Biology subjects

Zou, W.

Publications and source records attributed to Zou, W..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

TMEM63B functions as a mammalian thirst receptor

Thirst drives animals to reinstate water homeostasis by fluid intake. An increase of blood osmolality is thought to induce thirst by activating a thirst receptor expressed in the subfornical organ (SFO), but the molecular identity of this receptor remains elusive. Here, we provide behavioral and functional evidence to show that TMEM63B functions as a mammalian thirst receptor in the SFO and mediates osmotic and dehydrated thirst. First, we showed that TMEM63B is expressed in SFO excitatory neurons and required for the neuronal responses to hypertonic stimulation. Heterologously expressed TMEM63B is activated by hypertonic stimuli and point mutations can alter the reversal potential of the channel. More importantly, purified TMEM63B in liposomes establishes osmolarity-gated currents. Finally, Tmem63b knockout mice have profound deficits in thirst, and deleting TMEM63B within the SFO neurons recapitulated this phenotype. Taken together, these results provide a molecular basis for thirst 82and demonstrate TMEM63B is the long-sought mammalian thirst receptor.

neuroscience↗

bioRxiv2023

Seeding Activity of Skin Misfolded Tau as a Novel Biomarker for Tauopathies

Tauopathies are a group of age-related neurodegenerative diseases with a molecular hallmark of the prion-like propagation and accumulation of pathologically phosphorylated tau protein in the brain. They include Alzheimers disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Picks disease (PiD). Currently, in the peripheral tissues and body fluids there are no reliable diagnostic biomarkers available that are able to directly reflect the capability of propagation and spreading of the misfolded tau aggregates. Here, we revealed significantly increased amounts of phosphorylated tau in the skin of AD patients compared to those in other tauopathies and normal controls. Moreover, the seed-amplification assay (SAA) by the ultrasensitive real-time quaking-induced conversion (RT-QuIC) displayed that the prion-like seeding activity of pathological tau in the skin of cadavers with neuropathologically confirmed tauopathies including AD, PSP, CBD, PiD was dramatically higher than that in normal controls, yielding 75-80% sensitivity and 95-100% specificity, respectively, depending on different tau substrates used. The increased tau-seeding activity was also observed in biopsy skin samples from living AD and PSP patients. Moreover, analysis of the end products of skin-tau SAA confirmed that the increased seeding activity is accompanied with formation of tau aggregates that are of different physicochemical properties determined by the different tau-substrates used. Our study provides proof-of-concept that the skin tau-SAA can differentiate tauopathies from normal controls, suggesting that the seeding activity of the skin misfolded tau can serve as an accurate diagnostic biomarker of tauopathies.

neuroscience↗

bioRxiv2023

The Multifaceted Effects of Short-Term Acute Hypoxia Stress: Insights into the tolerance mechanism of Propsilocerus akamusi (Diptera: Chironomidae)

Plenty of freshwater species, especially macroinvertebrates that are essential to the provision of numerous ecosystem functions, encountered higher mortality due to acute hypoxia. However, within the family Chironomidae, a wide range of tolerance to hypoxia/anoxia is displayed and Propsilocerus akamusi depends on this great endurance to become a dominant species in eutrophic lakes. To further understand how P. akamusi responds to acute hypoxic stress, we used multi-omics analysis in combination with histomorphological characteristics and physiological indicators. To evaluate enzyme activity, the transcriptome and metabolome, and histomorphological characteristics, we set up two groups: a control group (DO 8.4mg/L) and a hypoxic group (DO 0.39mg/L). With blue-black chromatin, cell tightness, cell membrane invagination, and the production of apoptotic vesicles, tissue cells displayed typical apoptotic features. While lactate dehydrogenase (LDH), (Alcohol dehydrogenase) ADH, catalase (CAT), and Na+/K+ -ATPase (NKA) activities were dramatically enhanced under hypoxic stress, glycogen content, and superoxide dismutase (SOD) activities were significantly reduced compared to the control group. The above results were further supported by the joint analysis of the transcriptome and metabolome, which further revealed that in addition to carbohydrates, including glycogen, the energy metabolism of the fatty acid, trehalose, and glyoxylate cycles is also included. Furthermore, we also revealed the ethanol tested in hypoxic stress should derive from symbiodinium of P akamusi. Understanding the processes which enable P. akamusi to survive lengthy periods of hypoxia in eutrophic lakes might help to design sensitive biomonitoring procedures, and this species has the potential to be used as an effective eutrophication indicator.

biophysics↗

bioRxiv2023

A Single Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome

Cyanobacteriochrome (CBCR) GAF domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, becoming fluorescent. The third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803 binds phycocyanobilin (PCB) natively, yielding red/green photoswitching properties but also binds phycoerythrobilin (PEB). GAF3-PCB has low quantum yields but non-photoswitching GAF3-PEB is brighter, making it a promising platform for new genetically encoded fluorescent tools. GAF3, however, shows low PEB binding efficiency (chromophorylation) at [~]3% compared to total protein expressed in E. coli. Here we explored site-directed mutagenesis and plasmid-based methods to improve GAF3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. We found that a single mutation improved chromophorylation while tuning the emission over [~]30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised by [~]7-fold. Moreover, we show that protein-chromophore interactions can tune autoisomerization of PEB to PUB in a GAF domain, which will facilitate future engineering of similar GAF domain-derived fluorescent proteins.

biochemistry↗

bioRxiv2023

Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy

BackgroundCancer immunotherapy has transformed the clinical approach to patients with malignancies as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment (TME) to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. MethodsWe developed approaches to categorize solid tumors into "Desert", "Excluded" and "Inflamed" types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. ResultsWe show that categorization based on "manual" observation is predictive for clinical benefit from anti-programmed cell death ligand-1 (PD-L1) therapy in two large cohorts of patients with non-small cell lung cancer (NSCLC) or triple-negative breast cancer (TNBC). For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based read-outs and patient response to therapy. ConclusionsOur findings suggest tumor immunophenotype (IP) generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. What is already known on this topicClinical benefit from checkpoint inhibitor-targeted therapies is realized only in a subset of patients. Robust biomarkers to identify patients who may respond to such therapies are needed. What this study addsWe have developed manual and automated approaches to categorize tumors into immunophenotypes based on the spatial distribution of CD8+ T effector cells that predict clinical benefit from anti-PD-L1 immunotherapy for patients with advanced non-small cell lung cancer or triple-negative breast cancer. How this study might affect research, practice or policyTumor immunophenotypes should be further validated as predictive biomarker for checkpoint inhibitor-targeted therapies in prospective clinical studies.

pathology↗

bioRxiv2023

Avirulent ME49Δgra5 Protecting Hosts Against Toxoplasma gondii Infection and Breast Tumors

Toxoplasma gondii is the causative agent of toxoplasmosis, a zoonotic disease that poses a threat to human health and a considerable loss to livestock farming. At present, clinical therapeutic drugs mainly target T. gondii tachyzoites and fail to eradicate bradyzoites. Developing a safe and effective vaccine against toxoplasmosis is urgent and important. T. gondii possesses dense granule organelles that secrete immunogenic dense granule proteins (GRAs). GRA5 localizes to the parasitophorous vacuole membrane (PVM) in the tachyzoite stage and to the cyst wall in the bradyzoite stage. Here we report that gra5 knockout T. gondii strain is avirulent and fails to form cysts, but stimulates sero-conversion in mice. We next investigated the protective efficacy of ME49{Delta}gra5 vaccination against T. gondii infection. All the immunized mice survived the challenge infection with either wild-type RH, ME49, VEG tachyzoites or ME49 cysts. Remarkably, ME49{Delta}gra5 tachyzoites inoculation in situ attenuated the growth of murine breast tumor (4T1) in mice and prevented 4T1s lung metastasis. ME49{Delta}gra5 inoculation might upregulate the levels of Th1 cytokines and tumor-infiltrating T cells in tumor microenvironment (TME) and trigger anti-tumor responses by increasing the number of NK, B, T cells, macrophages, and dendritic cells (DCs) in spleen. Collectively, the results support the potential of ME49{Delta}gra5 as attenuated live vaccine against T. gondii infection and breast cancer. Author summaryThe zoonotic toxoplasmosis poses a great disease burden to humans and big loss to livestock farming. At present, clinical therapeutic drugs mainly target T. gondii tachyzoites but have no effect on bradyzoites. A safe and effective vaccine against toxoplasmosis is urgent and important. Dense granule proteins (GRAs) are a group of immunoactive proteins secreted by T. gondii dense granule organelles. GRA5 is located on the parasitophorous vacuole membrane (PVM) at the tachyzoite stage and on the cyst wall at the bradyzoite stage. We discovered that the gra5 knockout T. gondii strain became avirulent and failed to form cysts in mice. ME49{Delta}gra5 immunized mice survived the challenge infection of wild-type RH, ME49, and VEG tachyzoites, and ME49 cysts. Meanwhile, ME49{Delta}gra5 tachyzoites inoculation in situ remarkably attenuated the growth of murine breast tumor (4T1) in mice including the injected and distant tumors, as well as prevented 4T1s lung metastasis. This results support the potential of ME49{Delta}gra5 as attenuated live vaccine against T. gondii infection and breast cancer.

microbiology↗

bioRxiv2023

Distinct mutational processes shape selection of MHC class I and class II mutations across primary and metastatic tumors

Disruption of antigen presentation via loss of MHC expression is a strategy whereby cancer cells escape immune surveillance and develop resistance to immunotherapy. We developed the personalized genomics algorithm Hapster and accurately called somatic mutations within the MHC genes of 10,001 primary and 2,199 metastatic tumors, creating a catalog of 1663 nonsynonymous mutations that provide key insights into MHC mutagenesis. We found that MHC-I genes are among the most frequently mutated genes in both primary and metastatic tumors, while MHC-II mutations are more restricted. Recurrent deleterious mutations are found within haplotype and cancer-type specific hotspots associated with distinct mutational processes. Functional classification of MHC residues revealed significant positive selection for mutations disruptive to the B2M, peptide, and T-cell binding interfaces, as well as MHC chaperones. At the cohort level, all cancers with positive selection for MHC mutations are responsive to immune checkpoint inhibitors, underscoring the translational relevance of our findings.

cancer biology↗

bioRxiv2022

The Cation/H+ exchanger OsCAX2 is involved in cadmium tolerance and accumulation through vacuolar sequestration in rice

Excessive cadmium (Cd) in rice grains is a serious food safety problem. The development of Cd-safe varieties requires the identification of germplasms and genes with major effect on Cd accumulation but without negative effects on other important traits. Here, we reported that OsCAX2, a member of the rice Cation/H+ exchanger (CAX) family, is an important Cd transporter. OsCAX2 encodes a tonoplast-localized protein and is strongly upregulated by Cd, mainly expresses in root exodermis, parenchyma in cortex, endodermis and stele cells. Depletion of OsCAX2 resulted in enhanced Cd sensitivity and root-to-shoot translocation in rice, while overexpression of OsCAX2 significantly increased Cd tolerance and reduced Cd transport by promoting root Cd influx and vacuolar storage, which ultimately reduced Cd transport via xylem. OsCAX2 also had significant effects on tissues/organs distribution of Cd but had no effects on grain yield and agronomic traits. Importantly, the OsCAX2 overexpressing lines had more than 70% lower grain Cd accumulation, increased iron (Fe), zinc (Zn) and manganese (Mn) and reduced copper (Cu) accumulation. Therefore, OsCAX2 is an ideal gene for developing Cd-safe rice varieties via transgenic approach.

plant biology↗

bioRxiv2022

Arabidopsis ORP2A positively regulates glucose signaling by interacting with AtRGS1 and promoting AtRGS1 degradation

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. AtRGS1 (Regulator of G protein Signaling 1) with intrinsic GTPase-accelerating protein (GAP) activity could suppress G protein and glucose signal transduction in Arabidopsis. However, how AtRGS1 activity is regulated is currently poor understood. Here we identified a knockout mutant orp2a-1 (oxysterol-binding protein (OSBP)-related protein 2A) which shows phenotypes similar to agb1-2 (arabidopsis g-protein beta 1). With overexpression of ORP2A, transgenic lines display short hypocotyl, hypersensitivity to sugar and lower intracellular AtRGS1 level than control. Consistently, ORP2A shows interaction with AtRGS1 in vitro and vivo. Tissue specificity of ORP2A with two alternative protein forms imply its functions in organ size and shape controlling. Bioinformatic data and phenotypes of orp2a-1, agb1-2 and double mutant reveal genetic interactions in the regulation of G protein signaling and sugar response between ORP2A and G{beta}. Both alternative splicing forms of ORP2A locate in the ER, PM (Plasma Membrane) and EPCS (ER-PM Contact Sites), and interact with VAP27-1 mediated by a FFAT-like motif in vivo and vitro. ORP2A also displays differential phosphatidyl phosphoinositide binding activity mediated by its PH domain in vitro. Taken together, it is suggested that Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

plant biology↗

bioRxiv2022

Enhancing Mitochondrial Functions by Optogenetic Clustering

Known as the powerhouses of cells, mitochondria and its dynamics are important for their functions in cells. Herein, an optogenetic method that controlling mitochondria to form the clusters was developed. The plasmid named CRY2PHR-mCherry-Miro1TM was designed for the optogenetic system. The photoactivable protein CRY2PHR was anchored to mitochondria, via the specific organelle-targeting transmembrane domain Miro1TM. Under blue light illumination, CRY2PHR can form the oligomerization, called puncta. With the illuminated time extended, the puncta can interact, and the mitochondria were found to form clustering with reversibility and spatiotemporal controllability. The mitochondrial functions were found to enhance after the formation of optogenetic mitochondrial clusters. This method presented here provides a way to control mitochondrial clustering and raise mitochondrial functions up.

cell biology↗

bioRxiv2022

A Novel Selective ERK1/2 Inhibitor, Laxiflorin B, Targets EGFR Mutation Subtypes in Non-small-cell Lung Cancer

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways including the ErbB axis. ERK1/2 ectopic activation is responsible for various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, but specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel ERK covalent inhibitor, Laxiflorin B, a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. More importantly, mass spectrometry combined with computer simulation analysis revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 through D-ring, and Cys-178 of ERK1 though non-inhibitory binding of A-ring, respectively. Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity in a NSCLC tumor xenograft model in nude mice, and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 modification of Laxiflorin B, exhibited higher affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

pharmacology and toxicology↗

bioRxiv2022

Crowd-sourced benchmarking of single-sample tumour subclonal reconstruction

Tumours are dynamically evolving populations of cells. Subclonal reconstruction algorithms use bulk DNA sequencing data to quantify parameters of tumour evolution, allowing assessment of how cancers initiate, progress and respond to selective pressures. A plethora of subclonal reconstruction algorithms have been created, but their relative performance across the varying biological and technical features of real-world cancer genomic data is unclear. We therefore launched the ICGC-TCGA DREAM Somatic Mutation Calling -- Tumour Heterogeneity and Evolution Challenge. This seven-year community effort used cloud-computing to benchmark 31 containerized subclonal reconstruction algorithms on 51 simulated tumours. Each algorithm was scored for accuracy on seven independent tasks, leading to 12,061 total runs. Algorithm choice influenced performance significantly more than tumour features, but purity-adjusted read-depth, copy number state and read mappability were associated with performance of most algorithms on most tasks. No single algorithm was a top performer for all seven tasks and existing ensemble strategies were surprisingly unable to outperform the best individual methods, highlighting a key research need. All containerized methods, evaluation code and datasets are available to support further assessment of the determinants of subclonal reconstruction accuracy and development of improved methods to understand tumour evolution.

bioinformatics↗

bioRxiv2021

The Transcriptome Architecture of Polyomaviruses

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

microbiology↗

bioRxiv2021

OXPHOS Promotes Apoptotic Resistance and Persistence in TH17 cells

Apoptotic cell death is a cell-intrinsic, immune tolerance mechanism that regulates the magnitude and resolution of T cell-mediated responses. Evasion of apoptosis is critical for the generation of memory T cells, as well as autoimmune T cells, and knowledge of the mechanisms that enable resistance to apoptosis will provide insight into ways to modulate their activity during protective and pathogenic responses. IL-17-producing CD4 T cells (TH17s) are long-lived, memory cells. These features enable their role in host defense, chronic inflammatory disorders, and anti-tumor immunity. A growing number of reports now indicate that TH17s in vivo require mitochondrial oxidative phosphorylation (OXPHOS), a metabolic phenotype that is poorly induced in vitro. To elucidate the role of OXPHOS in TH17 processes, we developed a system to polarize TH17s that metabolically resembled their in vivo counterparts. We discovered that directing TH17s to use OXPHOS promotes mitochondrial fitness, glutamine anaplerosis, and an anti-apoptotic phenotype marked by high BCL-XL and low BIM. Through competitive co-transfer experiments and tumor studies, we further revealed how OXPHOS protects TH17s from cell death while enhancing their persistence in the periphery and tumor microenvironment. Together, our work demonstrates a non-classical role of metabolism in regulating TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

immunology↗

bioRxiv2021

Two microRNA regulatory circuits set start and end times for dendritic arborization of a nociceptive neuron

Choreographic dendritic arborization takes place within a defined time frame, but the timing mechanism is currently not known. Here, we report that the precisely timed lin-4-lin-14 regulatory circuit triggers an initial dendritic growth activity whereas the precisely timed lin-28-let-7-lin-41 regulatory circuit signals a subsequent developmental decline in dendritic growth ability, hence restricting dendritic arborization within a set time frame. Loss-of-function mutations in the lin-4 microRNA gene cause limited dendritic outgrowth whereas loss-of-function mutations in its direct target, the lin-14 transcription factor gene, cause precocious and excessive outgrowth. In contrast, loss-of-function mutations in the let-7 microRNA gene prevent a developmental decline in dendritic growth ability whereas loss-of-function mutations in its upstream regulator, the lin-28 RNA-binding protein gene, and its direct target, the lin-41 tripartite motif protein gene, cause further decline. lin-4 and let-7 regulatory circuits are expressed in the right place at the right time to set start and end times for PVD dendritic arborization. Replacing the endogenous lin-4 promoter at the lin-4 locus with a late-onset let-7 promoter delays PVD dendrite arborization whereas replacing the endogenous let-7 promoter at the let-7 locus with an early-onset lin-4 promoter causes precocious decline in dendritic growth ability in PVD neurons. Our results indicate that the lin-4-lin-14 and the lin-28-let-7-lin-41 regulatory circuits control the timing of dendrite arborization through antagonistic regulation of the DMA-1 receptor level on dendrites. The LIN-14 transcription factor directly represses the dma-1 gene expression through a transcriptional means whereas the LIN-41 tripartite motif protein indirectly activates the dma-1 gene expression through a post-transcriptional means.

neuroscience↗

bioRxiv2021

The kpc-1 (furin) 3'UTR promotes dendritic transport and local translation of mRNAs to regulate dendrite branching and self-avoidance of a nociceptive neuron

A recently reported Schizophrenia-associated genetic variant in the 3UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3UTR in neuronal development(1). We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3UTR participates in dendrite branching and self-avoidance. The kpc-1 3UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes local protein synthesis. We identify a secondary structural motif in the kpc-1 3UTR required for dendrite self-avoidance. Animals with dma-1 receptor over-expression exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship.

neuroscience↗

bioRxiv2021

The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the devastating ongoing coronavirus disease-2019 (COVID-19) pandemic which poses a great threat to global public health. The spike (S) polypeptide of SARS-CoV-2 consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary. The inclusion of the 4 amino acids (PRRA) at the S1/S2 boundary forms a furin cleavage site (FCS), 682RRAR{downarrow}S686, distinguishing SARS-CoV-2 from its closest relative, the SARS-CoV. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, the virus acquired various deletions surrounding the FCS. In the present study, we studied the viral transcriptome in SARS-CoV-2 infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability at the S1/S2 boundary using RNA-seq. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the S1/S2 boundary of the S gene: one is a deletion of 12 amino acids, 678TNSPRRAR{downarrow}SVAS689, which contains the FCS, another is a deletion of 5 amino acids, 675QTQTN679, which is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions revealed that the selective pressure for the FCS maintains the S gene stability in HAE-ALI but with exceptions, in which the FCS deletions are remained at a high rate. Thus, our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is donor-dependent.

microbiology↗

bioRxiv2020

Control of archetype BK polyomavirus miRNA expression

BK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide persistently infected. BKPyV infection is usually asymptomatic in healthy people; however, it causes polyomavirus-associated nephropathy in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. BKPyV has a circular, double-stranded DNA genome that is divided genetically into three parts: an early region, a late region, and a non-coding control region (NCCR). The NCCR contains the viral DNA replication origin and cis-acting elements regulating viral early and late gene expression. It was previously shown that a BKPyV miRNA expressed from the late strand regulates viral large T antigen expression and limits the replication capacity of archetype BKPyV. A major unanswered question in the field is how expression of the viral miRNA is regulated. Typically, miRNA is expressed from introns in cellular genes but there is no intron readily apparent in the BKPyV from which the miRNA could derive. Here we provide evidence for primary RNA transcripts that circle the genome more than once and include the NCCR. We identified splice junctions resulting from splicing of primary transcripts circling the genome more than once, and Sanger sequencing of RT-PCR products indicates that there are viral transcripts that circle the genome up to four times. Our data suggest that the miRNA is expressed from the intron of these greater-than-genome size primary transcripts.

microbiology↗