Autoantigens of Small Nerve Fibers and Human Coronavirus Antigens: Is There a Possibility for Molecular Mimicry?

In post-COVID-19 syndrome, clinical presentation of the nerve fiber dysfunction plays an important role. The possibility of autoantigen cross-mimicry of human coronaviruses and the peripheral nervous system needs to be investigated. The bioinformatic analysis was applied to search for possible common protein sequences located in the immunoreactive epitopes. Among the autoantigens of the human nervous system, fibroblast growth factor receptor protein 3, myelin protein P0, myelin protein P2, sodium channel protein type 9, alpha protein subunit, plexin-D1 protein and ubiquitin-carboxyl-terminal hydrolase protein of the L1 isoenzyme were selected. The original "Alignmentaj" analytical program was created. The UniProt database, Protein Data Bank, and AlphaFold databases were used. The analysis of protein sequence similarities of spike glycoproteins in human coronaviruses revealed common pentapeptides of the MERS-CoV-2 virus with the fibroblast growth factor receptor 3 and myelin protein P2. Among seasonal coronaviruses, common peptide sequences were identified in HCoV-HKU-1 virus with sodium channel protein type 9 subunit alpha and Plexin-D1, HCoV-OC43 with Plexin-D1, as well as HCoV-NL63 with Plexin-D1 and Ubiquitin carboxyl-terminal hydrolase isozyme L1. Some shared peptides belong to immunoreactive epitopes. The most important targets for the molecular similarities are the sodium channel subunits and fibroblast growth factor receptor 3, both for seasonal and highly pathogenic coronaviruses. The data obtained make it possible to identify new potential targets for the development of autoimmune reactions that may occur against the background of the infections with highly pathogenic as well as seasonal coronaviruses.

SARS-CoV-2 and Other Coronaviruses in Rats, Berlin, Germany, 2023.

We tested 130 rats captured in Berlin for coronaviruses. SARS-CoV-2 antibodies were detected in 1 rat, but all animals were negative by reverse transcription PCR, suggesting SARS-CoV-2 was not circulating in the rat population. However, alphacoronaviruses were found. Monitoring rodent populations helps to determine coronavirus occurrence, transmission, and zoonotic potential.

Whole genome characteristics of hedgehog coronaviruses from Poland and analysis of the evolution of the Spike protein for its interspecies transmission potential.

BACKGROUND: The hedgehogs have been recently identified as possible reservoir of Middle East respiratory syndrome coronavirus like (MERS-CoV-like). These viruses were classified as a distinct Betacoronavirus erinacei (BCoV-Eri) species within the MerBCoV-Eriirus subgenus. As coronaviruses are known for their ability to jump between different hosts, including humans, this can pose a particular threat to people in direct contact with hedgehogs, such as those working at animal asylums. Our previous studies have shown the presence of BCoV-Eri strains in animals collected in the wildlife rehabilitation centre. This study aimed to investigate the presence of CoV in subsequent hedgehogs collected from the urban area of Poland and their molecular characteristics. RESULTS: Monitoring for the presence of coronavirus infection in hedgehogs revealed five positive individuals. The presence of BCoV-Eri was found in a total of 20% of animals tested. Our analyses revealed no correlation between CoVs positivity and animal health conditions but a higher probability of such infection in juveniles and females. The whole genome of two Polish Hedgehog coronavirus 1 strains were sequenced and compared with available counterparts from European and Asian countries. Phylogenetic analysis showed that both CoV strains formed common cluster with other similar MerBCoV-Eriirus, but they were also found to be genetically variable and most changes in the S protein were identified. Our analysis revealed that some S protein sites of the Hedgehog coronavirus 1 strains evolved under positive selection pressure and of five such sites, three are in the S1 region while the other two in the S2 region of the Spike. CONCLUSIONS: BCoV-Eri is to some extent prevalent in wildlife asylums in Poland. Given that the S protein of BCoVs-Eri is highly variable and that some sites of this protein evolve under positive selection pressure, these strains could potentially acquire a favourable feature for cross-species transmission. Consequently, the threat to humans working in such asylums is particularly high. Adequate biosecurity safeguards, but also human awareness of such risks, are therefore essential.

Human coronaviruses activate and hijack the host transcription factor HSF1 to enhance viral replication.

Organisms respond to proteotoxic-stress by activating the heat-shock response, a cellular defense mechanism regulated by a family of heat-shock factors (HSFs); among six human HSFs, HSF1 acts as a proteostasis guardian regulating severe stress-driven transcriptional responses. Herein we show that human coronaviruses (HCoV), both low-pathogenic seasonal-HCoVs and highly-pathogenic SARS-CoV-2 variants, are potent inducers of HSF1, promoting HSF1 serine-326 phosphorylation and triggering a powerful and distinct HSF1-driven transcriptional-translational response in infected cells. Despite the coronavirus-mediated shut-down of the host translational machinery, selected HSF1-target gene products, including HSP70, HSPA6 and AIRAP, are highly expressed in HCoV-infected cells. Using silencing experiments and a direct HSF1 small-molecule inhibitor we show that, intriguingly, HCoV-mediated activation of the HSF1-pathway, rather than representing a host defense response to infection, is hijacked by the pathogen and is essential for efficient progeny particles production. The results open new scenarios for the search of innovative antiviral strategies against coronavirus infections.

Persistence of two coronaviruses and efficacy of steam vapor disinfection on two types of carpet.

BACKGROUND: Coronaviruses, a group of highly transmissible and potentially pathogenic viruses, can be transmitted indirectly to humans via fomites. To date, no study has investigated their persistence on carpet fibers. Establishing persistence is essential before testing the efficacy of a disinfectant. METHODS: The persistence of BCoV and HCoV OC43 on polyethylene terephthalate (PET) and nylon carpet was first determined using infectivity and RT-qPCR assays. Then, the disinfectant efficacy of steam vapor was evaluated against both coronaviruses on nylon carpet. RESULTS: Immediately after inoculation of carpet coupons, 32.50% of BCoV and 3.87% of HCoV OC43 were recovered from PET carpet, compared to 34.86% of BCoV and 24.37% of HCoV OC43 recovered from nylon carpet. After incubation at room temperature for 1 h, BCoV and HCoV OC43 showed a 3.6 and > 2.8 log10 TCID50 reduction on PET carpet, and a 0.6 and 1.8 log10 TCID50 reduction on nylon carpet. Based on first-order decay kinetics, the whole gRNA of BCoV and HCoV OC43 were stable with k values of 1.19 and 0.67 h- 1 on PET carpet and 0.86 and 0.27 h- 1 on nylon carpet, respectively. A 15-s steam vapor treatment achieved a > 3.0 log10 TCID50 reduction of BCoV and > 3.2 log10 TCID50 reduction of HCoV OC43 on nylon carpet. CONCLUSION: BCoV was more resistant to desiccation on both carpet types than HCoV OC43. Both viruses lost infectivity quicker on PET carpet than on nylon carpet. Steam vapor inactivated both coronaviruses on nylon carpet within 15 s.

Impact of a Functional Dairy Powder and Its Primary Component on the Growth of Pathogenic and Probiotic Gut Bacteria and Human Coronavirus 229E.

Milk boasts an array of potent bioactive compounds, such as lactoferrin (Lf), immunoglobulins, and functional proteins, all delivering substantial therapeutic benefits. In this study, Immune Powder (a functional dairy formulation) and its primary component called Fractionated Milk Protein (FMP) containing Lf, zinc, and immunoglobulins and formulated by Ausnutria Pty Ltd. were evaluated for their potential broad-spectrum pharmacological activity. In particular, this study investigated the antibacterial (against pathogenic Escherichia coli), prebiotic (promoting Lactobacillus delbrueckii growth), anti-inflammatory (inhibition of NO production in RAW264.7 macrophages), and antiviral (against human coronavirus 229E) effects of the samples. In addition, the impact of simulated gastric digestion on the efficacy of the samples was explored. LCMS-based proteomics was implemented to unveil cellular and molecular mechanisms underlying antiviral activity. The Immune Powder demonstrated antibacterial activity against E. coli (up to 99.74 ± 11.47% inhibition), coupled with prebiotic action (10.84 ± 2.2 viability fold-change), albeit these activities diminished post-digestion (p < 0.01). The Immune Powder effectively mitigated NO production in lipopolysaccharide-stimulated RAW264.7 macrophages, with declining efficacy post-digestion (p < 0.0001). The Immune Powder showed similar antiviral activity before and after digestion (p > 0.05) with up to 3-fold improvement. Likewise, FMP exhibited antibacterial potency pre-digestion at high concentrations (95.56 ± 1.23% inhibition at 125 mg/mL) and post-digestion at lower doses (61.82 ± 5.58% inhibition at 3906.25 µg/mL). FMP also showed enhanced prebiotic activity post-digestion (p < 0.0001), NO inhibition pre-digestion, and significant antiviral activity. The proteomics study suggested that the formulation and its primary component shared similar antiviral mechanisms by inhibiting scavenger receptor binding and extracellular matrix interaction.

Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses.

Several coronaviruses infect humans, with three, including the SARS-CoV2, causing diseases. While coronaviruses are especially prone to induce pandemics, we know little about their evolutionary history, host-to-host transmissions, and biogeography. One of the difficulties lies in dating the origination of the family, a particularly challenging task for RNA viruses in general. Previous cophylogenetic tests of virus-host associations, including in the Coronaviridae family, have suggested a virus-host codiversification history stretching many millions of years. Here, we establish a framework for robustly testing scenarios of ancient origination and codiversification versus recent origination and diversification by host switches. Applied to coronaviruses and their mammalian hosts, our results support a scenario of recent origination of coronaviruses in bats and diversification by host switches, with preferential host switches within mammalian orders. Hotspots of coronavirus diversity, concentrated in East Asia and Europe, are consistent with this scenario of relatively recent origination and localized host switches. Spillovers from bats to other species are rare, but have the highest probability to be towards humans than to any other mammal species, implicating humans as the evolutionary intermediate host. The high host-switching rates within orders, as well as between humans, domesticated mammals, and non-flying wild mammals, indicates the potential for rapid additional spreading of coronaviruses across the world. Our results suggest that the evolutionary history of extant mammalian coronaviruses is recent, and that cases of long-term virus-host codiversification have been largely over-estimated.

The SARS-CoV-2 virus, which caused the recent global coronavirus pandemic, is the latest in a string of coronaviruses that have caused serious outbreaks. This group of coronaviruses can also infect other mammals and likely jumped between species ­ including from non-humans to humans ­ over the course of evolution. Determining when and how viruses evolved to infect humans can help scientists predict and prevent outbreaks. However, tracking the evolutionary trajectory of coronaviruses is challenging, and there are conflicting views on how often coronaviruses crossed between species and when these transitions likely occurred. Some studies suggest that coronaviruses originated early on in evolution and evolved together with their mammalian hosts, only occasionally jumping to and from different species. While others suggest they appeared more recently, and rapidly diversified by regularly transferring between species. To determine which is the most likely scenario, Maestri, Perez-Lamarque et al. developed a computational approach using already available data on the genetics and evolutionary history of mammals and coronaviruses. This revealed that coronaviruses originated recently in bats from East Asia and Europe, and primarily evolved by rapidly transferring between different mammalian species. This has led to geographical hotspots of diverse coronaviruses in East Asia and Europe. Maestri, Perez-Lamarque et al. found that it was rare for coronaviruses to spill over from bats to other types of mammals. Most of these spillovers resulted from coronaviruses jumping from bats to humans or domesticated animals. Humans appeared to be the main intermediary host that coronaviruses temporarily infected as they transferred from bats to other mammals. These findings ­ that coronaviruses emerged recently in evolution, jumped relatively frequently between species, and are geographically restricted ­ suggest that future transmissions are likely. Gathering more coronavirus samples from across the world and using even more powerful analysis tools could help scientists understand more about how these viruses recently evolved. These insights may lead to strategies for preventing new coronaviruses from emerging and spreading among humans.

Microbiological and decomposition analysis of mass mink burial sites during the COVID-19 pandemic.

In 2020, Denmark buried approximately four million culled, farmed mink in mass graves treated with slaked lime due to widespread SARS-CoV-2 infections. After six months, environmental concerns prompted the exhumation of these cadavers. Our analysis encompassed visual inspections, soil pH measurements, and gas emission assessments of the grave environment. Additionally, we evaluated carcasses for decay status, cadaverine content, and the presence of various pathogens, including SARS-CoV-2 and mink coronavirus. Our findings revealed minimal microbial activity and limited carcass decomposition. Although viral RNA from SARS-CoV-2 and mink coronavirus, along with DNA from Aleutian mink disease virus, were detected, the absence of infectious SARS-CoV-2 in cell culture assays suggests slow natural degradation processes. This study provides critical insights for future considerations in managing mass burial scenarios during outbreaks of livestock-associated zoonotic pathogens.

Monitoring of Astroviruses, Brno-Hantaviruses, Coronaviruses, Influenza Viruses, Bornaviruses, Morbilliviruses, Lyssaviruses and Pestiviruses in Austrian Bats.

Here, we report the results of a monitoring study of bat viruses in Austria to strengthen the knowledge of circulating viruses in Austrian bat populations. In this study, we analyzed 618 oropharyngeal and rectal swab samples from 309 bats and 155 pooled tissue samples from dead bats. Samples were collected from 18 different bat species from multiple locations in Austria, from November 2015 to April 2018, and examined for astroviruses, bornaviruses, coronaviruses, hantaviruses, morbilliviruses, orthomyxoviruses (influenza A/C/D viruses), pestiviruses and rhabdoviruses (lyssaviruses) using molecular techniques and sequencing. Using RT-qPCR, 36 samples revealed positive or suspicious results for astroviruses, Brno-hantaviruses, and coronaviruses in nine different bat species. Further sequencing revealed correspondent sequences in five samples. In contrast, none of the tested samples was positive for influenza viruses A/C/D, bornaviruses, morbilliviruses, lyssaviruses, or pestiviruses.

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity.

Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.

Nanopore guided annotation of transcriptome architectures.

Nanopore direct RNA sequencing (DRS) enables the capture and full-length sequencing of native RNAs, without recoding or amplification bias. Resulting data sets may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails, on individual RNA molecules. The success of these analyses is highly dependent on the provision of high-resolution transcriptome annotations in combination with workflows that minimize misalignments and other analysis artifacts. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene-dense genomes of viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS data sets that inform resulting read alignments and developed the nanopore guided annotation of transcriptome architectures (NAGATA) software package (https://github.com/DepledgeLab/NAGATA). We demonstrate, using a combination of synthetic and original DRS data sets derived from adenoviruses, herpesviruses, coronaviruses, and human cells, that NAGATA outperforms existing transcriptome annotation software and yields a consistently high level of precision and recall when reconstructing both gene sparse and gene-dense transcriptomes. Finally, we apply NAGATA to generate the first high-resolution transcriptome annotation of the neglected pathogen human adenovirus type F41 (HAdV-41) for which we identify 77 distinct transcripts encoding at least 23 different proteins. IMPORTANCE: The transcriptome of an organism denotes the full repertoire of encoded RNAs that may be expressed. This is critical to understanding the biology of an organism and for accurate transcriptomic and epitranscriptomic-based analyses. Annotating transcriptomes remains a complex task, particularly in small gene-dense organisms such as viruses which maximize their coding capacity through overlapping RNAs. To resolve this, we have developed a new software nanopore guided annotation of transcriptome architectures (NAGATA) which utilizes nanopore direct RNA sequencing (DRS) datasets to rapidly produce high-resolution transcriptome annotations for diverse viruses and other organisms.

Coronaviruses (Coronaviridae) of bats in the northern Caucasus and south of western Siberia.

INTRODUCTION: Bats are natural reservoirs of coronaviruses (Coronaviridae), which have caused three outbreaks of human disease SARS, MERS and COVID-19 or SARS-2 over the past decade. The purpose of the work is to study the diversity of coronaviruses among bats inhabiting the foothills and mountainous areas of the Republics of Dagestan, Altai and the Kemerovo region. MATERIALS AND METHODS: Samples of bat oral swabs and feces were tested for the presence of coronavirus RNA by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It has been shown that the greater horseshoe bats (Rhinolophus ferrumequinum), inhabiting the Republic of Dagestan, are carriers of two different coronaviruses. One of the two coronaviruses is a member of the Sarbecovius subgenus of the Betacoronavirus genus, which includes the causative agents of SARS and COVID-19. The second coronavirus is assigned to the Decacovirus subgenus of the Alphacoronavirus genus and is most similar to viruses identified among Rhinolophus spp. from European and Middle Eastern countries. In the Altai Republic and Kemerovo region, coronaviruses belonging to the genus Alphacoronavirus, subgenus Pedacovirus, were found in the smooth-nosed bats: Ikonnikov`s bat (Myotis ikonnikovi) and the eastern bat (Myotis petax). The virus from the Altai Republic from M. ikonnikovi is close to viruses from Japan and Korea, as well as viruses from Myotis spp. from European countries. The virus from the Kemerovo region from M. petax groups with coronaviruses from Myotis spp. from Asian countries and is significantly different from coronaviruses previously discovered in the same natural host.

Host-Virus Cophylogenetic Trajectories: Investigating Molecular Relationships between Coronaviruses and Bat Hosts.

Bats, with their virus tolerance, social behaviors, and mobility, are reservoirs for emerging viruses, including coronaviruses (CoVs) known for genetic flexibility. Studying the cophylogenetic link between bats and CoVs provides vital insights into transmission dynamics and host adaptation. Prior research has yielded valuable insights into phenomena such as host switching, cospeciation, and other dynamics concerning the interaction between CoVs and bats. Nonetheless, a distinct gap exists in the current literature concerning a comparative cophylogenetic analysis focused on elucidating the contributions of sequence fragments to the co-evolution between hosts and viruses. In this study, we analyzed the cophylogenetic patterns of 69 host-virus connections. Among the 69 host-virus links examined, 47 showed significant cophylogeny based on ParaFit and PACo analyses, affirming strong associations. Focusing on two proteins, ORF1ab and spike, we conducted a comparative analysis of host and CoV phylogenies. For ORF1ab, the specific window ranged in multiple sequence alignment (positions 520-680, 770-870, 2930-3070, and 4910-5080) exhibited the lowest Robinson-Foulds (RF) distance (i.e., 84.62%), emphasizing its higher contribution in the cophylogenetic association. Similarly, within the spike region, distinct window ranges (positions 0-140, 60-180, 100-410, 360-550, and 630-730) displayed the lowest RF distance at 88.46%. Our analysis identified six recombination regions within ORF1ab (positions 360-1390, 550-1610, 680-1680, 700-1710, 2060-3090, and 2130-3250), and four within the spike protein (positions 10-510, 50-560, 170-710, and 230-730). The convergence of minimal RF distance regions with combination regions robustly affirms the pivotal role of recombination in viral adaptation to host selection pressures. Furthermore, horizontal gene transfer reveals prominent instances of partial gene transfer events, occurring not only among variants within the same host species but also crossing host species boundaries. This suggests a more intricate pattern of genetic exchange. By employing a multifaceted approach, our comprehensive strategy offers a nuanced understanding of the intricate interactions that govern the co-evolutionary dynamics between bat hosts and CoVs. This deeper insight enhances our comprehension of viral evolution and adaptation mechanisms, shedding light on the broader dynamics that propel viral diversity.

Recombinant porcine interferon delta 8 inhibits swine acute diarrhoea syndrome coronavirus infection in vitro and in vivo.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which originates from zoonotic transmission of bat coronaviruses in the HKU2 lineage, causes severe illness in pigs and carries a high risk of spreading to humans. At present, there are no licenced therapeutics for the treatment of SADS-CoV. In this study, we examined the effectiveness of recombinant porcine interferon delta 8 (IFN-δ8) against SADS-CoV both in vitro and in vivo. In vitro experiments showed that IFN-δ8 inhibited SADS-CoV proliferation in a concentration-dependent manner, with complete inhibition occurring at a concentration of 5 µg/mL. In vivo experiments demonstrated that two 50 µg/kg doses of IFN-δ8 injected intraperitoneally protected piglets against lethal challenge, blocked viral shedding, attenuated intestinal damage, and decreased the viral load in the jejunum and ileum. Further findings suggested that IFN-δ8 inhibited SADS-CoV infection by increasing the expression of IFN-stimulated genes. These results indicate that IFN-δ8 shows promise as a biological macromolecule drug against SADS-CoV infection.

Exploring TRIM proteins' role in antiviral defense against influenza A virus and respiratory coronaviruses.

Numerous tripartite motif (TRIM) proteins, identified as E3 ubiquitin ligases, participate in various viral infections through ubiquitylation, ISGylation, and SUMOylation processes. Respiratory viruses, particularly influenza A virus (IAV) and respiratory coronaviruses (CoVs), have severely threatened public health with high morbidity and mortality, causing incalculable losses. Research on the regulation of TRIM proteins in respiratory virus infections is crucial for disease prevention and control. This review introduces TRIM proteins, summarizes recent discoveries regarding their roles and molecular mechanisms in IAV and CoVs infections, discusses current research gaps, and explores potential future trends in this rapidly developing field. It aims to enhance understanding of virus-host interactions and inform the development of new molecularly targeted therapies.

Structural basis for mouse receptor recognition by bat SARS2-like coronaviruses.

The animal origin of SARS-CoV-2 remains elusive, lacking a plausible evolutionary narrative that may account for its emergence. Its spike protein resembles certain segments of BANAL-236 and RaTG13, two bat coronaviruses considered possible progenitors of SARS-CoV-2. Additionally, its spike contains a furin motif, a common feature of rodent coronaviruses. To explore the possible involvement of rodents in the emergence of SARS-CoV-2 spike, we examined the crystal structures of the spike receptor-binding domains (RBDs) of BANAL-236 and RaTG13 each complexed with mouse receptor ACE2. Both RBDs have residues at positions 493 and 498 that align well with two virus-binding hotspots on mouse ACE2. Our biochemical evidence supports that both BANAL-236 and RaTG13 spikes can use mouse ACE2 as their entry receptor. These findings point to a scenario in which these bat coronaviruses may have coinfected rodents, leading to a recombination of their spike genes and a subsequent acquisition of a furin motif in rodents, culminating in the emergence of SARS-CoV-2.

TMPRSS2 and glycan receptors synergistically facilitate coronavirus entry.

The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.