Biology subjects

Hou, Y.

Publications and source records attributed to Hou, Y..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

Fibrinolysis influences SARS-CoV-2 infection in ciliated cells

Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host proteases, may cleave the furin site of SARS-CoV-2 S protein and {gamma} subunits of epithelial sodium channels ({gamma} ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU (encoding urokinase plasminogen activator), SCNN1G ({gamma}ENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated edematous respiratory condition.

bioinformatics↗

bioRxiv2020

High-throughput Single-cell CNV Detection Reveals Clonal Evolution During Hepatocellular Carcinoma Recurrence

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution, but classical DNA amplification-based methods are low-throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN). The method has a throughput of up to 1,800 cells per run for copy number variation (CNV) detection. Also, it has a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy based on evaluation in cell lines and tumour tissues. We used this approach to profile the tumour clones in paired primary and relapsed tumour samples of hepatocellular carcinoma (HCC). We identified 3 clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumour containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumour, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

cell biology↗

bioRxiv2020

Massively parallel sequencing of 165 ancestry-informative SNPs and forensic biogeographical ancestry inference in three southern Chinese Sinitic/Tai-Kadai populations

Ancestry informative markers (AIMs), which are distributed throughout the human genome, harbor significant allele frequency differences among diverse ethnic groups. The use of sets of AIMs to reconstruct population history and genetic relationships is attracting interest in the forensic community, because biogeographic ancestry information for a casework sample can potentially be predicted and used to guide the investigative process. However, subpopulation ancestry inference within East Asia remains in its infancy due to a lack of population reference data collection and incomplete validation work on newly developed or commercial AIM sets. In the present study, 316 Chinese persons, including 85 Sinitic-speaking Haikou Han, 120 Qiongzhong Hlai and 111 Daozhen Gelao individuals belonging to Tai-Kadai-speaking populations, were analyzed using the Precision ID Ancestry Panel (165 AISNPs). Combined with our previous 165-AISNP data (375 individuals from 6 populations), the 1000 Genomes Project and forensic literature, comprehensive population genetic comparisons and ancestry inference were further performed via ADMIXTURE, TreeMix, PCA, f-statistics and N-J tree. Although several nonpolymorphic loci were identified in the three southern Chinese populations, the forensic parameters of this ancestry inference panel were better than those for the 23 STR-based Huaxia Platinum System, which is suitable for use as a robust tool in forensic individual identification and parentage testing. The results based on the ancestry assignment and admixture proportion evaluation revealed that this panel could be used successfully to assign individuals at a continental scale but also possessed obvious limitations in discriminatory power in intercontinental individuals, especially for European-Asian admixed Uyghurs or in populations lacking reference databases. Population genetic analyses further revealed five continental population clusters and three East Asian-focused population subgroups, which is consistent with linguistic affiliations. Ancestry composition and multiple phylogenetic analysis further demonstrated that the geographically isolated Qiongzhong Hlai harbored a close phylogenetic relationship with Austronesian speakers and possessed a homogenous Tai-Kadai-dominant ancestry, which could be used as the ancestral source proxy in population history reconstruction of Tai-Kadai-speaking populations and as one of the representatives for forensic database establishment. In summary, more population-specific AIM sets focused on East Asian subpopulations, comprehensive algorithms and high-coverage population reference data should be developed and validated in the next step.

genetics↗

bioRxiv2020

Multimodal Single-Cell Omics Analysis of COVID-19 Sex Differences in Human Immune Systems

Sex differences in the risk of SARS-CoV-2 infection have been controversial and the underlying mechanisms of COVID-19 sexual dimorphism remain understudied. Here we inspected sex differences in SARS-CoV-2 positivity, hospitalization, admission to the intensive care unit (ICU), sera immune profiling, and two single-cell RNA-sequencing (snRNA-seq) profiles from nasal tissues and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with varying degrees of disease severity. Our propensity score-matching observations revealed that male individuals have a 29% increased likelihood of SARS-CoV-2 positivity, with a hazard ration (HR) 1.32 (95% confidence interval [CI] 1.18-1.48) for hospitalization and HR 1.51 (95% CI 1.24-1.84) for admission to ICU. Sera from male patients at hospital admission had decreased lymphocyte count and elevated inflammatory markers (C-reactive protein, procalcitonin, and neutrophils). We found that SARS-CoV-2 entry factors, including ACE2, TMPRSS2, FURIN and NRP1, have elevated expression in nasal squamous cells from males with moderate and severe COVID-19. Cell-cell network proximity analysis suggests possible epithelium-immune cell interactions and immune vulnerability underlying a higher mortality in males with COVID-19. Monocyte-elevated expression of Toll like receptor 7 (TLR7) and Bruton tyrosine kinase (BTK) is associated with severe outcomes in males with COVID-19. These findings provide basis for understanding immune responses underlying sex differences, and designing sex-specific targeted treatments and patient care for COVID-19.

systems biology↗

bioRxiv2020

Methyltransferase SMYD5 Exaggerates IBD by Downregulating Mitochondrial Functions via Post-translational Control of PGC-1α Stability

Background and AimsThe expression and role of methyltransferase SET and MYND domain-containing protein 5 (SMYD5) in inflammatory bowel diseases (IBD) is completely unknown. Here, we investigated the role and the underlying mechanism of epithelial SMYD5 in IBD pathogenesis and progression. MethodsThe expression and subcellular localization of SMYD5 and peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) were examined by Western blot analysis, immunofluorescence staining, and immunohistochemistry in intestinal epithelial cells (IECs) and in colon tissues from human IBD patients and mice with experimental colitis. Mice with Smyd5 conditional knockout in IECs and littermate controls were subjected to DSS-induced experimental colitis and the disease severity and inflammation were assessed. SMYD5-regulated mitochondrial biogenesis was examined by RT-qPCR and transmission electron microscopy and mitochondrial oxygen consumption rate was measured in a Seahorse Analyzer system. The interaction between SMYD5 and PGC-1 was determined by co-immunoprecipitation assay. PGC-1 degradation and turnover (half-life) were analyzed by cycloheximide chase assay. SMYD5-mediated PGC-1 methylation was measured via in vitro methylation followed by mass spectrometry to identify the specific lysine residues that were methylated. ResultsUp-regulated SMYD5 and down-regulated PGC-1 were observed in IECs from IBD patients and mice with experimental colitis. However, Smyd5 depletion in IECs protected mice from DSS-induced colitis. SMYD5 was critically involved in regulating mitochondrial biology such as mitochondrial biogenesis, respiration, and apoptosis. Mechanistically, SMYD5 regulated mitochondrial functions in a PGC-1 dependent manner. Further, SMYD5 mediated lysine methylation of PGC-1 and facilitated its ubiquitination and proteasomal degradation. ConclusionSMYD5 attenuates mitochondrial functions in IECs and promotes IBD progression by enhancing the proteasome-mediated degradation of PGC-1 protein in methylation-dependent manner. Strategies to decrease SMYD5 expression and/or increase PGC-1 expression in IECs might be a promising therapeutic approach to treat patients with IBD.

cell biology↗

bioRxiv2020

Molecular Parallelisms and Divergences Between Human and Canine Mammary Cancer

Canine mammary cancer is poorly characterized at the genomic level. Dog really can be an appropriate experimental model for human cancers from the genomic and evolutionary perspective or not? Here, we perform a cross-species cancer genomics analysis, independent evolution of cancer from normal tissues, which provide us an excellent opportunity to address an evolutionary perspective of cancer. As, evolutionary theories are critical for understanding tumorigenesis at the level of species as well as at the level of cells and tissues, for the development of effective therapies. Analysis of canine mammary cancer reveals a diversity of histological types as compare to human breast cancer. Our systematic analysis of 24 canine mammary tumors with whole-genome sequences, reveals 185 protein-coding cancer genes carried exonic mutations. Cross-species comparative analysis of 1080 human breast cancers identifies higher median mutation frequency in human breast cancer and canine mammary cancer shows lower across exonic regions (2.67 and 0.187 average no. of mutations per tumor per megabase (Mb), respectively). A comparison of somatic mutations in the PIK3CA gene, reveals common recurrence of the conserved mutations, in both species. However, the Ka/Ks ratio in the human PIK3CA gene 2.37 is higher and 1.43 in dogs is lower. To address the mutation accumulation and antagonistic pleiotropy theory, we investigated Ka/Ks value 237 aging-related genes from human and canine, the aging-related genes do not show selection in canine mammary cancer. It demonstrates new aspects of cancer genes that are evolving in different species instantaneously. These findings may suggest, the same organs in different mammals impose different selective pressures on the same set of genes in cancer. In both species, some genes may experience strong selective pressures, but do not converge genetically or the conserved genes do not show the same selection pressure in both species. However, human breast cancer shows transcriptomic similarity with canine mammary carcinoma but the other subtypes are quite different. We found canine mammary tumor can be used as a model for inter and intra-tumor heterogeneity. These findings provided insight into mammary cancer across species and possessed potential clinical significance. Collectively, these studies suggest a convergence of some genetic changes in mammary cancer between species but also distinctly different paths to tumorigenesis.

genomics↗

bioRxiv2020

Comprehensive characterization of protein-protein interaction network perturbations by human disease mutations

Technological and computational advances in genomics and interactomics have made it possible to identify rapidly how disease mutations perturb interaction networks within human cells. In this study, we investigate at large-scale the effects of network perturbations caused by disease mutations within the human three-dimensional (3D), structurally-resolved macromolecular interactome. We show that disease-associated germline mutations are significantly enriched in sequences encoding protein-protein interfaces compared to mutations identified in healthy subjects from the 1000 Genomes and ExAC projects; these interface mutations correspond to protein-protein interaction (PPI)-perturbing alleles including p.Ser127Arg in PCSK9 at the PCSK9-LDLR interface. In addition, somatic missense mutations are significantly enriched in PPI interfaces compared to non-interfaces in 10,861 human exomes across 33 cancer subtypes/types from The Cancer Genome Atlas. Using a binomial statistical model, we computationally identified 470 PPIs harboring a statistically significant excess number of missense mutations at protein-protein interfaces (termed putative oncoPPIs) in pan-cancer analysis. We demonstrate that the oncoPPIs, including histone H4 complex in individual cancer types, are highly correlated with patient survival and drug resistance/sensitivity in human cancer cell lines and patient-derived xenografts. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay. We further showed that ALOX5 p.Met146Lys at the ALOX5-MAD1L1 interface and RXRA p.Ser427Phe at the RXRA-PPARG interface promote significant tumor cell growth using cell line-based functional assays, providing a functional proof-of-concept. In summary, if broadly applied, this human 3D interactome network analysis offers a powerful tool for prioritizing alleles with mutations altering PPIs that may contribute to the pathobiology of human diseases, and may offer disease-specific targets for genotype-informed therapeutic discovery.

systems biology↗

bioRxiv2020

Caloric Restriction Remodels Energy Metabolic Pathways of Gut Microbiota and Promotes Host Autophagy

Calorie restriction (CR) can improve the metabolic balance of adults and elevate the relative abundance of probiotic bacteria in the gut while promoting longevity. However, the interaction between remodeled intestinal flora and metabolic improvement, as well as the mechanism for probiotic bacterial increase, are still unclear. In this study, using a metabolomics platform, we demonstrate for the first time, that CR leads to increased levels of malate and its related metabolites in biological samples. Next, we investigated the effects of CR on the gut microbial genome and the expression of mRNA related to energy metabolism which revealed a partially elevated TCA cycle and a subsequently promoted glyoxylate cycle, from which large amounts of malate can be produced to further impact malate related pathways in the host liver. Through the identification of key "hungry" metabolites produced by the gut microbiota that function in the promotion of autophagy in the host, further insight has been gained about a functional metabolic network important for both host-microbial symbiosis and maintenance of host health.

systems biology↗

bioRxiv2020

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Despite various genetic and epigenetic signatures are found to be related with active origins, it remains elusive how the selection of origins is determined. The classic Rosette model proposes that the origins clustered in a chromatin domain are preferentially and simultaneously fired, but direct imaging evidence has been lacking due to insufficient spatial resolution. Here, we applied dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We found that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase, in contrary to the Rosette model. Intriguingly, while both active and dormant origins are distributed homogeneously in the TAD during the G1 phase, active origins relocate to the TAD periphery before entering the S phase. We proved that such origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observed that the replication machinery protein PCNA forms immobile clusters around the TADs at the G1/S transition, which explains why origins at the TAD periphery are preferentially fired. Thus, we propose a "Chromatin Re-organization Induced Selective Initiation" (CRISI) model that the transcription-coupled chromatin structural re-organization determines the selection of replication origins, which transcends the scope of specific genetic and epigenetic signatures for origin efficiency. Our in situ super-resolution imaging unveiled coordination among DNA replication, transcription, and chromatin organization inside individual TADs, providing new insights into the biological functions of sub-domain chromatin structural dynamics.

genomics↗

bioRxiv2020

Genome Diversity in Ukraine

The main goal of this collaborative effort is to provide genome wide data for the previously underrepresented population in Eastern Europe, and to provide cross-validation of the data from genome sequences and genotypes of the same individuals acquired by different technologies. We collected 97 genome-grade DNA samples from consented individuals representing major regions of Ukraine that were consented for the public data release. DNBSEQ-G50 sequences, and genotypes by an Illumina GWAS chip were cross-validated on multiple samples, and additionally referenced to one sample that has been resequenced by Illumina NovaSeq6000 S4 at high coverage. The genome data has been searched for genomic variation represented in this population, and a number of variants have been reported: large structural variants, indels, CNVs, SNPs and microsatellites. This study provides the largest to-date survey of genetic variation in Ukraine, creating a public reference resource aiming to provide data for historic and medical research in a large understudied population. While most of the common variation is shared with other European populations, this survey of population variation contributes a number of novel SNPs and structural variants that have not been reported in the gnomAD/1KG databases representing global distribution of genomic variation. These endemic variants will become a valuable resource for designing future population and clinical studies, help address questions about ancestry and admixture, and will fill a missing place in the puzzle characterizing human population diversity in Eastern Europe. Our results indicate that genetic diversity of the Ukrainian population is uniquely shaped by the evolutionary and demographic forces, and cannot be ignored in the future genetic and biomedical studies. This data will contribute a wealth of new information bringing forth different risk and/or protective alleles. The newly discovered low frequency and local variants can be added to the current genotyping arrays for genome wide association studies, clinical trials, and in genome assessment of proliferating cancer cells.

genomics↗

bioRxiv2020

The DNA mimic protein BCAS0292 is involved in the regulation of virulence of Burkholderia cenocepacia

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. B. cenocepacia possesses a large genome encoding multiple virulence systems, stress response proteins and a large locus that responds to low oxygen. We previously identified BCAS0292, an acidic protein encoded on replicon 3. Deletion of the BCAS0292 gene resulted in altered abundance of >1000 proteins; 46 proteins became undetectable while 556 proteins showed >1.5-fold reduced abundance, suggesting BCAS0292 is a global regulator. Moreover, the {triangleup}BCAS0292 mutant showed a range of pleiotropic effects: virulence, host-cell attachment and motility were reduced, antibiotic susceptibility was altered and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. Structural analysis revealed BCAS0292 presents a dimeric {beta}-structure with a negative electrostatic surface. Further, the {Delta}BCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. We propose that BCAS0292 acts as a DNA-mimic, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

microbiology↗

bioRxiv2020

Prime editing primarily induces undesired outcomes in mice

Genome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

molecular biology↗

bioRxiv2020

Mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay

Polymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here we analyzed 26 PMG patients employing array-based DNA-methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q.

neuroscience↗

bioRxiv2020

An Antioxidant Enzyme Therapeutic for COVID-19

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. We report herein a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

biochemistry↗

bioRxiv2020

Dual-Mechanism Estrogen Receptor Inhibitors Reveal an Ensemble of Binding Modes for Robust Breast Cancer Antagonism

Efforts to improve estrogen receptor-a (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, non-canonical structural perturbations of the receptor ligand-binding domain (LBD) to drive antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Solution structural and coregulator peptide binding analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access non-consensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity. Significance StatementTo address the unmet clinical need for effectively suppressing estrogen receptor (ER) activity with both de novo resistance and in advanced ER-positive breast cancers that are resistant to standard-of-care antiestrogens, we have developed dual-mechanism ER inhibitors (DMERIs) that employ two distinct ER targeting moieties. These DMERI elicited non-canonical structural perturbations of the receptor ligand-binding domain and stabilized multiple antagonist sub-states within the dimer to generate highly efficacious antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. This work reveals new conformational modes by which the activity of ER can be effectively suppressed to block breast cancer proliferation.

cancer biology↗

bioRxiv2020

Inter-determination of blood metabolite levels and gut microbiome supported by Mendelian randomization

The gut microbiome has been implicated in a variety of physiological states, but controversy over causality remains unresolved. Here, we performed bidirectional Mendelian randomization (MR) analyses on 3,432 Chinese individuals with whole genome, whole metagenome, anthropometric, and blood metabolic trait data. We identified 58 causal relationships between the gut microbiome and blood metabolites, and replicated 43 of them. Increased relative abundances of fecal Oscillibacter and Alistipes were causally linked to decreased triglyceride concentration. Conversely, blood metabolites such as glutamic acid appeared to decrease fecal Oxalobacter, and members of Proteobacteria were influenced by metabolites such as 5-methyltetrahydrofolic acid, alanine, glutamate, and selenium. Two-sample MR with data from Biobank Japan partly corroborated results with triglyceride and with uric acid, and also provided causal support for published fecal bacterial markers for cancer and cardiovascular diseases. This study illustrates the value of human genetic information to help prioritize gut microbial features for mechanistic and clinical studies.

genetics↗

bioRxiv2020

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of COVID-19 Spike psedotype virus

BackgroundThe novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating. The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PurposeThe objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. MethodsIn our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. ResultsResults showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD =(7.31{+/-}0.62)e-7 M and (4.82{+/-}0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. ConclusionsCQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.

biochemistry↗

bioRxiv2020

Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

biochemistry↗