Biology subjects

Qu, K.

Publications and source records attributed to Qu, K..

31 records · Page 2Linked to original sources

bioRxiv2020

Maturation of the matrix and viral membrane of HIV-1

Gag - the main structural protein of HIV-1 - is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1, providing a basis to understand MAs role in virus assembly. Unexpectedly, we found that MA rearranges during maturation, to form a new, hexameric lattice in which the acyl chain of a phospholipid extends out of the membrane to bind a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only achieves assembly of the viral capsid surrounding the genome, but extends to repurpose the membrane-bound MA lattice for an entry or post-entry function, and causes partial removal of 2,500 acyl chains from the viral membrane.

microbiology↗

bioRxiv2020

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

microbiology↗

bioRxiv2020

The native structure of the full-length, assembled influenza A virus matrix protein, M1.

Influenza A virus causes millions of severe illnesses during annual epidemics. The most abundant protein in influenza virions is the matrix protein M1 that mediates virus assembly by forming an endoskeleton beneath the virus membrane. The structure of full-length M1, and how it oligomerizes to mediate assembly of virions, is unknown. Here we have determined the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We found that the C-terminal domain of M1 is disordered in solution, but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerising M1 into linear strands which coat the interior surface of the assembling virion membrane. In the M1 polymer, five histidine residues, contributed by three different M1 monomers, form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore provide mechanisms for influenza virus assembly and disassembly.

biochemistry↗

bioRxiv2020

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

biochemistry↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗

bioRxiv2020

Tocilizumab treatment in severe COVID-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis

ABSTRACTDespite the current devastation of the COVID-19 pandemic, several recent studies have suggested that the immunosuppressive drug Tocilizumab can powerfully treating inflammatory responses that occur in this disease. Here, by employing single-cell analysis of the immune cell composition of severe-stage COVID-19 patients and these same patients in post Tocilizumab-treatment remission, we have identified a monocyte subpopulation specific to severe disease that contributes to inflammatory storms in COVID-19 patients. Although Tocilizumab treatment attenuated the strong inflammatory immune response, we found that immune cells including plasma B cells and CD8+ T cells still exhibited an intense humoral and cell-mediated anti-virus immune response in COVID-19 patients after Tocilizumab treatment. Thus, in addition to providing a rich, very high-resolution data resource about the immune cell distribution at multiple stages of the COVID-19 disease, our work both helps explain Tocilizumab’s powerful therapeutic effects and defines a large number of potential new drug targets related to inflammatory storms.Competing Interest StatementJingwen Fang is the executive officer of HanGen BiotechView Full Text

genomics↗

bioRxiv2019

WEDGE: recovery of gene expression values for sparse single-cell RNA-seq datasets using matrix decomposition

The low capture rate of expressed RNAs from single-cell sequencing technology is one of the major obstacles to downstream functional genomics analyses. Recently, a number of imputation methods have emerged for single-cell transcriptome data, however, recovering missing values in very sparse expression matrices remains a substantial challenge. Here, we propose a new algorithm, WEDGE (WEighted Decomposition of Gene Expression), to impute gene expression matrices by using a biased low-rank matrix decomposition method (bLRMD). WEDGE successfully recovered expression matrices, reproduced the cell-wise and gene-wise correlations, and improved the clustering of cells, performing impressively for applications with multiple cell type datasets with high dropout rates. Overall, this study demonstrates a potent approach for imputing sparse expression matrix data, and our WEDGE algorithm should help many researchers to more profitably explore the biological meanings embedded in their scRNA-seq datasets.

bioinformatics↗

bioRxiv2019

Functional classification of noncoding RNAs associated with distinct histone modifications by PIRCh-seq

Many long noncoding RNAs (lncRNAs) regulate gene transcription through binding to histone modification complexes. Therefore, a comprehensive study of nuclear RNAs in a histone modification-specific manner is critical to understand their regulatory mechanisms. Here we develop a method named Profiling Interacting RNAs on Chromatin by deep sequencing (PIRCh-seq), in which we profile chromatin-associated transcriptome in 5 different cell types using antibodies recognizing histone H3 and 6 distinct histone modifications associated with active or repressive chromatin states. PIRCh-seq identified chromatin-associated RNAs with substantially less contamination by nascent transcripts, as compared to existing methods. We classified chromatin-enriched lncRNAs into 6 functional groups based on the patterns of their association with specific histone modifications. LncRNAs were enriched with different chromatin modifications in different cell types, suggesting lncRNAs regulation may also be cell type-specific. By integrating profiles of RNA secondary structure and RNA m6A modification, we found that RNA bases which bind to chromatin tend to be more single stranded. We discovered hundreds of allele-specific RNA-chromatin interactions, nominating specific single nucleotide variants that alter RNA association with chromatin. These results provide a unique resource to globally study the functions of chromatin-associated lncRNAs and elucidate the basic mechanisms of chromatin-RNA interaction.

cell biology↗

bioRxiv2019

APEC: An accesson-based method for single-cell chromatin accessibility analysis

The development of sequencing technologies has promoted the survey of genome-wide chromatin accessibility at single-cell resolution; however, comprehensive analysis of single-cell epigenomic profiles remains a challenge. Here, we introduce an accessibility pattern-based epigenomic clustering (APEC) method, which classifies each individual cell by groups of accessible regions with synergistic signal patterns termed "accessons". By integrating with other analytical tools, this python-based APEC package greatly improves the accuracy of unsupervised single-cell clustering for many different public data sets. APEC also predicts gene expressions, identifies significant differential enriched motifs, discovers super enhancers, and projects pseudotime trajectories. Furthermore, we adopted a fluorescent tagmentation-based single-cell ATAC-seq technique (ftATAC-seq) to investigated the per cell regulome dynamics of mouse thymocytes. Associated with ftATAC-seq, APEC revealed a detailed epigenomic heterogeneity of thymocytes, characterized the developmental trajectory and predicted the regulators that control the stages of maturation process. Overall, this work illustrates a powerful approach to study single-cell epigenomic heterogeneity and regulome dynamics.

bioinformatics↗

bioRxiv2019

Landscape and dynamics of the transcriptional regulatory network during natural killer cell differentiation

Natural killer (NK) cells are essential in controlling cancer and infection. However, little is known about the dynamics of the transcriptional regulatory machinery during NK cell differentiation. In this study, we applied assay of transposase accessible chromatin with sequencing (ATAC-seq) technique in a self-developed in vitro NK cell differentiation system. Analysis of ATAC-seq data illustrated two distinct transcription factor (TF) clusters that dynamically regulate NK cell differentiation. Moreover, two TFs from the second cluster, FOSL2 and EGR2, were identified as novel essential TFs that control NK cell maturation and function. Knocking down either of these two TFs significantly impacted NK cell transformation. Finally, we constructed a genome-wide transcriptional regulatory network that provides an understanding of the regulatory dynamics during NK cell differentiation.

bioinformatics↗

bioRxiv2018

The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

neuroscience↗

bioRxiv2018

Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS with loop-mediated isothermal amplification assay

The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detect nirS gene using those of Pseudomonas aeruginosa to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lacking nirS gene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detecting nirS gene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, the nirS gene of P. aeruginosa in spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

microbiology↗

bioRxiv2018

CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type-I-interferon signaling platform. Here, we determined cryoEM structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs, at resolutions sufficient to build and refine atomic models. The structures identify the filament forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.\n\neTOCStructures of MDA5 bound to double-stranded RNA reveal a flexible, predominantly hydrophobic filament forming interface. The filaments have variable helical twist. Structures determined with ATP and transition state analogs show how the ATPase cycle is coupled to changes in helical twist, the mode of RNA binding and the length of the RNA footprint of MDA5.\n\nHighlightsO_LICryoEM structures of MDA5-dsRNA filaments determined for three catalytic states\nC_LIO_LIFilament forming interfaces are flexible and predominantly hydrophobic\nC_LIO_LIMutation of filament-forming residues can cause loss or gain of IFN-{beta} signaling\nC_LIO_LIATPase cycle is coupled to changes in filament twist and size of the RNA footprint\nC_LI

immunology↗