Biology subjects

Feng, H.

Publications and source records attributed to Feng, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Serine proteinase inhibitors from Nicotiana benthamiana, a non-preferred host plant, inhibit the growth of Myzus persicae (green peach aphid)

The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA-Red, G002, and G006). Comparison of gene expression levels in aphid guts and whole aphids showed that several proteases, including a highly expressed serine protease, are significantly overexpressed in the guts. Furthermore, we identified three genes encoding serine protease inhibitors (SerPIN-II1, 2, and 3) in Nicotiana benthamiana, which is a non-preferred host for M. persicae. Using virus-induced gene silencing (VIGS) with a tobacco rattle virus (TRV) vector and overexpression with a turnip mosaic virus (TuMV) vector, we demonstrated that N. benthamiana SerPIN-II1 and SerPIN-II2 cause reduced survival and growth, but do not affect aphid protein content. Similarly, SerPIN-II3 overexpression reduced survival and growth, and serpin-II3 knockout mutations, which we generated using CRISPR/Cas9, increased survival and growth. Whereas protein content was significantly increased in aphids fed on SerPIN-II3 overexpressing plants, it was decreased in aphids fed on serpin-II3 mutants. Together, our results show that three PIN-IIs from N. benthamiana, a non-preferred host plant, effectively inhibit M. persicae survival and growth, thereby representing a new resource for the development of aphid-resistant crop plants.

plant biology↗

bioRxiv2022

Discovering Haematoma-Stimulated Circuits for Secondary Brain Injury after Intraventricular Haemorrhage by Spatial Transcriptome Analysis

Central nervous system (CNS) diseases, such as neurodegenerative disorders and brain diseases caused by acute injuries, are important yet challenging to study due to disease leisions locations and other complexities. Utilizing the powerful spatial transcriptome analysis together with novel algorithms we developed for the study, we report here for the first time a 3D trajectory map of gene expression changes in the brain following acute neural injury using a mouse model of intraventricular haemorrhage (IVH). IVH is a common and representative complication after various acute brain injuries with severe mortality and mobility implications. Our data identified three main 3D global pseudospace-time trajectory bundles which represent the main neural circuits from the lateral ventricle to the hippocampus and primary cortex induced by experimental intraventricular haematoma stimulation. Further analysis indicated a rapid response in the primary cortex, as well as a direct and integrated effect on the hippocampus after IVH. These results are informative in understanding the pathophysiological changes, including the spatial and temporal patterns of gene expression changes, in IVH patients after acute brain injuries and strategizing more effective clinical management regimens. The bioinformatics strategies will also be useful for the study of other CNS diseases.

bioinformatics↗

bioRxiv2022

Trans-cellular control of synapse properties by a cell type-specific splicing regulator

The recognition of synaptic partners and specification of synaptic properties are fundamental for the function of neuronal circuits. Terminal selector transcription factors coordinate the expression of terminal gene batteries that specify cell type-specific properties. Moreover, pan-neuronal alternative splicing regulators have been implicated in directing neuronal differentiation. However, the cellular logic of how splicing regulators instruct specific synaptic properties remains poorly understood. Here, we combine genome-wide mapping of mRNA targets and cell type-specific loss-of-function studies to uncover the contribution of the nuclear RNA binding protein SLM2 to hippocampal synapse specification. Focusing on hippocampal pyramidal cells and SST-positive GABAergic interneurons, we find that SLM2 preferentially binds and regulates alternative splicing of transcripts encoding synaptic proteins, thereby generating cell type-specific isoforms. In the absence of SLM2, cell type-specification, differentiation, and viability are unaltered and neuronal populations exhibit normal intrinsic properties. By contrast, cell type-specific loss of SLM2 results in highly selective, non-cell autonomous synaptic phenotypes, altered synaptic transmission, and associated defects in a hippocampus-dependent memory task. Thus, alternative splicing provides a critical layer of gene regulation that instructs specification of neuronal connectivity in a trans-synaptic manner.

neuroscience↗

bioRxiv2022

Deep-learning-assisted Sort-Seq enables high-throughput profiling of gene expression characteristics with high precision

As an essential physiological process, gene expression determines the function of each cell. However, owing to the complex nondeterministic and nonlinear nature of gene expression, the steady-state intracellular protein abundance of a clonal population forms a distribution. The characteristics of this distribution, including expression strength and noise, are closely related to cellular behavior. Therefore, quantitative description of these characteristics is an important goal in biology. This task, however, has so far relied on arrayed methods, which are time-consuming and labor-intensive. To address this issue, we propose a deep-learning-assisted Sort-Seq approach (dSort-Seq) in this work, enabling high-throughput profiling of expression properties with high precision. We demonstrated the validity of dSort-Seq for large-scale assaying of the dose-response relationships of biosensors. In addition, we comprehensively investigated the contribution of transcription and translation to noise production in E. coli, from which we discovered that the expression noise is strongly coupled with the mean expression level instead of translation strength, even in the case of weak transcription. We also discovered that the transcriptional interference caused by overlapping RpoD-binding sites contributes to noise production, which suggested the existence of a simple and feasible noise control strategy in E. coli. Overall, dSort-Seq is able to efficiently determine the strength-noise landscape, which has promising applications in studies related to gene expression.

genetics↗

bioRxiv2022

CRISPRi-FGP: web-based genome-scale CRISPRi sgRNA design and validation tool in prokaryotes

CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications. To address this issue, we introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. In addition, GLiDe provides the capability to design sgRNAs for newly discovered organisms. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system. Availability and ImplementationFreely available on the web at: https://www.thu-big.net/sgRNA_design/. AUTHOR SUMMARYCRISPRi screening is a powerful strategy for functional genomic study in prokaryotes. However, its applicability has been hindered by false positive results arising from off-target binding of sgRNAs. To tackle this challenge head-on, we present GLiDe, a web-based tool meticulously crafted for the genome-scale CRISPRi sgRNA library design in prokaryotes. GLiDe integrates a stringent quality control mechanism grounded in prior experimental insights. It has a built-in database and accepts uploaded reference files for new organisms. Moreover, we have demonstrated that our tool outperforms existing tools in CRISPRi sgRNA design for prokaryotes through cytometry assay. In summary, GLiDe emerges as a robust solution for sgRNA library design, poised to significantly advance functional genomic studies.

bioinformatics↗

bioRxiv2022

Thermodynamic Parameters Contributions of Single Internal Mismatches In RNA/DNA Hybrid Duplexes

Nucleic acid is a fundamental component of life. The thermodynamic is a basic physical quantity for nucleic acid and has been adopted in varieties of biotechnological applications. However, the parameters predicting thermodynamic parameters of RNA/DNA hybrid duplexes is incomplete and thus lead to the inability to proper predicting the properties. To address this problem, we measured for 99 RNA/DNA hybrid duplexes with 11 nucleotide length in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using SciPy non-linear curve fit. A dataset of 228 duplexes were constructed combining with literature experimental thermodynamic parameters for fitting a full set of all 113 possible Watson-Crick and single internal mismatch involving nearest-neighbor parameters using singular value decomposition. The new nearest-neighbor parameters predict thermodynamic parameters of RNA/DNA hybrid duplexes within an average error of 0.26 kcal/mol (3.55%) in free energy at 37{degrees}C ({Delta}G{degrees}37), 5.05 kcal/mol (6.70%) in enthalpy ({Delta}H{degrees}), 16.3 cal/mol{middle dot}K (7.50%) in entropy ({Delta}S{degrees}) and 1.14{degrees}C (2.91%) in melting temperature (Tm). The highest accuracy of all parameters reported. We observed a trend of increased stability for mismatch involving pairs of rG/dT >> rU/dG > rG/dA > rG/dG {approx} rA/dC {approx} rA/dG > rC/dA {approx} rC/dT > rU/dT {approx} rU/dC >> rA/dA >> rC/dC, which can be explained by different hydrogen bond conditions in mismatch pairs. We further compared the stability with RNA/RNA and DNA/DNA duplexes and found a backbone structure depended pattern in these three kinds of nucleic acid duplexes. This nearest-neighbor parameter set could enhance thermodynamical understanding of RNA/DNA combination and have potential usage in predicting on- and off-target RNA-DNA binding activity when applied to biotechnological applications such as CRISPR/Cas systems, RNAi and qPCR.

molecular biology↗

bioRxiv2022

Development of Highly Efficient CRISPR-Mediated Gene Editing in the Rotifer Brachionus manjavacas

Rotifers have been studied in the laboratory and field for over 100 years and are an emerging modern model system for investigation of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance, and have a long been used in studies of microevolution, ecological dynamics, and ecotoxicology. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least one CRISPR-induced mutation, with many apparently mutated at both alleles or mosaic. In addition, we achieved precise CRISPR-mediated knockin of a stop codon cassette in the mlh3 locus, with half of injected mothers producing 33% or more F2 offspring with an insertion of the cassette. These results demonstrate the efficacy of the CRISPR/Cas9 system in rotifers to provide insight into the function of specific genes and further advance rotifers as a model system for biological discovery.

genomics↗

bioRxiv2022

A novel brain penetrant PDGFRα inhibitor HY-008 is effective against glioblastoma

BackgroundGlioblastoma multiforme (GBM) is the most common primary intracranial malignant tumor in adults, with poor prognosis and high recurrence. Routine treatments of GBM show unsatisfactory efficiency in improving patients survival because of limited area of surgical resection and drug resistance. New therapeutic agents are needed to improve GBM treatment efficiency, but the blood-brain barrier (BBB) permeability is a major hurdle. Here, we report HY-008 as a promising therapeutic drug targeting PDGFR signaling with high BBB permeability and efficient inhibiting effects both in vitro and in vivo. MethodsThrough structural modification and medicinal chemistry efforts, HY-007 and HY-008 were developed. The brain and plasma pharmacokinetic profiles of these two compounds were assessed. The inhibitory efficiency of HY-007 and HY-008 on GBM cell survival and PDGFR signaling were evaluated. The efficacy of HY-007 and HY-008 as a single agent or HY-008 in combination with temozolomide (TMZ) was investigated using transformed mouse astrocyte and glioma stem-like cell (GSC) orthotopic xenograft models. ResultsHY-007 and HY-008 both had good brain permeability and desirable PK profiles with mild hERG inhibition, while HY-008 is more brain permeable than HY-007. In vitro, HY-007 and HY-008 both significantly inhibited viability of the established GBM cells with PDGF-A overexpression and transformed mouse astrocytes with PDGF-A/PDGFR overexpression by targeting the PDGFR signaling activated Erk1/2 and Akt. In vivo, HY-007 and HY-008 both effectively inhibited orthotopic GBM tumor xenograft growth and prolonged the survival of mice, and HY-008 showed less toxicity and better therapeutic effect. In addition, HY-008 increased sensitivity of TMZ, exhibited treatment efficiency both as a single agent and in combination with TMZ, providing significant survival benefits for GBM tumor xenograft-bearing mice. ConclusionsOur data demonstrate that HY-008 is a promising therapeutic agent in GBM treatment and a combination HY-008 with TMZ could serves as a potential efficient therapeutic option for improving GBM clinical treatment.

cancer biology↗

bioRxiv2022

Horizontally transferred genes as RNA interference targets for aphid and whitefly control

RNA interference (RNAi)-based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi, or plants, are attractive targets for RNAi-mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings, and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco-adapted isolate of Myzus persicae (green peach aphid) and bioinformaticaly identified 30 HTGs. We then used plant-mediated virus-induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal HTGs did not affect aphid survivorship, but decreased aphid reproduction. Importantly, although there was uptake of plant-expressed RNA by Coccinella septempunctata (seven-spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid-targeted VIGS constructs. In other experiments, we targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant-mediated RNA interference.

plant biology↗

bioRxiv2022

Treg cells drive MYCN-mediated immunosuppression and tumor aggressiveness in high-risk neuroblastoma

Solid tumors, especially those with aberrant MYCN activation, harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance1,2, yet the underlying mechanisms are elusive and effective strategies to tackle this challenge are lacking. Here we demonstrated the crucial role of T regulatory (Treg) cells in MYCN-mediated immune repression and tumor aggression using high-risk neuroblastoma (NB) as a model system. Human MYCN-activated NB attracts CD4+ Treg cells, which are also found enriched in MYCN-high primary patient samples. Zebrafish MYCN-overexpressing neural crests recruit Cd4+ cells before tumor formation and induce an immunosuppressive microenvironment, thereby promoting tumor onset and progression. Strikingly, disruption of Treg cells through depletion of forkhead box protein 3a restores anti-tumor immunity and impairs NB development. Together, our studies establish Treg cells as the key driver of MYCN-mediated immunosuppression and tumor aggressiveness, providing mechanistic insights and therapeutic implications.

cancer biology↗

bioRxiv2022

Heterosynaptic Plasticity of the Visuo-auditory Projection Requires Cholecystokinin released from Entorhinal Cortex Afferents

The entorhinal cortex is involved in establishing enduring visuo-auditory associative memory in the neocortex. Here we explored the mechanisms underlying this synaptic plasticity related to projections from the visual and entorhinal cortices to the auditory cortex, using optogenetics of dual pathways. High-frequency laser stimulation (HFLS) of the visuo-auditory projection did not induce long-term potentiation (LTP). However, after pairing with sound stimulus, the visuo-auditory inputs were potentiated following either infusion of cholecystokinin (CCK) or HFLS of the entorhino-auditory CCK-expressing projection. Combining retrograde tracing and RNAscope in situ hybridization, we show that CCK expression is higher in entorhinal cortex neurons projecting to the auditory cortex than in those originating from the visual cortex. In the presence of CCK, potentiation in the neocortex occurred when the pre-synaptic input arrived 200 ms before post-synaptic firing, even after just five trials of pairing. Behaviorally, inhibition of CCK signaling blocked the generation of associative memory. Our results indicate that neocortical visuo-auditory association is formed through heterosynaptic plasticity, which depends on release of CCK in the neocortex mostly from entorhinal afferents.

neuroscience↗

bioRxiv2022

Analysis of sex differential genes at whole genome level reveals their organ- and chromosome-dependent expressions, regulations, and interactions

Males and females possess genomes that are almost identical but differ in morbidity, prevalence, severity, response to therapies and mortality of many diseases. We comprehensively analyzed gene expression data in seven tissues from BXD recombinant inbred (RI) mouse strains. We found that there were considerable differences in the numbers and functions among different tissues and between autosomal and sex chromosomes. Among sex differential genes, those on autosomal chromosomes mainly function to regulate metabolic pathways related to their host organs, while those on sex chromosomes mainly regulate nuclear proteins required for DNA replication or transcription during early development. Kidney possessed the fewest sex differential genes on sex chromosomes. The sexually dimorphic genes expressed on X chromosomes were all related to perception, while genes on the autosomal chromosomes were related to metabolism. These patterns of gene expression do not translate into similarity of protein structures, rather they are grouping with function.

cell biology↗

bioRxiv2022

Allelic Transcription Factor binding shape transcriptional kinetics in human cell lines

Gene expression from bulk RNA-seq studies is an average measurement between two chromosomes and across cell populations. Both allelic and cell-to-cell heterogeneity in gene expression results from promoter bursting patterns that repeatedly alternate between an activated and inactivated state. Increased cell-to-cell heterogeneity in gene expression has been associated with aging and stem cell pluripotency. However, studies of bursting kinetics and their molecular mechanism are relatively limited in human cells compared to other species due to laborious single-molecule experiments. Here, we systematically investigate the regulatory effect of genetic variants and transcription factor (TF) binding on transcriptional kinetics at the single chromosome level with GM12878. We found that the transcription initiation rate and burst frequency correlate most with eQTL effect sizes among transcriptional kinetics, which suggests that eQTLs affect average gene expression mainly through altering burst kinetics. We further found that [~]90% of the variance of burst frequency can be explained by TF occupancy in phase with the core promoter. We identified and replicated several examples where eQTL or GWAS catalog loci perturb TF binding affinity and are consequently associated with the change of burst kinetics.

genetics↗

bioRxiv2022

Rapid Development of a Mucosal Nanoparticle Flu Vaccine by Genetic Engineering of Bacteriophage T4 using CRISPR-Cas

Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local mucosal as well as systemic immune responses and provided complete protections against divergent influenza viruses in a mouse model. Potentially, our platform can be customized for any respiratory pathogen to rapidly generate mucosal vaccines against future emerging epidemics and pandemics.

immunology↗

bioRxiv2022

Processing UMI Datasets at High Accuracy and Efficiency with the Sentieon ctDNA Analysis Pipeline

Liquid biopsy enables identification of low allele frequency (AF) tumor variants and novel clinical applications such as minimum residual disease (MRD) monitoring. However, challenges remain, primarily due to limited sample volume and low read count of low-AF variants. Because of the low AFs, some clinically significant variants are difficult to distinguish from errors introduced by PCR amplification and sequencing. Unique Molecular Identifiers (UMIs) have been developed to further reduce base error rates and improve the variant calling accuracy, which enables better discrimination between background errors and real somatic variants. While multiple UMI-aware ctDNA analysis pipelines have been published and adopted, their accuracy and runtime efficiency could be improved. In this study, we present the Sentieon ctDNA pipeline, a fast and accurate solution for small somatic variant calling from ctDNA sequencing data. The pipeline consists of four core modules: alignment, consensus generation, variant calling, and variant filtering. We benchmarked the ctDNA pipeline using both simulated and real datasets, and found that the Sentieon ctDNA pipeline is more accurate than alternatives.

bioinformatics↗

bioRxiv2022

Structure-based prediction and characterization of photo-crosslinking in native protein-RNA complexes

UV-crosslinking of protein and RNA in direct contacts has been widely used to study protein-RNA complexes despite our poor understanding on the mechanisms of photo-crosslinking. This knowledge gap is due to the challenge to precisely map the crosslink sites in protein and RNA simultaneously in their native sequence and structural contexts. Here we developed PxR3D-map, a computational method to analyze protein-RNA interactions and photo-crosslinking by bridging crosslinked nucleotides and amino acids mapped using different assays with protein-RNA complex structures. PxR3D-map reliably predicts crosslink sites using structural information characterizing protein-RNA interaction interfaces. We found that photo-crosslinking is facilitated by base stacking with not only aromatic residues, but also dipeptide bonds that involve glycine, and distinct mechanisms are utilized by different types of RNA-binding domains. Our work suggests protein-RNA photocrosslinking is highly selective in the cellular environment, which can guide interpretation of data generated by UV-crosslinking-based assays and further technology development.

biophysics↗

bioRxiv2022

A toxin-mediated policing system in Bacillus improves population fitness via penalizing non-cooperating phenotypic cheaters

Microbial cooperation is vulnerable to exploitation by social cheaters. Although the strategies for controlling genotypic cheaters have been well investigated, the mechanism and significance of preventing phenotypic cheating remain largely unknown. Here, we revealed the molecular mechanism and ecological significance of a policing system for punishing phenotypic cheaters in the community of a plant beneficial strain Bacillus velezensis SQR9. Coordinated activation of extracellular matrix (ECM) production and autotoxin bacillunoic acids (BAs) biosynthesis/self-immunity, punished public goods-nonproducing cheaters in strain SQR9s community. Spo0A was identified to be the co-regulator for triggering both ECM production and BAs synthesis/immunity, which activates acetyl-CoA carboxylase (ACC) to produce malonyl-CoA, an essential precursor for BAs biosynthesis, thereby stimulating BAs production and self-immunity. Elimination of phenotypic cheaters by this policing system, significantly enhanced population fitness under different stress conditions and in plant rhizosphere. This study provides insights into our understanding of maintenance and evolution of microbial cooperation.

microbiology↗

bioRxiv2022

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

Although picornaviruses are conventionally considered nonenveloped, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX. Authors SummaryMechanisms underlying nonlytic release of canonical nonenveloped viruses from infected cells are poorly understood. We show here that release of hepatitis A virus from cells in exosome-like vesicles requires nonredundant activities of two distinct Bro1-domain proteins associated with host cell machinery (ESCRT) for endosomal sorting, HD-PTP and ALIX. We demonstrate both Bro1 domain proteins are recruited to the viral capsid by the pX segment of the 1D capsid protein, and that they act in a non-redundant manner to mediate virus release. Fusing pX to a self-assembling nanocage protein resulted in ESCRT-dependent release mediated by a short pX peptide sequence conserved in hepatoviruses from bats to humans. Mutations within the pX sequence ablate release and result in noncytolytic virus accumulating intracellularly. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows nonlytic release of quasi-enveloped virus is an ancient evolutionary trait of hepatoviruses.

microbiology↗