CASTOR1 phosphorylation predicts poor survival in male patients with KRAS-mutated lung adenocarcinoma.

BACKGROUND: Lung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. We have previously shown a tumor suppressive role of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), which is targeted for degradation upon phosphorylation at S14 (pCASTOR1) in multiple types of cancer. This study focuses on the predictive value of pCASTOR1 in lung adenocarcinoma (LUAD) patients with KRAS mutations. RESULTS: Employing a newly developed pCASTOR1 specific antibody, we found that tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells (P < 0.05). Higher pCASTOR1 scores predicted poorer overall survival (OS) (HR = 3.3, P = 0.0008) and relapse-free survival (RFS) (HR = 3.0, P = 0.0035) in male patients with KRAS mutations. pCASTOR1 remained an independent predictor for OS (HR = 4.1, P = 0.0047) and RFS (HR = 3.5, P = 0.0342) after controlling for other factors. Notably, in early-stage LUAD, elevated pCASTOR1 scores were associated with significantly worse OS (HR = 3.3, P = 0.0176) and RFS (HR = 3.1, P = 0.0277) in male patients with KRAS mutations, akin to late-stage patients. CONCLUSION: Elevated pCASTOR1 scores serve as biomarkers predicting poorer OS and RFS in male LUAD patients with KRAS mutations, offering potential clinical utility in optimizing treatment strategies for this subgroup.

Lactylation of RNA m6A demethylase ALKBH5 promotes innate immune response to DNA herpesviruses and mpox virus.

RNA N6-methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) plays a crucial role in regulating innate immunity. Lysine acylation, a widespread protein modification, influences protein function, but its impact on ALKBH5 during viral infections has not been well characterized. This study investigates the presence and regulatory mechanisms of a previously unidentified lysine acylation in ALKBH5 and its role in mediating m6A modifications to activate antiviral innate immune responses. We demonstrate that ALKBH5 undergoes lactylation, which is essential for an effective innate immune response against DNA herpesviruses, including herpes simplex virus type 1 (HSV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), and mpox virus (MPXV). This lactylation attenuates viral replication. Mechanistically, viral infections enhance ALKBH5 lactylation by increasing its interaction with acetyltransferase ESCO2 and decreasing its interaction with deacetyltransferase SIRT6. Lactylated ALKBH5 binds interferon-beta (IFN-ß) messenger RNA (mRNA), leading to demethylation of its m6A modifications and promoting IFN-ß mRNA biogenesis. Overexpression of ESCO2 or depletion of SIRT6 further enhances ALKBH5 lactylation to strengthen IFN-ß mRNA biogenesis. Our results identify a posttranslational modification of ALKBH5 and its role in regulating antiviral innate immune responses through m6A modification. The finding provides an understanding of innate immunity and offers a potential therapeutic target for HSV-1, KSHV, and MPXV infections.

The loss of hepatitis B virus receptor NTCP/SLC10A1 in human liver cancer cells is due to epigenetic silencing.

Human Na+-taurocholate cotransporting polypeptide (hNTCP) is predominantly expressed in hepatocytes, maintaining bile salt homeostasis and serving as a receptor for hepatitis B virus (HBV). hNTCP expression is downregulated during hepatocellular carcinoma (HCC) development. In this study, we investigated the molecular mechanisms underlying hNTCP dysregulation using HCC tissues and cell lines, and primary human hepatocytes (PHHs). Firstly, we observed a significant reduction of hNTCP in HCC tumors compared to adjacent and normal tissues. Additionally, hNTCP mRNA levels were markedly lower in HepG2 cells compared to PHHs, which was corroborated at the protein level by immunoblotting. Sanger sequencing confirmed identical sequences for hNTCP promoter, exons, and mRNA coding sequences between PHH and HepG2 cells, indicating no mutations or splicing alterations. We then assessed the epigenetic status of hNTCP. The hNTCP promoter, with low CG content, showed no significant methylation differences between PHH and HepG2 cells. Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) revealed a loss of activating histone posttranslational modification (PTM) H3K27ac near the hNTCP transcription start site (TSS) in HepG2 cells. This loss was also confirmed in HCC tumor cells compared to adjacent and background cells. Treating HepG2 cells with histone deacetylase inhibitors enhanced H3K27ac accumulation and glucocorticoid receptor (GR) binding at the hNTCP TSS, significantly increasing hNTCP mRNA and protein levels, and rendering the cells susceptible to HBV infection. In summary, histone PTM-related epigenetic mechanisms play a critical role in hNTCP dysregulation in liver cancer cells, providing insights into hepatocarcinogenesis and its impact on chronic HBV infection. IMPORTANCE: HBV is a hepatotropic virus that infects human hepatocytes expressing the viral receptor hNTCP. Without effective antiviral therapy, chronic HBV infection poses a high risk of liver cancer. However, most liver cancer cell lines, including HepG2 and Huh7, do not support HBV infection due to the absence of hNTCP expression, and the mechanism underlying this defect remains unclear. This study demonstrates a significant reduction of hNTCP in hepatocellular carcinoma samples and HepG2 cells compared to normal liver tissues and primary human hepatocytes. Despite identical hNTCP genetic sequences, epigenetic analyses revealed a loss of the activating histone modification H3K27ac near the hNTCP transcription start site in cancer cells. Treatment with histone deacetylase inhibitors restored H3K27ac levels, reactivated hNTCP expression, and rendered HepG2 cells susceptible to HBV infection. These findings highlight the role of epigenetic modulation in hNTCP dysregulation, offering insights into hepatocarcinogenesis and its implications for chronic HBV infection.

Understanding YTHDF2-mediated mRNA degradation by m6A-BERT-Deg.

N6-methyladenosine (m6A) is the most abundant mRNA modification within mammalian cells, holding pivotal significance in the regulation of mRNA stability, translation and splicing. Furthermore, it plays a critical role in the regulation of RNA degradation by primarily recruiting the YTHDF2 reader protein. However, the selective regulation of mRNA decay of the m6A-methylated mRNA through YTHDF2 binding is poorly understood. To improve our understanding, we developed m6A-BERT-Deg, a BERT model adapted for predicting YTHDF2-mediated degradation of m6A-methylated mRNAs. We meticulously assembled a high-quality training dataset by integrating multiple data sources for the HeLa cell line. To overcome the limitation of small training samples, we employed a pre-training-fine-tuning strategy by first performing a self-supervised pre-training of the model on 427 760 unlabeled m6A site sequences. The test results demonstrated the importance of this pre-training strategy in enabling m6A-BERT-Deg to outperform other benchmark models. We further conducted a comprehensive model interpretation and revealed a surprising finding that the presence of co-factors in proximity to m6A sites may disrupt YTHDF2-mediated mRNA degradation, subsequently enhancing mRNA stability. We also extended our analyses to the HEK293 cell line, shedding light on the context-dependent YTHDF2-mediated mRNA degradation.

KSHV vIL-6 promotes SIRT3-induced deacetylation of SERBP1 to inhibit ferroptosis and enhance cellular transformation by inducing lipoyltransferase 2 mRNA degradation.

Ferroptosis, a defensive strategy commonly employed by the host cells to restrict pathogenic infections, has been implicated in the development and therapeutic responses of various types of cancer. However, the role of ferroptosis in oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-induced cancers remains elusive. While a growing number of non-histone proteins have been identified as acetylation targets, the functions of these modifications have yet to be revealed. Here, we show KSHV reprogramming of host acetylation proteomics following cellular transformation of rat primary mesenchymal precursor. Among them, SERPINE1 mRNA binding protein 1 (SERBP1) deacetylation is increased and required for KSHV-induced cellular transformation. Mechanistically, KSHV-encoded viral interleukin-6 (vIL-6) promotes SIRT3 deacetylation of SERBP1, preventing its binding to and protection of lipoyltransferase 2 (Lipt2) mRNA from mRNA degradation resulting in ferroptosis. Consequently, a SIRT3-specific inhibitor, 3-TYP, suppresses KSHV-induced cellular transformation by inducing ferroptosis. Our findings unveil novel roles of vIL-6 and SERBP1 deacetylation in regulating ferroptosis and KSHV-induced cellular transformation, and establish the vIL-6-SIRT3-SERBP1-ferroptosis pathways as a potential new therapeutic target for KSHV-associated cancers.

reguloGPT: Harnessing GPT for Knowledge Graph Construction of Molecular Regulatory Pathways.

Motivation: Molecular Regulatory Pathways (MRPs) are crucial for understanding biological functions. Knowledge Graphs (KGs) have become vital in organizing and analyzing MRPs, providing structured representations of complex biological interactions. Current tools for mining KGs from biomedical literature are inadequate in capturing complex, hierarchical relationships and contextual information about MRPs. Large Language Models (LLMs) like GPT-4 offer a promising solution, with advanced capabilities to decipher the intricate nuances of language. However, their potential for end-to-end KG construction, particularly for MRPs, remains largely unexplored. Results: We present reguloGPT, a novel GPT-4 based in-context learning prompt, designed for the end-to-end joint name entity recognition, N-ary relationship extraction, and context predictions from a sentence that describes regulatory interactions with MRPs. Our reguloGPT approach introduces a context-aware relational graph that effectively embodies the hierarchical structure of MRPs and resolves semantic inconsistencies by embedding context directly within relational edges. We created a benchmark dataset including 400 annotated PubMed titles on N6-methyladenosine (m6A) regulations. Rigorous evaluation of reguloGPT on the benchmark dataset demonstrated marked improvement over existing algorithms. We further developed a novel G-Eval scheme, leveraging GPT-4 for annotation-free performance evaluation and demonstrated its agreement with traditional annotation-based evaluations. Utilizing reguloGPT predictions on m6A-related titles, we constructed the m6A-KG and demonstrated its utility in elucidating m6A's regulatory mechanisms in cancer phenotypes across various cancers. These results underscore reguloGPT's transformative potential for extracting biological knowledge from the literature. Availability and implementation: The source code of reguloGPT, the m6A title and benchmark datasets, and m6A-KG are available at: https://github.com/Huang-AI4Medicine-Lab/reguloGPT.

Understanding YTHDF2-mediated mRNA Degradation By m6A-BERT-Deg.

N6-methyladenosine (m6A) is the most abundant mRNA modification within mammalian cells, holding pivotal significance in the regulation of mRNA stability, translation, and splicing. Furthermore, it plays a critical role in the regulation of RNA degradation by primarily recruiting the YTHDF2 reader protein. However, the selective regulation of mRNA decay of the m6A-methylated mRNA through YTHDF2 binding is poorly understood. To improve our understanding, we developed m6A-BERT-Deg, a BERT model adapted for predicting YTHDF2-mediated degradation of m6A-methylated mRNAs. We meticulously assembled a high-quality training dataset by integrating multiple data sources for the HeLa cell line. To overcome the limitation of small training samples, we employed a pre-training-fine-tuning strategy by first performing a self-supervised pre-training of the model on 427,760 unlabeled m6A site sequences. The test results demonstrated the importance of this pre-training strategy in enabling m6A-BERT-Deg to outperform other benchmark models. We further conducted a comprehensive model interpretation and revealed a surprising finding that the presence of co-factors in proximity to m6A sites may disrupt YTHDF2-mediated mRNA degradation, subsequently enhancing mRNA stability. We also extended our analyses to the HEK293 cell line, shedding light on the context-dependent YTHDF2-mediated mRNA degradation.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

Hyperinflammation is the hallmark of Kaposi's sarcoma (KS), the most common cancer in AIDS patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. However, the role and mechanism of induction of inflammation in KS remain unclear. In a screening for inhibitors of KSHV-induced oncogenesis, over half of the identified candidates were anti-inflammatory agents including dexamethasone functions by activating glucocorticoid receptor (GR) signaling. Here, we examined the mechanism mediating KSHV-induced inflammation. We found that numerous inflammatory pathways were activated in KSHV-transformed cells. Particularly, interleukin-1 alpha (IL-1α) and IL-1 receptor antagonist (IL-1Ra) from the IL-1 family were the most induced and suppressed cytokines, respectively. We found that KSHV miRNAs mediated IL-1α induction while both miRNAs and vFLIP mediated IL-1Ra suppression. Furthermore, GR signaling was inhibited in KSHV-transformed cells, which was mediated by vFLIP and vCyclin. Dexamethasone treatment activated GR signaling, and inhibited cell proliferation and colony formation in soft agar of KSHV-transformed cells but had a minimal effect on matched primary cells. Consequently, dexamethasone suppressed the initiation and growth of KSHV-induced tumors in mice. Mechanistically, dexamethasone suppressed IL-1α but induced IL-1Ra expression. Treatment with recombinant IL-1α protein rescued the inhibitory effect of dexamethasone while overexpression of IL-1Ra caused a weak growth inhibition of KSHV-transformed cells. Furthermore, dexamethasone induced IκBα expression resulting in inhibition of NF-κB pathway and IL-1α expression. These results reveal an important role of IL-1 pathway in KSHV-induced inflammation and oncogenesis, which can be inhibited by dexamethasone-activated GR signaling, and identify IL-1-mediated inflammation as a potential therapeutic target for KSHV-induced malignancies.

NAT10-dependent N4-acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation.

N-acetyltransferase 10 (NAT10) is an N4-acetylcytidine (ac4C) writer that catalyzes RNA acetylation at cytidine N4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac4C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac4C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac4C sites in PAN RNA in the context of KSHV infection abolishes PAN ac4C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac4C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac4C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac4C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac4C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.

METTL16 controls Kaposi's sarcoma-associated herpesvirus replication by regulating S-adenosylmethionine cycle.

Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) consists of latent and lytic replication phases, both of which are important for the development of KSHV-related cancers. As one of the most abundant RNA modifications, N6-methyladenosine (m6A) and its related complexes regulate KSHV life cycle. However, the role of METTL16, a newly discovered RNA methyltransferase, in KSHV life cycle remains unknown. In this study, we have identified a suppressive role of METTL16 in KSHV lytic replication. METTL16 knockdown increased while METTL16 overexpression reduced KSHV lytic replication. METTL16 binding to and writing of m6A on MAT2A transcript are essential for its splicing, maturation and expression. As a rate-limiting enzyme in the methionine-S-adenosylmethionine (SAM) cycle, MAT2A catalyzes the conversion of L-methionine to SAM required for the transmethylation of protein, DNA and RNA, transamination of polyamines, and transsulfuration of cystathionine. Consequently, knockdown or chemical inhibition of MAT2A reduced intracellular SAM level and enhanced KSHV lytic replication. In contrast, SAM treatment was sufficient to inhibit KSHV lytic replication and reverse the effect of the enhanced KSHV lytic program caused by METTL16 or MAT2A knockdown. Mechanistically, METTL16 or MAT2A knockdown increased while SAM treatment decreased the intracellular reactive oxygen species level by altering glutathione level, which is essential for efficient KSHV lytic replication. These findings demonstrate that METTL16 suppresses KSHV lytic replication by modulating the SAM cycle to maintain intracellular SAM level and redox homeostasis, thus illustrating the linkage of KSHV life cycle with specific m6A modifications, and cellular metabolic and oxidative conditions.

Molecular and immune signatures, and pathological trajectories of fatal COVID-19 lungs defined by in situ spatial single-cell transcriptome analysis.

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. In this study, we investigated the spatial single-cell molecular and cellular features of postmortem COVID-19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole-slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS-CoV-2. Compared with the non-COVID-19 control, COVID-19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-ß-SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single-cell atlas of cellular and molecular signatures of fatal COVID-19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

COVID-19db linkage maps of cell surface proteins and transcription factors in immune cells.

The highly contagious SARS-CoV-2 and its associated disease (COVID-19) are a threat to global public health and economies. To develop effective treatments for COVID-19, we must understand the host cell types, cell states and regulators associated with infection and pathogenesis such as dysregulated transcription factors (TFs) and surface proteins, including signaling receptors. To link cell surface proteins with TFs, we recently developed SPaRTAN (Single-cell Proteomic and RNA-based Transcription factor Activity Network) by integrating parallel single-cell proteomic and transcriptomic data based on Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and gene cis-regulatory information. We apply SPaRTAN to CITE-seq data sets from patients with varying degrees of COVID-19 severity and healthy controls to identify the associations between surface proteins and TFs in host immune cells. Here, we present COVID-19db of Immune Cell States (https://covid19db.streamlit.app/), a web server containing cell surface protein expression, SPaRTAN-inferred TF activities, and their associations with major host immune cell types. The data include four high-quality COVID-19 CITE-seq data sets with a toolset for user-friendly data analysis and visualization. We provide interactive surface protein and TF visualizations across major immune cell types for each data set, allowing comparison between various patient severity groups for the discovery of potential therapeutic targets and diagnostic biomarkers.

Immune evasion strategy involving propionylation by the KSHV interferon regulatory factor 1 (vIRF1).

Post-translational modifications (PTMs) are essential for host antiviral immune response and viral immune evasion. Among a set of novel acylations, lysine propionylation (Kpr) has been detected in both histone and non-histone proteins. However, whether protein propionylation occurs in any viral proteins and whether such modifications regulate viral immune evasion remain elusive. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) can be propionylated in lysine residues, which is required for effective inhibition of IFN-ß production and antiviral signaling. Mechanistically, vIRF1 promotes its own propionylation by blocking SIRT6's interaction with ubiquitin-specific peptidase 10 (USP10) leading to its degradation via a ubiquitin-proteasome pathway. Furthermore, vIRF1 propionylation is required for its function to block IRF3-CBP/p300 recruitment and repress the STING DNA sensing pathway. A SIRT6-specific activator, UBCS039, rescues propionylated vIRF1-mediated repression of IFN-ß signaling. These results reveal a novel mechanism of viral evasion of innate immunity through propionylation of a viral protein. The findings suggest that enzymes involved in viral propionylation could be potential targets for preventing viral infections.

KSHV hijacks FoxO1 to promote cell proliferation and cellular transformation by antagonizing oxidative stress.

Reactive oxygen species (ROS) are a group of a highly short-lived molecules that control diverse behaviors of cells. Normal cells maintain ROS balance to ensure their functions. Because of oncogenic stress, cancer cells often have excessive ROS, also known as oxidative stress, which are often counteracted by enhanced antioxidant systems to maintain redox homeostasis. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with Kaposi's sarcoma (KS), which manifests hyper inflammation and oxidative stress as the hallmarks. We have previously shown that excessive ROS can disrupt KSHV latency by inducing viral lytic replication, leading to cell death. Paradoxically, most KS tumor cells are latently infected by KSHV in a highly inflammatory and oxidative stress tumor microenvironment, which is in part due to the activation of alternative complement and TLR4 pathways, indicating the existence of an enhanced antioxidant defense system in KS tumor cells. In this study, we show that KSHV upregulates antioxidant genes, including SOD2 and CAT by hijacking the forkhead box protein O1 (FoxO1), to maintain intracellular ROS level. Moreover, the fine-tuned balance of ROS level in KSHV-transformed cells is essential for cell survival. Consequently, KSHV-transformed cells are extremely sensitive to exogenous ROS insult such as treatment with a low level of hydrogen peroxide (H2 O2 ). Either chemical inhibition or knockdown of FoxO1 by short interfering RNAs decreases the expression of antioxidant genes and subsequently increases the intracellular ROS level in KSHV-transformed cells, resulting in the inhibition of cell proliferation and colony formation in soft agar. Mechanistically, KSHV-encoded microRNAs and vFLIP upregulate FoxO1 by activating the NF-κB pathway. These results reveal a novel mechanism by which an oncogenic virus counteracts oxidative stress by upregulating FoxO1, which is essential for KSHV-induced cell proliferation and cellular transformation. Therefore, FoxO1 might be a potential therapeutic target for KSHV-related malignancies.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1ß (IL-1ß), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1ß, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.

Infectivity of pseudotyped SARS-CoV-2 variants of concern in different human cell types and inhibitory effects of recombinant spike protein and entry-related cellular factors.

Since the report of the first COVID-19 case in 2019, SARS-CoV-2 variants of concern (VOCs) have continued to emerge, manifesting diverse infectivity, evasion of host immunity and pathology. While ACE2 is the predominant receptor of SARS-CoV-2, TMPRSS2, Kim-1, NRP-1, CD147, furin, CD209L, and CD26 have also been implicated as viral entry-related cofactors. To understand the variations in infectivity and pathogenesis of VOCs, we conducted infection analysis in human cells from different organ systems using pseudoviruses of VOCs including Alpha, Beta, Gamma, and Delta. Recombinant spike S1, RBD, ACE2, Kim-1, and NRP-1 proteins were tested for their ability to block infection to dissect their roles in SARS-CoV-2 entry into cells. Compared with wild type SARS-CoV-2 (WT), numerous VOCs had significant increases of infectivity across a wide spectrum of cell types. Recombinant ACE2 protein more effectively inhibited the infection of VOCs including Delta and Omicron (BA.1 and BA.2) than that of WT. Interestingly, recombinant S1, RBD, Kim-1, and NRP-1 proteins inhibited the infection of all pseudoviruses in a manner dependent on the levels of ACE2 expression in different cell types. These results provide insights into the diverse infectivity of SARS-CoV-2 VOCs, which might be helpful for managing the emergence of new VOCs.

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp).

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.

Ultrarapid and ultrasensitive detection of SARS-CoV-2 antibodies in COVID-19 patients via a 3D-printed nanomaterial-based biosensing platform.

Rapid detection of antibodies during infection and after vaccination is critical for the control of infectious outbreaks, understanding immune response, and evaluating vaccine efficacy. In this manuscript, we evaluate a simple ultrarapid test for SARS-CoV-2 antibodies in COVID-19 patients, which gives quantitative results (i.e., antibody concentration) in 10-12 s using a previously reported nanomaterial-based three-dimensional (3D)-printed biosensing platform. This platform consists of a micropillar array electrode fabricated via 3D printing of aerosolized gold nanoparticles and coated with nanoflakes of graphene and specific SARS-CoV-2 antigens, including spike S1, S1 receptor-binding domain (RBD) and nucleocapsid (N). The sensor works on the principle of electrochemical transduction, where the change of sensor impedance is realized by the interactions between the viral proteins attached to the sensor electrode surface and the antibodies. The three sensors were used to test samples from 17 COVID-19 patients and 3 patients without COVID-19. Unlike other serological tests, the 3D sensors quantitatively detected antibodies at a concentration as low as picomole within 10-12 s in human plasma samples. We found that the studied COVID-19 patients had higher concentrations of antibodies to spike proteins (RBD and S1) than to the N protein. These results demonstrate the enormous potential of the rapid antibody test platform for understanding patients' immunity, disease epidemiology and vaccine efficacy, and facilitating the control and prevention of infectious epidemics.