Biology subjects

Wu, X.

Publications and source records attributed to Wu, X..

At least 451 records · Page 25Linked to original sources

bioRxiv2022

DLGAP1-AS2-Mediated Phosphatidic Acid Synthesis Confers Chemoresistance via Activation of YAP Signaling

Squamous cell carcinomas (SCCs) constitute a group of human malignancies that originate from the squamous epithelium. Most SCC patients experience treatment failure and relapse and have a poor prognosis due to de novo and acquired resistance to first-line chemotherapeutic agents. To identify chemoresistance mechanisms and explore novel chemosensitizer targets, we performed whole-transcriptome sequencing of paired resistant/parental SCC cells. We identified DLGAP1 antisense RNA 2 (D-AS2) as a crucial noncoding RNA that contributes to chemoresistance in SCC. Mechanistically, D-AS2 associates with histones to regulate the distal elements of FAM3 metabolism regulating signaling molecule D (FAM3D) and reduces extracellular FAM3D protein secretion. FAM3D interacts with Gi-coupled G protein-coupled receptor (GPCR) formyl peptide receptor (FPR) 1 and FPR2 to suppress phospholipase D (PLD) activity; thus, reduced FAM3D activates PLD signaling. Moreover, activated PLD promotes phosphatidic acid (PA) production and subsequent yes-associated protein (YAP) nuclear translocation. Accordingly, in vivo administration of a D-AS2-targeting antisense oligonucleotide sensitizes SCC to cisplatin treatment. In summary, our study reveals that D-AS2/FAM3D-mediated PLD/PA lipid signaling is essential in SCC chemoresistance and that D-AS2 can be targeted to sensitize SCC to cytotoxic chemotherapeutic agents. SignificanceThis study identifies D-AS2 as a targetable lipid-related lncRNA that activates YAP signaling via PLD/PA axis to trigger chemoresistance in SCC.

cancer biology↗

bioRxiv2022

Solution structure of the phosphatidylinositol 3-phosphate binding domain from the Legionella effector SetA

Legionella pneumophila is a facultative intracellular pathogen that causes Legionnaires disease or Pontiac fever in humans upon accidental inhalation of Legionella-contaminated aerosols. During infection, L. pneumophila secretes more than 300 effectors into the host for the biogenesis of a replication-permissive niche, known as the Legionella containing vacuole (LCV). Among these, a large number of effectors harbor protein domains that recognize specific phosphoinositide (PI) lipids and mediate the anchoring of these effectors to the surface of LCV or other host membrane-bound organelles. The Legionella effector SetA contains a unique C-terminal domain (SetA-CTD) that has been shown to specifically bind with phosphatidylinositol-3-phosphate (PI(3)P) and target SetA to endosomes and LCVs. Here, we report the NMR solution structure of SetA-CTD, which mainly comprises a four -helix bundle. The structure reveals a basic pocket at one end of the -helix bundle for PI(3)P binding and two hydrophobic loops for membrane insertion. Mutations of key residues involved in lipid binding result in the loss of SetA in membrane association and endosomal localization. Structural comparison with other PI(3)P-binding domains highlights a general theme applied by multiple families of phosphoinositide-binding domains across species.

biophysics↗

bioRxiv2022

Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering

Myosin 10 (Myo10) is a member of the MyTH4/FERM domain family of unconventional, actin-based motor proteins. Studies have implicated Myo10 in supporting cell adhesion via its integrin-binding FERM domain, and spindle positioning and spindle pole integrity via its microtubule-binding MyTH4 domain. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from a Myo10 knockout mouse. Most notably, both of these knockout cells exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is PCM fragmentation, which creates y-tubulin-positive, centriole-negative microtubule asters that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing centrosome clustering. These results argue, therefore, that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Finally, we present evidence that the defect in spindle pole integrity in Myo10 knockout cells is likely due to a defect in pole stability rather than pole maturation, and that Myo10 promotes supernumerary centrosome clustering at least in part by promoting cell adhesion during mitosis.

cell biology↗

bioRxiv2022

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

The complement component C3 is a fundamental plasma protein for host defense. However, recent work has demonstrated the critical importance of local C3 expression in cell survival. Here we analyzed the effects of local versus peripheral sources of C3 expression in a model of bacterial pneumonia. While mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient in liver-deficient C3 remain protected, comparable to wildtype mice. Human lung transcriptome analysis showed secretory epithelial cells are a major source of C3. Mice with a C3 gene ablation from lung epithelial cells had worse pulmonary injury compared to wild type, despite maintaining normal circulating C3 levels. Finally, in human cellular and mouse pneumonia models, we show that C3 reduces epithelial cell death mediated through the alternative pathway component Factor B. Thus, our findings suggest that a locally-derived C3-Factor B pathway protects the lung mucosal barrier. One Sentence SummaryLung-derived C3 mitigates severe bacterial pneumonia suggesting a novel cytoprotective role at mucosal barrier surfaces independent of circulating C3.

immunology↗

bioRxiv2022

Emodin Combined with Multiple Low-frequency Low-intensity Ultrasound to Relieve Osteomyelitis Through Sonoantimicrobial Chemotherapy

Treatment of osteomyelitis is still challenging as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) through synergistic antibacterial and anti-biofilm effects. The efficiency of different intensities of ultrasound, single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 5 min every 4 h, three times) against bacteria and biofilm was compared, contributing to develop the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) have significant synergetic bactericidal and anti-biofilm effects and EM+M-LFLIU exhibits superior performance in anti-biofilm. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of bacterial membrane and wall, down-regulate the expression of oxidative stress, membrane wall synthesis, bacterial virulence and other related genes (agrB, PBP3, sgtB, GMK, zwf, msrA). In vivo study, micro-CT, H&E staining, ELISA assay and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis of MRSA infection. Our work proffers an original treating bacterial osteomyelitis approach that weakens drug-resistant bacterial and suppresses biofilm formation through SACT, which may provide new prospects for clinical treatment.

microbiology↗

bioRxiv2022

A Genome-wide CRISPR Screen Identifies WDFY3 as a Novel Regulator of Macrophage Efferocytosis

AbstractsPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages. HighlightsO_LIFunctional readout for pooled genome-wide CRISPR screen in primary macrophages. C_LIO_LIWDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo. C_LIO_LIWDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization. C_LIO_LIWDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. C_LIO_LIC-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake. C_LI

cell biology↗

bioRxiv2022

Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels

Neuronal KCNQ channels mediate the muscarine-regulated M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that mutations have on channel function remain poorly defined, largely because of our limited understanding of the voltage sensing mechanisms that trigger channel gating. Here, we present measurements of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-causing mutations using mutagenesis, cysteine accessibility, and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of 8-9 amino acids in the S4 become exposed upon opening of KCNQ2 channels. VCF shows that the voltage dependence and kinetics of S4 movement and channel opening/closing closely correlate, suggesting an activation scheme in which channel opening does not require multiple voltage-sensor movements. VCF and kinetic modeling reveal different mechanisms by which epilepsy-causing mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

biophysics↗

bioRxiv2022

HuR-dependent expression of Wisp1 is necessary for TGF????-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGF{beta}-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGF{beta}-induced myofibroblast activation upon HuR inhibition. To delineate HuR-dependent mechanisms, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify eleven mRNA transcripts that showed enriched HuR binding following TGF{beta} treatment as well as significant co-expression correlation with HuR, SMA, and periostin using single-cell RNA-sequencing from ischemic-zone isolated fibroblasts. Of these, Wnt1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 is able to partially rescue myofibroblast contractile function following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGF{beta}. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGF{beta}, in part through post-transcriptional regulation of Wisp1. HIGHLIGHTSO_LIThe RNA binding protein HuR is highly expressed in cardiac fibroblasts and its expression strongly correlates with markers of active myofibroblasts C_LIO_LIHuR inhibition reduces migration, contraction, and ECM production activity of cardiac fibroblasts C_LIO_LIExpression of the secreted matricellular protein, Wisp1, is increased in a HuR-dependent manner following TGF{beta} treatment C_LIO_LIRecombinant Wisp1 rescues myofibroblast contractile function following HuR inhibition C_LIO_LIHuR-dependent expression of Wisp1 is necessary for myofibroblast activation C_LI

molecular biology↗

bioRxiv2022

OKseqHMM: a genome-wide replication fork directionality analysis toolkit

MotivationDuring each cell division, tens of thousands of DNA replication origins are coordinately activated to ensure the complete duplication of the entire human genome. However, the progression of replication forks can be challenged by numerous factors. One such factor is transcription-replication conflicts (TRC), which can either be co-directional or head-on with the latter being revealed as more dangerous for genome integrity. ResultsIn order to study the direction of replication fork movement and TRC, we developed a bioinformatics tool, called OKseqHMM, to directly measure the genome-wide replication fork directionality (RFD) as well as replication initiation and termination from data obtained by Okazaki fragment sequencing (OK-Seq) and related techniques. Availability and ImplementationWe have gathered and analyzed OK-seq data from a large number of organisms including yeast, mouse and human, to generate high-quality RFD profiles and determine initiation zones and termination zones by using Hidden Markov Model (HMM) algorithm (all tools and data are available at https://github.com/CL-CHEN-Lab/OK-Seq). In addition, we have extended our analysis to data obtained by related techniques, such as eSPAN and TrAEL-seq, which also contain RFD information. Our works, therefore, provide an important tool and resource for the community to further study TRC and genome instability, in a wide range of cell line models and growth conditions, which is of prime importance for human health. ContactChun-Long Chen (Institut Curie), chunlong.chen@curie.fr CategoryGenome analysis

bioinformatics↗

bioRxiv2022

Highly efficient multiplex genome editing in dicot plants using improved CRISPR/Cas systems

CRISPR/Cas9-mediated gene editing provides a powerful tool for dissecting gene function and improving important traits in crops. However, there are still persisting challenges to obtain high homozygous/bi-allelic (ho/bi) mutations in dicot plants. Here, we develop an improved CRISPR/Cas9 system harboring a calreticulin-like gene promoter, which can boost targeted mutations in dicots. Additionally, the pDC45_dsg construct, combining a 35Spro-tRNA_sgRNA-EU unit and PCE8pro-controlled Cas9, can achieve more than 80.0% ho/bi mutations at target sites in allotetraploid tobacco. We construct pDC45_Fast system that can simultaneously fulfill gene editing and shorten the life span of T0 generation tobacco and tomato. This study provides new tools for improving targeted gene mutagenesis in dicots, and makes manipulations of genes in Solanum more feasible.

plant biology↗

bioRxiv2022

Sensitization of Chrysosplenetin against Artemisinin Resistant Plasmodium berghei K173 via Blocking Host ABC Transporters Potentially Mediated by NF-κBB p52 or PXR/CAR Signaling Pathway

This study investigated if artemisinin-chrysosplenetin combination (ART-CHR) improved ART antimalarial efficacy against resistant Plasmodium berghei K173 via depressing host ABC transporter and potential molecular mechanism. Parasitaemia% and inhibition% were calculated and gene/protein expressions of ABC transporters or PXR/CAR/NF-{kappa}B p52 were detected by Western-blot and RT-qPCR. In vitro transcription of PXR/CAR was studied by dual-luciferase reporter assay. Our data indicated that ART-CHR improved ART efficacy against resistant parasites. P-gp inhibitor verapamil and CHR showed a stronger effect in killing resistant parasites while vehicle and Bcrp inhibitor novobiocin did not. ART activated intestinal ABCB1/ABCG2 and CHR inhibited them. ART decreased Bcrp protein whereas CHR increased it. ART ascended ABCC1/ABCC4/ABCC5 mRNA but ART-CHR descended them. CHR as well as rifampin (RIF) or 5-fluorouracil (5-FU) increased transcription levels of PXR/CAR while showed a versatile regulation on in vivo hepatic and enternal PXR/CAR in Mdr1a+/+ (WT) or Mdr1a-/- (KO) mice infected with sensitive or resistant parasites. Oppositely, hepatic and enteric N-{kappa}B p52 mRNA was conformably decreased in WT but increased in KO-resistant mice. NF-{kappa}B pathway should potentially involved in the mechanism of CHR on inhibiting ABC transporters and ART resistance while PXR/CAR play a more complicated role in this mechanism.

molecular biology↗

bioRxiv2022

Sulfane sulfur post-translationally modifies the global regulator AdpA to influence actinorhodin production and morphological differentiation of Streptomyces coelicolor

The transcription factor AdpA is a key regulator controlling both secondary metabolism and morphological differentiation in Streptomyces. Due to its critical functions, its expression undergoes multi-level regulations at transcriptional, post-transcriptional, and translational levels, yet no post-translational regulation has been reported. Sulfane sulfur, such as organic polysulfide (RSnH, n[≥]2), is common inside microorganisms, but its physiological functions are largely unknown. Herein, we discovered that sulfane sulfur post-translationally modifies AdpA in S. coelicolor via specifically reacting with Cys62 of AdpA to form a persulfide (Cys62-SSH). This modification decreases the affinity of AdpA to its self-promoter PadpA, allowing increased expression of adpA, further promoting the expression of its target genes actII-4 and wblA. ActII-4 activates actinorhodin biosynthesis and WblA regulates morphological development. Bioinformatics analyses indicated that AdpA-Cys62 is highly conserved in Streptomyces, suggesting the prevalence of such modification in this genus. Thus, our study unveils a new type of regulation on the AdpA activity and sheds a light on how sulfane sulfur stimulates the production of antibiotics in Streptomyces. IMPORTANCEStreptomyces produce myriad of polyketide compounds having (potential) clinical applications. While the database of polyketide gene clusters are quickly expanding, the regulation mechanisms of them are rarely known. Sulfane sulfur species are commonly present in microorganisms with unclear functions. Herein, we discovered that sulfane sulfur increases actinorhodin (ACT) production in S. coelicolor. The underlying mechanism is sulfane sulfur specifically reacts with AdpA, a global transcription factor controlling both ACT gene cluster and morphological differentiation related genes, to form sulfhydrated AdpA. This modification changes dynamics of AdpA-controlled gene network and leads to high expression of ACT biosynthetic genes. Given the wide prevalence of AdpA and sulfane sulfur in Streptomyces, this mechanism may represent a common regulating pattern of polyketide gene clusters. Thus, this finding provides a new strategy for mining and activating valuable polyketide gene clusters.

microbiology↗

bioRxiv2022

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding into the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention or rearrangements between descendants of whole genome duplications (WGDs), which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its 3D chromatin architecture at the onset of zygotic activation, and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes, and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

evolutionary biology↗

bioRxiv2022

Structural basis of peptidomimetic agonism revealed by small molecule GLP-1R agonists Boc5 and WB4-24

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of them are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric non-peptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryo-electron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3 and 7, one arm of both compounds inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8-D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts showing an interaction feature substantially similar to a previously known small molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by non-peptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine tuning of pharmacological efficacy in the development of future small molecule therapeutics targeting GLP-1R. SignificanceGLP-1R agonists are efficacious in the treatment of type 2 diabetes and obesity. While most clinically used agents require subcutaneous injection, Boc5, as the first orthosteric non-peptidic agonist of GLP-1R, suffers from poor oral bioavailability that hinders its therapeutic development. The cryo-electron microscopy structures of Boc5 and its closely related analog WB4-24 presented here reveal a previously unknown binding pocket located deeper in the transmembrane domain for non-peptidic GLP-1R agonists. Molecular interaction with this site may facilitate a broad spectrum of in vivo agonistic activities, in addition to that with the upper helical bundles presumably responsible for biased signaling. These findings deepen our understanding of peptidomimetic agonism at GLP-1R and may help design better drug leads against this important target.

molecular biology↗

bioRxiv2021

SoyFGB v2.0: a unique access to variations of Chinese Soybean Gene Bank (CNSGB) germplasm

In Chinese National Soybean GeneBank (CNSGB), we have collected more than 30,000 soybean accessions. However, data sharing for soybean remains an especially sensitive question, and how to share the genome variations within rule frame has been bothering the soybean germplasm workers for a long time. Here we release a big data source named Soybean Functional Genomics & Breeding database (SoyFGB v2.0) (https://sfgb.rmbreeding.cn/), which embed a core collection of 2,214 soybean resequencing genome (2K-SG) from the CNSGB germplasm. This source presents a unique example which may help elucidating the following three major functions for multiple genome data mining with general interests for plant researchers. 1) On-line analysis tools are provided by the Analysis module for haplotype mining in high-throughput genotyped germplasms with different methods. 2) Variations for 2K-SG are provided in SoyFGB v2.0 by Browse module which contains two functions of SNP and InDel. Together with the Gene (SNP & InDel) function embedded in Search module, the genotypic information of 2K-SG for targeting gene / region is accessible. 3) Scaled phenotype data of 42 traits, including 9 quality and 33 quantitative traits are provided by SoyFGB v2.0. With the scaled-phenotype data search and seed request tools under a control list, the germplasm information could be shared without direct downloading the unpublished phenotypic data or information of sensitive germplasms. In a word, the mode of data mining and sharing underlies SoyFGB v2.0 may inspire more ideas for works on genome resources of not only soybean but also the other plants.

genomics↗

bioRxiv2021

Construction of a potent pan-vaccine based on the evolutionary tendency of SARS-CoV-2 spike protein.

SARS-CoV-2 continued to spread globally along with different variants. Here, we systemically analyzed viral infectivity and immune-resistance of SARS-CoV-2 variants to explore the underlying rationale of viral mutagenesis. We found that the Beta variant harbors both high infectivity and strong immune resistance, while the Delta variant is the most infectious with only a mild immune-escape ability. Remarkably, the Omicron variant is even more immune-resistant than the Beta variant, but its infectivity increases only in Vero E6 cells implying a probable preference for the endocytic pathway. A comprehensive analysis revealed that SARS-CoV-2 spike protein evolved into distinct evolutionary paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance, resulting in a narrow spectrum of the current single-strain vaccine. In light of these findings and the phylogenetic analysis of 2674 SARS-CoV-2 S-protein sequences, we generated a consensus antigen (S6) taking the most frequent mutations as a pan-vaccine against heterogeneous variants. As compared to the ancestry SWT vaccine with significantly declined neutralizations to emerging variants, the S6 vaccine elicits broadly neutralizing antibodies and full protections to a wide range of variants. Our work highlights the importance and feasibility of a universal vaccine strategy to fight against antigen drift of SARS-CoV-2.

microbiology↗

bioRxiv2021

Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a model of RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) N{varepsilon}-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro and in vivo, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and potentially protected their neonatal offspring from lethal challenge similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

bioengineering↗

bioRxiv2021

Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

The frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the [Formula] pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

bioengineering↗