which the MLS switches between two RNA conformations with distinct protein partners during the transition from Mbp mRNA transport to Mbp translation at the myelin sheath. Such regulation of RNA behavior by structure switching may generalize to other eukaryotic mRNAs whose behaviors shift across space and time. Significance StatementIn the brain, oligodendrocyte cells generate myelin, a type of insulation that wraps around neuronal axons in order to facilitate fast electrical signaling. A critical step in myelination is the local translation of MBP (myelin basic protein) in the myelin sheath. This requires the transport of Mbp mRNA, an incompletely understood phenomenon that we revisit using two recent approaches for mRNA structure and function. We refine a 127-nt region that is necessary and sufficient for mRNA transport to the myelin sheath. A proteomic screen reveals that this myelin localization signal (MLS) associates with a translation-suppressing protein called hnRNP-F, suggesting a model where Mbp mRNA switches between two states, one for transport and one for translation at the myelin sheath.

cell biology↗

bioRxiv2025

Structural basis of lipid-mediated gating in a two-pore domain potassium channel

TOK1 is the first identified member of the K2P channel family and contains additional N- and C-terminal domains, displaying a configuration distinct from that of canonical K2P channels. Although recent advances in structural studies of K2P channels have elucidated their architectures, the structural basis of TOK1 has remained unknown, limiting our understanding of its unique configuration. Here, we present the cryo-electron microscopy (cryo-EM) structure of TOK1, unveiling its distinctive domain architecture. Furthermore, the structures of TOK1 in three distinct states provide mechanistic insights into its regulation through lipid binding and dissociation. Phosphorylation of TOK1 induces the formation of an additional lipid-binding site, leading to channel inactivation. Conversely, upon activation, the phospholipid dissociates, allowing ion permeation. Our comprehensive study, integrating cryo-EM structural analysis, molecular dynamics simulations, electrophysiological recordings, and mass spectrometry, elucidates the distinctive features of TOK1, an atypical K2P channel, and provides a framework for understanding lipid-mediated regulation within this family.

molecular biology↗

bioRxiv2025

Class I myosins direct circumferential F-actin flows to define cell chirality

Eukaryotic cells possess intrinsic chirality in their structure, motility, and intracellular dynamics, which are designated cell chirality. Cell chirality participates in the left-right asymmetric morphogenesis and tissue integrity. However, the mechanisms of cell chirality formation remain elusive. In Drosophila, two evolutionarily conserved myosin I genes, Myosin 1D (Myo1D) and Myosin 1C (Myo1C), respectively, dictate the dextral and sinistral chirality of the cells and body. Here, we reported that Myo1D and Myo1C respectively directed the clockwise and counterclockwise circumferential flow of F-actin in Drosophila macrophages. Both induced the corresponding circular cytoplasm flows and depended on Myosin2 (Myo2). In a modified in vitro motility assay using near-physiological actin concentrations, Myo1D triggered the self-organization of the F-actin ring (chiral F-actin ring) that rotated clockwise; conversely, Myo1C induced the random flow of F-actin. The chiral F-actin ring implied that the F-actin bundle was parallelly and annularly polarized concerning its barbed pointed end. Considering that Myo1D and Myo1C are localized to the dorsal plasma membrane of macrophages, Myo1D and Myo1C might organize the parallelly polarized F-actin in macrophages. Our results suggest that Myo2 might drive the clockwise circumferential flow of F-actin along its parallel and annular polarity induced by Myo1D, which may be a molecular basis of cell and organ chirality.

cell biology↗

bioRxiv2025

Elucidating Chiral Myosin-Induced Actin Dynamics: From Single Filament Behavior to Collective Structures

The myosin superfamily comprises over 70 classes, each with multiple subclasses, and shows substantial diversity in properties such as velocity, ATPase activity, duty ratio, and directionality. This functional diversity enables the specialized roles of each myosin in various organisms, organs, and cell types. Recent studies have revealed that certain myosins induce chiral curved motions of actin filaments. However, this newly identified property remains largely unexplored. Here, we investigated this chiral motion in vitro using Chara corallina myosin XI (CcXI), which drives fast counterclockwise (CCW) movement of actin filaments. This chiral motion arises from asymmetric displacement at the filaments leading tip, and its curvature depends on the surface density of myosin. Surprisingly, at near-physiological actin concentrations, actin filaments exhibiting chiral curved motion undergo collective motion, spontaneously forming a novel structure--termed the actin chiral ring (ACR)--that exhibits persistent CCW rotation. ACRs display remarkable stability, continuing to rotate at their formation site until ATP is depleted, while maintaining their structure even after rotation ceases. This stability is unprecedented among reported collective motions of cytoskeletal proteins driven by various motors. Our findings demonstrate that myosins with chiral activity can autonomously organize actin filaments into stable, chiral structures through collective motion, providing new insights into actin self-organization by unconventional myosins. This study advances our understanding of the diverse functional roles of unconventional myosins and introduces a new paradigm for cytoskeletal organization. Significance StatementMyosins are motor proteins that move along actin filaments and support various intracellular functions. Recently, some myosins have been found to drive actin filaments along chiral curved paths, but the mechanisms and significance of this behavior remain poorly understood. Here, we analyzed this activity and discovered that it not only drives chiral curved motion of single actin filaments, but also organizes them into stable, unidirectionally rotating ring structures through collective motion. These rings, termed actin chiral rings (ACRs), spontaneously emerge at near-physiological actin concentrations. Our findings uncover a previously unrecognized organizing principle of actin self-assembly driven by myosin with chiral activity and provide a new framework for understanding how cytoskeletal chirality is made.

cell biology↗

bioRxiv2025

A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases

Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence ({lambda}max = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues.

cancer biology↗

bioRxiv2024

Virological characteristics of the SARS-CoV-2 XEC variant

The SARS-CoV-2 JN.1 variant (BA.2.86.1.1), arising from BA.2.86.1 with spike protein (S) substitution S:L455S, outcompeted the previously predominant XBB lineages by the beginning of 2024. Subsequently, JN.1 subvariants including KP.2 (JN.1.11.1.2) and KP.3 (JN.1.11.1.3), which acquired additional S substitutions (e.g., S:R346T, S:F456L, and S:Q493E), have emerged concurrently. As of October 2024, KP.3.1.1 (JN.1.11.1.3.1.1), which acquired S:31del, outcompeted other JN.1 subvariants including KP.2 and KP.3 and is the most predominant SARS-CoV-2 variant in the world. Thereafter, XEC, a recombinant lineage of KS.1.1 (JN.13.1.1.1) and KP.3.3 (JN.1.11.1.3.3), was first identified in Germany on August 7, 2024. XEC acquired two S substitutions, S:T22N and S:F59S, compared with KP.3 through recombination, with a breakpoint at genomic position 21,738-22,599. We estimated the relative effective reproduction number (Re) of XEC using a Bayesian multinomial logistic model based on genome surveillance data from the USA, the United Kingdom, France, Canada, and Germany, where this variant has spread as of August 2024. In the USA, the Re of XEC is 1.13-fold higher than that of KP.3.1.1. Additionally, the other countries under investigation herein showed higher Re for XEC. These results suggest that XEC has the potential to outcompete the other major lineage including KP.3.1.1. We then assessed the virological properties of XEC using pseudoviruses. Pseudovirus infection assay showed that the infectivity of KP.3.1.1 and XEC was significantly higher than that of KP.3. Although S:T22N did not affect the infectivity of the pseudovirus based on KP.3, S:F59S significantly increased it. Neutralization assay was performed using three types of human sera: convalescent sera after breakthrough infection (BTI) with XBB.1.5 or KP.3.3, and convalescent sera after JN.1 infection. In all serum groups, XEC as well as KP.3.1.1 showed immune resistance when compared to KP.3 with statistically significant differences. In the cases of XBB.1.5 BTI sera and JN.1 infection sera, the 50% neutralization titers (NT50s) of XEC and KP.3.1.1 were comparable. However, we revealed that the NT50 of XEC was significantly (1.3-fold) lower than that of KP.3.1.1. Moreover, both S:T22N and S:F59S significantly (1.5-fold and 1.6-fold) increased the resistance to KP.3.3 BTI sera. Here we showed that XEC exhibited higher pseudovirus infectivity and higher immune evasion than KP.3. Particularly, XEC exhibited more robust immune resistance to KP.3.3 BTI sera than KP.3.1.1. Our data suggest that the higher Re of XEC than KP.3.1.1 is attributed to this property and XEC will be a predominant SARS-CoV-2 variant in the world in the near future.

microbiology↗

bioRxiv2024

Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1

The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPAR{gamma} is mediated by Transportin-1, but PPAR{gamma} lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPAR{gamma} in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPAR{gamma}, indicating that PPAR{gamma} recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPAR{gamma}.

molecular biology↗

bioRxiv2024

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

The introduction of combined antiretroviral therapy (cART) has greatly improved the quality of life of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Nonetheless, the ever-present desire to seek out a full remedy for HIV-1 infections makes the discovery of novel antiviral medication compelling. Owing to this, a new late-stage inhibitor, Lenacapavir/Sunlenca, an HIV multi-phase suppressor, was clinically authorized in 2022. Besides unveiling cutting-edge antivirals inhibiting late-stage proteins or processes, newer therapeutics targeting host restriction factors hold promise for the curative care of HIV-1 infections. Notwithstanding, bone marrow stromal antigen 2 (BST2)/Tetherin/CD317/HM1.24, which entraps progeny virions is an appealing HIV-1 therapeutic candidate. In this study, a novel drug screening system was established, using the Jurkat/Vpr-HiBiT T cells, to identify drugs that could obstruct HIV-1 release; the candidate compounds were selected from the Ono Pharmaceutical compound library. Jurkat T cells expressing Vpr-HiBiT were infected with NL4-3, and the amount of virus release was quantified indirectly by the amount of Vpr-HiBiT incorporated into the progeny virions. Subsequently, the candidate compounds that suppressed viral release were used to synthesize the heterocyclic compound, HT-7, which reduces HIV-1 release with less cellular toxicity. Notably, HT-7 increased cell surface BST2 coupled with HIV-1 release reduction in Jurkat cells but not Jurkat/KO-BST2 cells. Seemingly, HT-7 impeded simian immunodeficiency virus (SIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) release. Concisely, these results suggest that the reduction in viral release, following HT-7 treatment, resulted from the modulation of cell surface expression of BST2 by HT-7. ImportanceA collection of scientific strategies has been revealed to find long-term cure for HIV-1 infection. One of these techniques, the therapeutic approach, involves harnessing late events that are not targeted by current medication. The regulator of HIV-1 assembly and release, the HIV-1 Gag protein, has emerged as a prospective inhibitor. We set up a high-efficiency, economically viable, and facile screening system for the identification of late-stage inhibitors. Herein, we discovered a heterocyclic compound that inhibits HIV-1 release. This newly high- performance testing technique can be employed in virological research for investigating HIV- 1 late-stage processes.

microbiology↗

bioRxiv2024

Virological characteristics of the SARS-CoV-2 KP.2 variant

The JN.1 variant (BA.2.86.1.1), arising from BA.2.86(.1) with the S:L455S substitution, exhibited increased fitness and outcompeted the previous dominant XBB lineage by the biggening of 2024. JN.1 subsequently diversified, leading to the emergence of descendants with spike (S) protein substitutions such as S:R346T and S:F456L. Particularly, the KP.2 (JN.1.11.1.2) variant, a descendant of JN.1 bearing both S:R346T and S:F456L, is rapidly spreading in multiple regions as of April 2024. Here, we investigated the virological properties of KP.2. KP.2 has three substitutions in the S protein including the two above and additional one substitution in non-S protein compared with JN.1. We estimated the relative effective reproduction number (Re) of KP.2 based on the genome surveillance data from the USA, United Kingdom, and Canada where >30 sequences of KP.2 has been reported, using a Bayesian multinomial logistic model. The Re of KP.2 is 1.22-, 1.32-, and 1.26-times higher than that of JN.1 in USA, United Kingdom, and Canada, respectively. These results suggest that KP.2 has higher viral fitness and potentially becomes the predominant lineage worldwide. Indeed, as of the beginning of April 2024, the estimated variant frequency of KP.2 has already reached 20% in United Kingdom. The pseudovirus assay showed that the infectivity of KP.2 is significantly (10.5-fold) lower than that of JN.1. We then performed a neutralization assay using monovalent XBB.1.5 vaccine sera and breakthrough infection (BTI) sera with XBB.1.5, EG.5, HK.3 and JN.1 infections. In all cases, the 50% neutralization titer (NT50) against KP.2 was significantly lower than that against JN.1. Particularly, KP.2 shows the most significant resistance to the sera of monovalent XBB.1.5 vaccinee without infection (3.1-fold) as well as those who with infection (1.8-fold). Altogether, these results suggest that the increased immune resistance ability of KP.2 partially contributes to the higher Re more than previous variants including JN.1.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron HK.3 variant harboring the "FLip" substitution

In November 2023, SARS-CoV-2 XBB descendants, including EG.5.1 (XBB.1.9.2.5.1), the currently predominant lineage, are circulating worldwide according to Nextstrain. EG.5.1 has a characteristic amino acid substitution in the spike protein (S), S:F456L, which contributes to its escape from humoral immunity. EG.5.1 has further evolved, and its descendant lineage harboring S:L455F (i.e., EG.5.1+S:L455F) emerged and was named HK.3 (XBB.1.9.2.5.1.1.3). HK.3 was initially discovered in East Asia and is rapidly spreading worldwide. Notably, the XBB subvariants bearing both S:L455F and S:F456L substitutions, including HK.3, are called the "FLip" variants. These FLip variants, such as JG.3 (XBB.1.9.2.5.1.3.3), JF.1 (XBB.1.16.6.1) and GK.3 (XBB.1.5.70.3), have emerged convergently, suggesting that the acquisition of these two substitutions confers a growth advantage to XBB in the human population. Here, we investigated the virological properties of HK.3 as a representative of the FLip variants.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 BA.2.86 variant

In late 2023, a lineage of SARS-CoV-2 emerged and was named the BA.2.86 variant. BA.2.86 is phylogenetically distinct from other Omicron sublineages identified so far, displaying an accumulation of over 30 amino acid mutations in its spike protein. Here, we performed multiscale investigations to reveal the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Experimental studies showed that four clinically-available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 is significantly lower than that of BA.2 in both in vitro cell cultures and in vivo, it is suggested that the attenuated pathogenicity of BA.2.86 is due to its decreased replication capacity.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

microbiology↗

bioRxiv2023

Virological characteristics correlating with SARS-CoV-2 spike protein fusogenicity

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike (S) protein is essential in mediating membrane fusion of the virus with the target cells. Several reports demonstrated that SARS-CoV-2 S protein fusogenicity is reportedly closely associated with the intrinsic pathogenicity of the virus determined using hamster models. However, the association between S protein fusogenicity and other virological parameters remains elusive. In this study, we investigated the virological parameters of eleven previous variants of concern (VOCs) and variants of interest (VOIs) correlating with S protein fusogenicity. S protein fusogenicity was found to be strongly correlated with S1/S2 cleavage efficiency and plaque size formed by clinical isolates. However, S protein fusogenicity was less associated with pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics. Taken together, our results suggest that S1/S2 cleavage efficiency and plaque size could be potential indicators to predict the intrinsic pathogenicity of newly emerged SARS-CoV-2 variants.

microbiology↗

bioRxiv2023

AtDREB2G is a novel regulator of riboflavin biosynthesis under low-temperature stress and abscisic acid treatment in Arabidopsis thaliana

Riboflavin (RF) serves as a precursor to FMN and FAD, crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the Dehydration-Responsive Element Binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G as a novel positive regulator of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.

plant biology↗

bioRxiv2023

Antiviral efficacy of the SARS-CoV-2 XBB breakthrough infection sera against Omicron subvariants including EG.5

As of July 2023, EG.5.1 (a.k.a. XBB.1.9.2.5.1), a XBB subvariant bearing the S:Q52H and S:F456L substitutions, alongside the S:F486P substitution (Figure S1A), has rapidly spread in some countries. On July 19, 2023, the WHO classified EG.5 as a variant under monitoring. First, we showed that EG.5.1 exhibits a higher effective reproduction number compared with XBB.1.5, XBB.1.16, and its parental lineage (XBB.1.9.2), suggesting that EG.5.1 will spread globally and outcompete these XBB subvariants in the near future. We then addressed whether EG.5.1 evades from the antiviral effect of the humoral immunity induced by breakthrough infection (BTI) of XBB subvariants and performed a neutralization assay using XBB BTI sera. However, the 50% neutralization titer (NT50) of XBB BTI sera against EG.5.1 was comparable to those against XBB.1.5/1.9.2 and XBB.1.16. Moreover, the sensitivity of EG.5.1 to convalescent sera of XBB.1- and XBB.1.5-infected hamsters was similar to those of XBB.1.5/1.9 and XBB.1.16. These results suggest that the increased Re of EG.5.1 is attributed to neither increased infectivity nor immune evasion from XBB BTI, and the emergence and spread of EG.5 is driven by the other pressures. We previously demonstrated that Omicron BTI cannot efficiently induce antiviral humoral immunity against the variant infected. In fact, the NT50s of the BTI sera of Omicron BA.1, BA.2, and BA.5 against the variant infected were 3.0-, 2.2-, and 3.4-fold lower than that against the ancestral B.1.1 variant, respectively. However, strikingly, we found that the NT50 of the BTI sera of XBB1.5/1.9 and XBB.1.16 against the variant infected were 8.7- and 8.3-fold lower than that against the B.1.1 variant. These results suggest that XBB BTI cannot efficiently induce antiviral humoral immunity against XBB subvariants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/552415v1_figs1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@f95376org.highwire.dtl.DTLVardef@d66fa8org.highwire.dtl.DTLVardef@3c8841org.highwire.dtl.DTLVardef@15824c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1.C_FLOATNO Virological features of EG.5.1 and XBB BTI (A) Frequency of mutations of interest in the representative XBB sublineages. Only mutations with a frequency >0.5 in at least one but not all the representative sublineages are shown. Note that the S proteins of XBB.1.5 and XBB.1.9.2 are identical. (B) Estimated epidemic dynamics of the representative XBB sublineages in countries where >50 sequences of EG.5.1, XBB.1.5, XBB.1.9.2, and XBB.1.16 were detected from April 1, 2023 to July 13, 2023. Countries are ordered according to the number of detected sequences of EG.5.1. Line, posterior mean; ribbon, 95% Bayesian confidence interval. The dynamics for EG.5.1 is highlighted by a red arrowhead. (C) Estimated relative Re of the representative XBB sublineages in the six countries. The relative Re of XBB.1.5 is set to 1 (horizontal dashed line). Violin, posterior distribution; dot, posterior mean; line, 95% Bayesian confidence interval. (D) Lentivirus-based pseudovirus assay. HOS-ACE2-TMPRSS2 cells were infected with pseudoviruses bearing each S protein. The amount of input virus was normalized to the amount of HIV-1 p24 capsid protein. The percentage infectivity of XBB.1.5/1.9.2, XBB.1.5/1.9.2+Q52H, XBB.1.5/1.9.2+F456L, and EG.5.1 compared to that of XBB.1.5/1.9.2 are shown. The horizontal dash line indicates the mean value of the percentage infectivity of the XBB.1.5/1.9.2. Assays were performed in quadruplicate. The presented data are expressed as the average {+/-} SD. Each dot indicates the result of an individual replicate. (E-G) Neutralization assay. Assays were performed with pseudoviruses harboring the S proteins of B.1.1, BA.1, BA.2, BA.5, BQ.1.1, XBB.1, XBB.1.5/1.9.2, XBB.1.16, EG.5.1, XBB.1.5/1.9.2+Q52H, and XBB.1.5/1.9.2+F456L. The following sera were used: convalescent sera from fully vaccinated individuals who had been infected with XBB.1.5 (one 3-dose vaccinated. 1 donor in total), XBB.1.9 (one 3-dose vaccinated donor, one 4-dose vaccinated donor and one 5-dose vaccinated donor. 3 donors in total), and XBB.1.16 (one 2-dose vaccinated donor, two 3-dose vaccinated donors, and one 4-dose vaccinated donor. 4 donors in total) (E); sera from hamster infected with XBB.1 (left) or XBB.1.5 (right) (F); and convalescent sera from fully vaccinated individuals who had been infected with BA.1 (thirteen 2-dose vaccinated, 13 donors in total) (left)1, BA.2 (nine 2-dose vaccinated and four 3-dose vaccinated donors. 13 donors in total) (middle)19, and BA.5 (one 2-dose vaccinated, thirteen 3-dose vaccinated donors, and one 4-dose vaccinated. 15 donors in total) (right)19 (G). Each dot indicates the result of an individual replicate. Assays for each serum sample were performed in triplicate to determine the 50% neutralization titer (NT50). Each dot represents one NT50 value, and the geometric mean and 95% confidence interval are shown. The number in parenthesis indicates the mean of NT50 values. The horizontal dash line indicates the detection limit (120-fold). In D, statistically significant differences (**, P < 0.001, ***, P < 0.0001) versus XBB.1.5/1.9.2 were determined by two-sided Students t tests. Blue asterisks indicate decreased percentage of infectivity. In E and G, statistically significant differences versus B.1.1 were determined by two-sided Wilcoxon signed-rank tests. The fold change between B.1.1 and the variant indicated is shown in red. Background information on the convalescent donors is summarized in Table S1. In F, statistically significant differences (*, P < 0.01, **, P < 0.001) between B.1.1 and XBB.1 (left) or XBB.1.5/1.9.2 (right) were determined by two-sided Wilcoxon signed-rank tests and indicated with asterisks. Red asterisks indicate decreased NT50s. C_FIG

microbiology↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

microbiology↗

bioRxiv2022

Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.

microbiology↗