Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

The HHV-6B U20 glycoprotein binds ULBP1, masking it from recognition by NKG2D and interfering with natural killer cell activation.

Introduction: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. Methods: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. Results: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.

Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication.

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

Immune evasion: An imperative and consequence of MYC deregulation.

MYC has been implicated in the pathogenesis of a wide range of human tumors and has been described for many years as a transcription factor that regulates genes with pleiotropic functions to promote tumorigenic growth. However, despite extensive efforts to identify specific target genes of MYC that alone could be responsible for promoting tumorigenesis, the field is yet to reach a consensus whether this is the crucial function of MYC. Recent work shifts the view on MYC's function from being a gene-specific transcription factor to an essential stress resilience factor. In highly proliferating cells, MYC preserves cell integrity by promoting DNA repair at core promoters, protecting stalled replication forks, and/or preventing transcription-replication conflicts. Furthermore, an increasing body of evidence demonstrates that MYC not only promotes tumorigenesis by driving cell-autonomous growth, but also enables tumors to evade the host's immune system. In this review, we summarize our current understanding of how MYC impairs antitumor immunity and why this function is evolutionarily hard-wired to the biology of the MYC protein family. We show why the cell-autonomous and immune evasive functions of MYC are mutually dependent and discuss ways to target MYC proteins in cancer therapy.

Amino acid metabolic reprogramming in the tumor microenvironment and its implication for cancer therapy.

Amino acids are essential building blocks for proteins, crucial energy sources for cell survival, and key signaling molecules supporting the resistant growth of tumor cells. In tumor cells, amino acid metabolic reprogramming is characterized by the enhanced uptake of amino acids as well as their aberrant synthesis, breakdown, and transport, leading to immune evasion and malignant progression of tumor cells. This article reviews the altered amino acid metabolism in tumor cells and its impact on tumor microenvironment, and also provides an overview of the current clinical applications of amino acid metabolism. Innovative drugs targeting amino acid metabolism hold great promise for precision and personalized cancer therapy.

Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

Identification of RNA-binding protein hnRNP C targeting the 3'UTR of the TAP-associated glycoprotein tapasin in melanoma.

Deregulation or loss of the human leukocyte antigen class I (HLA-I) molecules on tumor cells leading to inhibition of CD8+ T cell recognition is an important tumor immune escape strategy, which could be caused by a posttranscriptional control of molecules in the HLA-I pathway mediated by RNA-binding proteins (RBPs). So far, there exists only limited information about the interaction of RBPs with HLA-I-associated molecules, but own work demonstrated a binding of the heterogeneous ribonucleoprotein C (hnRNP C) to the 3' untranslated region (UTR) of the TAP-associated glycoprotein tapasin (tpn). In this study, in silico analysis of pan-cancer TCGA datasets revealed that hnRNP C is higher expressed in tumor specimens compared to corresponding normal tissues, which is negatively correlated to tpn expression, T cell infiltration and the overall survival of tumor patients. Functional analysis demonstrated an upregulation of tpn expression upon siRNA-mediated downregulation of hnRNP C, which is accompanied by an increased HLA-I surface expression. Thus, hnRNP C has been identified to target tpn and its inhibition could improve the HLA-I surface expression on melanoma cells suggesting its use as a possible biomarker for T-cell-based tumor immunotherapies.

SIGLEC15, negatively correlated with PD-L1 in HCC, could induce CD8+ T cell apoptosis to promote immune evasion.

Functional roles of SIGLEC15 in hepatocellular carcinoma (HCC) were not clear, which was recently found to be an immune inhibitor with similar structure of inhibitory B7 family members. SIGLEC15 expression in HCC was explored in public databases and further examined by PCR analysis. SIGLEC15 and PD-L1 expression patterns were examined in HCC samples through immunohistochemistry. SIGLEC15 expression was knocked-down or over-expressed in HCC cell lines, and CCK8 tests were used to examine cell proliferative ability in vitro. Influences of SIGLEC15 expression on tumor growth were examined in immune deficient and immunocompetent mice respectively. Co-culture system of HCC cell lines and Jurkat cells, flow cytometry analysis of tumor infiltrated immune cells and further sequencing analyses were performed to investigate how SIGLEC15 could affect T cells in vitro and in vivo. We found SIGLEC15 was increased in HCC tumor tissues and was negatively correlated with PD-L1 in HCC samples. In vitro and in vivo models demonstrated inhibition of SIGLEC15 did not directly influence tumor proliferation. However, SIGLEC15 could promoted HCC immune evasion in immune competent mouse models. Knock-out of Siglec15 could inhibit tumor growth and reinvigorate CD8+ T cell cytotoxicity. Anti-SIGLEC15 treatment could effectively inhibit tumor growth in mouse models with or without mononuclear phagocyte deletion. Bulk and single-cell RNA sequencing data of treated mouse tumors demonstrated SIGLEC15 could interfere CD8+ T cell viability and induce cell apoptosis. In all, SIGLEC15 was negatively correlated with PD-L1 in HCC and mainly promote HCC immune evasion through inhibition of CD8+ T cell viability and cytotoxicity.

The Structure Characteristics and Function of Non B Cell-Derived Immunoglobulin.

According to classical immunology theory, immunoglobulin (Ig) is exclusively produced by differentiated B lymphocytes, which exhibit a typical tetrapeptide chain structure and are predominantly present on the surface of B cells and in bodily fluids. B-Ig is one of the critical effector molecules for humoral immune responses specifically recognising antigens and eliminating them. However, mounting evidence has demonstrated that Ig is widely expressed in non B lineage cells, especially malignant ones (referred to as non B-Ig). Interestingly, non B-Ig mainly resides in the cytoplasm and secretion, but to some extent on the cell surface. Furthermore non B-Ig not only displays a tetrapeptide chain structure but also shows free heavy chains and free light chains (FLCs). Additionally, Ig derived from non B cancer cell typically displays unique glycosylation modifications. Functionally, non B-Ig demonstrated diversity and versatility, showing antibody activity and cellular biological activity, such as promoting cell proliferation and survival, and it is implicated in cancer progression and some immune-related diseases, such as renal diseases.

Single-cell spatial multiomics reveals tumor microenvironment vulnerabilities in cancer resistance to immunotherapy.

Heterogeneous resistance to immunotherapy remains a major challenge in cancer treatment, often leading to disease progression and death. Using CITE-seq and matched 40-plex PhenoCycler tissue imaging, we performed longitudinal multimodal single-cell analysis of tumors from metastatic melanoma patients with innate resistance, acquired resistance, or response to immunotherapy. We established the multimodal integration toolkit to align transcriptomic features, cellular epitopes, and spatial information to provide deeper insights into the tumors. With longitudinal analysis, we identified an "immune-striving" tumor microenvironment marked by peri-tumor lymphoid aggregates and low infiltration of T cells in the tumor and the emergence of MITF+SPARCL1+ and CENPF+ melanoma subclones after therapy. The enrichment of B cell-associated signatures in the molecular composition of lymphoid aggregates was associated with better survival. These findings provide further insights into the establishment of microenvironmental cell interactions and molecular composition of spatial structures that could inform therapeutic intervention.

Respiratory syncytial virus NS1 inhibits anti-viral Interferon-α-induced JAK/STAT signaling, by limiting the nuclear translocation of STAT1.

Human respiratory viruses are the most prevalent cause of disease in humans, with the highly infectious RSV being the leading cause of infant bronchiolitis and viral pneumonia. Responses to type I IFNs are the primary defense against viral infection. However, RSV proteins have been shown to antagonize type I IFN-mediated antiviral innate immunity, specifically dampening intracellular IFN signaling. Respiratory epithelial cells are the main target for RSV infection. In this study, we found RSV-NS1 interfered with the IFN-α JAK/STAT signaling pathway of epithelial cells. RSV-NS1 expression significantly enhanced IFN-α-mediated phosphorylation of STAT1, but not pSTAT2; and neither STAT1 nor STAT2 total protein levels were affected by RSV-NS1. However, expression of RSV-NS1 significantly reduced ISRE and GAS promoter activity and anti-viral IRG expression. Further mechanistic studies demonstrated RSV-NS1 bound STAT1, with protein modeling indicating a possible interaction site between STAT1 and RSV-NS1. Nuclear translocation of STAT1 was reduced in the presence of RSV-NS1. Additionally, STAT1's interaction with the nuclear transport adapter protein, KPNA1, was also reduced, suggesting a mechanism by which RSV blocks STAT1 nuclear translocation. Indeed, reducing STAT1's access to the nucleus may explain RSV's suppression of IFN JAK/STAT promoter activation and antiviral gene induction. Taken together these results describe a novel mechanism by which RSV controls antiviral IFN-α JAK/STAT responses, which enhances our understanding of RSV's respiratory disease progression.

MCP5, a methyl-accepting chemotaxis protein regulated by both the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, is required for the immune evasion of Borrelia burgdorferi.

Borrelia (or Borreliella) burgdorferi, the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well established as essential for its enzootic cycle, the function of methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B. burgdorferi remains unclear. In this study, we demonstrate that MCP5, one of the most abundant MCPs in B. burgdorferi, is differentially expressed in response to environmental signals as well as at different stages of the pathogen's enzootic cycle. Specifically, the expression of mcp5 is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, which are critical for the spirochete's colonization of the tick vector and mammalian host, respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 could not establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are defective in adaptive immunity, indicating the essential role of MCP5 in mammalian infection. However, the mcp5 mutant could establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting a crucial role of MCP5 in evading host innate immunity. In the tick vector, the mcp5 mutants survived feeding but failed to transmit to mice, highlighting the importance of MCP5 in transmission. Our findings reveal that MCP5, regulated by the Rrp1 and Rrp2 pathways, is critical for the establishment of infection in mammalian hosts by evading host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts, underscoring its potential as a target for intervention strategies.

Variable Surface Antigens of Plasmodium falciparum: Protein Families with Divergent Roles.

Malaria caused by Plasmodium falciparum (Pf) is an illness that contributes significantly to the global health burden. Pf makes significant alterations to the host cell to meet its metabolic demands and escape the immune response of the host. These include the export of a large number of parasite proteins to the infected Red Blood Cells (iRBC). Variable Surface Antigens (VSAs), which are highly polymorphic protein families with important roles in immune evasion, form an important component of the exported proteins. A total of five protein families constitute the VSAs, viz. PfEMP1 (Pf erythrocyte membrane protein 1), RIFIN (repetitive interspersed family), STEVOR (sub-telomeric open reading frame), SURFIN (surface-associated interspersed gene family), and PfMC-2TM (Pf Maurer's cleft two transmembrane). With orthologues present in various simian-infecting species, VSAs take up a variety of domain topologies and organizational structures while exhibiting differential expressions throughout the parasite life cycle. Their expression varies across clinical isolates and laboratory strains, which suggests their crucial role in host cell survival and defense. Members of VSAs are reported to contribute significantly to disease pathogenesis through immune evasion processes like cytoadherence, iRBC sequestration in the host vasculature, rosetting, reduced erythrocyte deformability, and direct immunosuppression. In this study, we have gathered information on various aspects of VSAs, like their orthologues, domain architecture, surface topology, functions and interactions, and three-dimensional structures, while emphasizing discoveries in the field. Considering the vast repertoire of Plasmodial VSAs with new emergent functions, a lot remains unknown about these families and, hence, malaria biology.

ETS homologous factor, controlled by lysine-specific demethylase 5B, suppresses clear cell renal cell carcinoma by inducing Filamin-B.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) remains a deadly disease with a poor prognosis. Here, we identified the ETS homologous factor (EHF) and its target Filamin-B (FLNB) as molecules related to immune evasion in ccRCC. We also explored the upstream modifier that manipulates EHF in ccRCC. DESIGN: Cell proliferation and apoptosis assay, wound healing assay, and Transwell assay were designed to analyze the effects of EHF or FLNB knockdown on the biological activity of ccRCC cells. The growth of differently treated ccRCC cells was assessed by orthotopic tumors. ccRCC cells with different treatments were co-cultured with macrophages, and the role of the lysine-specific demethylase 5B (KDM5B)/EHF/FLNB axis on macrophage polarization or ccRCC progression was characterized by detecting the expression of M2 macrophage markers in the co-culture system or tumor tissues of tumor-bearing mice. RESULTS: The expression of EHF and FLNB was higher, while KDM5B was lower in HK2 cells than in ccRCC cells. EHF overexpression inhibited the biological behavior of ccRCC cells and tumor growth in mice. EHF activated FLNB transcription. Knockdown of FLNB supported the biological activity of ccRCC cells and tumor growth and reversed M2 macrophage polarization in tumor tissues of mice in the presence of EHF. KDM5B inhibited EHF expression by H3K4me3 demethylation, and EHF knockdown potentiated M2 macrophage polarization and tumor growth in vivo repressed by KDM5B knockdown. CONCLUSIONS: KDM5B inhibited the expression of EHF by repressing H3K4me3 modification and the transcription of FLNB by EHF to promote immune evasion and progression of ccRCC.

Exploring the evolving roles and clinical significance of circRNAs in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) represents the predominant malignancies in the head and neck region, and has limited therapeutic alternatives. Circular RNAs (circRNAs), a substantial category of non-coding RNA molecules, exert influential roles in human disease development and progression, employing various mechanisms such as microRNA sponging, interaction with RNA-binding proteins, and translational capabilities. Accumulating evidence highlights the differential expression of numerous circRNAs in HNSCC, and numerous dysregulated circRNAs underscore their crucial involvement in malignant advancement and resistance to treatment. This review aims to comprehensively outline the characteristics, biogenesis, and mechanisms of circRNAs, elucidating their functional significance in HNSCC. In addition, we delve into the clinical implications of circRNAs, considering their potential as biomarkers or targets for diagnosis, prognosis, and therapeutic applications in HNSCC. The discussion extends to exploring future challenges in the clinical translation of circRNAs, emphasizing the need for further research.

Antibody drugs targeting SARS-CoV-2: Time for a rethink?

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) heavily burdens human health. Multiple neutralizing antibodies (nAbs) have been issued for emergency use or tested for treating infected patients in the clinic. However, SARS-CoV-2 variants of concern (VOC) carrying mutations reduce the effectiveness of nAbs by preventing neutralization. Uncoding the mutation profile and immune evasion mechanism of SARS-CoV-2 can improve the outcome of Ab-mediated therapies. In this review, we first outline the development status of anti-SARS-CoV-2 Ab drugs and provide an overview of SARS-CoV-2 variants and their prevalence. We next focus on the failure causes of anti-SARS-CoV-2 Ab drugs and rethink the design strategy for developing new Ab drugs against COVID-19. This review provides updated information for the development of therapeutic Ab drugs against SARS-CoV-2 variants.

The Pathogenesis of Foot-and-Mouth Disease Virus Infection: How the Virus Escapes from Immune Recognition and Elimination.

Foot-and-mouth disease virus (FMDV) is a highly contagious and economically devastating pathogen that affects cloven-hoofed animals worldwide. FMDV infection causes vesicular lesions in the mouth, feet, and mammary glands, as well as severe systemic symptoms such as fever, salivation, and lameness. The pathogenesis of FMDV infection involves complex interactions between the virus and the host immune system, which determine the outcome of the disease. FMDV has evolved several strategies to evade immune recognition and elimination, such as antigenic variation, receptor switching, immune suppression, and subversion of innate and adaptive responses. This review paper summarizes the current knowledge on the pathogenesis of FMDV infection and the mechanisms of immune evasion employed by the virus. It also discusses the challenges and opportunities for developing effective vaccines and therapeutics against this important animal disease.

Epigenetic regulation of major histocompatibility complexes in gastrointestinal malignancies and the potential for clinical interception.

BACKGROUND: Gastrointestinal malignancies encompass a diverse group of cancers that pose significant challenges to global health. The major histocompatibility complex (MHC) plays a pivotal role in immune surveillance, orchestrating the recognition and elimination of tumor cells by the immune system. However, the intricate regulation of MHC gene expression is susceptible to dynamic epigenetic modification, which can influence functionality and pathological outcomes. MAIN BODY: By understanding the epigenetic alterations that drive MHC downregulation, insights are gained into the molecular mechanisms underlying immune escape, tumor progression, and immunotherapy resistance. This systematic review examines the current literature on epigenetic mechanisms that contribute to MHC deregulation in esophageal, gastric, pancreatic, hepatic and colorectal malignancies. Potential clinical implications are discussed of targeting aberrant epigenetic modifications to restore MHC expression and 0 the effectiveness of immunotherapeutic interventions. CONCLUSION: The integration of epigenetic-targeted therapies with immunotherapies holds great potential for improving clinical outcomes in patients with gastrointestinal malignancies and represents a compelling avenue for future research and therapeutic development.

Research progress on the nonstructural protein 1 (NS1) of influenza a virus.

Influenza A virus (IAV) is the leading cause of highly contagious respiratory infections, which poses a serious threat to public health. The non-structural protein 1 (NS1) is encoded by segment 8 of IAV genome and is expressed in high levels in host cells upon IAV infection. It is the determinant of virulence and has multiple functions by targeting type Ι interferon (IFN-I) and type III interferon (IFN-III) production, disrupting cell apoptosis and autophagy in IAV-infected cells, and regulating the host fitness of influenza viruses. This review will summarize the current research on the NS1 including the structure and related biological functions of the NS1 as well as the interaction between the NS1 and host cells. It is hoped that this will provide some scientific basis for the prevention and control of the influenza virus.