Biology subjects

Bhalla, N.

Publications and source records attributed to Bhalla, N..

At least 19 recordsLinked to original sources

bioRxiv2026

NPP-21/TPR is required for developmental control of spindle checkpoint strength in C. elegans

The spindle checkpoint ensures accurate chromosome segregation by monitoring whether chromosomes, via kinetochores, are properly attached to the spindle. If chromosomes fail to establish bipolar attachment, the checkpoint delays the cell cycle to enable error correction. In C. elegans early embryos, activation of the spindle checkpoint produces a longer mitotic delay in primordial germ cells than somatic cells. We show that the conserved nucleoporin and spindle matrix component, NPP-21/TPR, is required for the stronger spindle checkpoint in germline cells. A checkpoint-proficient NPP-21::GFP transgene localizes to a spindle-like structure during mitosis and is enriched in germline cells, consistent with a cell-fate specific function for this protein. Finally, NPP-21 controls spindle checkpoint strength in germline cells via two, potentially linked, mechanisms: concentrating PCH-2 around mitotic chromosomes and promoting the localization of the checkpoint effector, Mad2, to unattached kinetochores. These experiments demonstrate a developmental role for NPP-21, and the spindle matrix, in controlling spindle checkpoint strength in immortal germline cells in C. elegans.

cell biology↗

bioRxiv2025

In-silico evidence of non-operonic fusion transcripts in Mycobacterium tuberculosis: algorithm optimization and signatures of genome plasticity.

BackgroundThe genome of Mycobacterium tuberculosis (Mtb) is known for its stable nature. It also contains transposases, redundant genes, repetitive DNA sequences, integrases, and remnants of lysogenized mycobacteriophages. These factors can result in intragenomic recombination, resulting in the formation of fusion transcripts. The present study aimed to identify signatures of long-distance gene fusion transcripts in RNA-seq data of clinical Mtb isolates. MethodologyThree approaches based on separate principles (split read alignment, repurposing STAR chimera, and transcript de novo assembly). The intersections of fusion calls between the three approaches that showed maximum performance were used for detecting fusions with real RNA-seq datasets of Mtb. ResulsThe junction of the split reads approach and the repurposed STAR chimera showed a high performance (F1 > 0.9). Sequence characteristics, clustering, and gene burden of operonic and long-distance gene fusions were consistent between the two independent real datasets, showing robustness of the optimized strategy. Fusion transcripts showed lineage specificity and signatures of indirect involvement of transposases, and transposition accessory genes (Rv1199c, Rv2512c, Rv3115, Rv0395, Rv2808, and Rv3327) in intragenomic recombination, resulting in the formation of fusion transcripts. The fusions mainly were within transposases, PPE, PE_PGRS family proteins, and some isolated fusions were of genes involved in the MoCo pathway, vesicle transport, and lipid turnover. ConclusionsThe observed fusions are likely driven by natural recombination, resulting in the formation of fusion proteins, coregulating proteins, or disruption. The study shows that the Mtb genome, especially of clinical isolates, may not be as stable as believed. ImportanceThe Mtb genome is believed to be stable, clonal, and immune to HGT, and thus, only SNPs and INDELs are thought to drive evolution. However, the drastic differences in phenotypes such as growth kinetics, virulence, and metabolic rate observed in clinical isolates compared to laboratory strains cannot be entirely attributable to SNPs and INDELs. The Mtb genome contains transposases and other accessory genes that can drive intragenomic recombination, bringing distant genes closer. As a result, there is a possibility of the occurrence of fusion transcripts. Growing evidence and our previous contributions also suggest changes in gene repertoires and gene copy numbers, which are also likely driven by intragenomic recombination events. This study presents optimization of a robust and easy-to-implement fusion calling algorithm using traditional bioinformatic calls. Using the same, we report fusion transcripts of non-operonic genes in the RNA-seq data of clinical Mtb isolates.

genomics↗

bioRxiv2025

Macrophages of diabetes and tuberculosis co-morbid conditions show perturbed differentiation and metabolic signatures

Diabetes negatively impacts the host immune system, leading to allergies, recurring infections, and lameness. Its impact on the distribution and molecular phenotypes of the bone marrow-derived macrophages (BMDMs) needs attention and, more importantly, in co-morbid conditions like diabetes and tuberculosis (DM-TB). In this study, total bone marrow cells were harvested at 3 w.p.i., from the low aerosol dose (100-120 CFU) of Mycobacterium tuberculosis H37Rv infected control and NA-STZ induced diabetic C57BL/6 mice. The bone marrow cells were differentiated into BMDMs, and CD11c, CD11b and F4/80 marker expressions were monitored using flow cytometry. BMDMs stimulated with Mtb H37Rv lysate were subjected to global transcriptome profiling to identify the perturbed metabolic and molecular landscape. The BMDMs of the DM-TB comorbid mice showed low CD11c, CD11b and higher F4/80 expression. The macrophages of the DM group showed a skewed pro-inflammatory response, characterized by perturbed complement response, NF-kB pathway and fatty acid metabolism, whereas macrophages of the DM-TB group showed deregulated ceramide and amino acid metabolism. Macrophages from the DM and DM-TB group stimulated with Mtb H37Rv lysate, showed a deregulated transcript signature that impacts humoral response. The dampened immune response observed in the DM-TB group macrophages highlights the importance of identifying novel targets for developing tailored drugs for comorbid conditions.

immunology↗

bioRxiv2025

Diabetes mellitus and tuberculosis comorbidity induce unique microbial gut dysbiosis and associated metabolome.

BackgroundPatients with diabetes and tuberculosis (DMTB) often exhibit atypical disease progression and poor treatment outcomes. Alterations in gut microbiota may contribute to the impaired immune response in diabetes. Understanding the microbiota composition and associated metabolite profiles in DMTB could provide insights for developing targeted adjunctive therapies to improve patient prognosis. MethodNicotinamide (NA) and streptozotocin (STZ) induced diabetic C57BL/6 mice were aerosol infected with 100-120 CFU of Mycobacterium tuberculosis H37Rv. At different time points (2 days pre-infection and 3- and 8 weeks post-infection; wpi), faecal samples were collected for microbiome and metabolome profiling using 16S rRNA sequencing and gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), respectively. ResultsDiabetic mice showed gut dysbiosis, with 16 bacterial genera significantly altered between DMTB and TB groups at 3 wpi, which reduced to 11 at 8 wpi. Among the 93 identified faecal metabolites, 11 and 10 metabolites were significantly deregulated (log2FC>{+/-}1.0, p<0.05) in the DMTB group compared to TB group at 3 and 8 wpi, respectively. A negative correlation between faecal valeric acid levels in DMTB with the abundances of Coriobacteriaceae, Granulicatella, Veillonella, Achromobacter, Erysipelatrichaceae, Atopobiaceae, Citrobacter, and Lactiplantibacillus were observed. Stronger correlations between microbiota and metabolite were observed in DMTB comorbidity compared to TB. Additionally, correlation analysis also revealed a phylogenetic distance-dependent synchrony in amino acid metabolism across five clades, each containing three or more bacterial genera. ConclusionThis study reveals significant differences in the gut microbiome and metabolome between DMTB and TB groups. The identified microbes and metabolic products may serve as potential prebiotic or probiotic candidates for improving recovery in DMTB comorbid conditions.

microbiology↗

bioRxiv2024

Copy number variation analysis of 9,482 Mycobacterium tuberculosis isolates identifies lineage-specific molecular determinants.

BackgroundClinical manifestations of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) show lineage-specific differences contributed by genetic polymorphism such as phylo-single nucleotide variations (PhyloSNPs) and insertion or deletions (INDELs). Intragenomic rearrangement events, such as gene duplications and deletions, may cause gene copy number differences in Mtb, contributing to lineage-specific phenotypic variations, if any, which need better understanding. ResultsThe relative gene copy number differences in high-quality publicly available whole genome sequencing datasets of 9,482 clinical Mtb isolates were determined by repurposing and modifying an RNA-seq data analysis pipeline. The pipeline included various steps, viz., alignment of reads, sorting by coordinate, GC bias correction, and variant stabilising transformation. The strategy showed maximum separation of lineage-specific clusters in two principal components, capturing [~]54% variability. Unsupervised hierarchical clustering of the top 100 genes and pairwise comparisons between Mtb lineages revealed an overlapping subset of genes (n=42) having significantly perturbed copy numbers (Benjamin Hochberg adjusted P-value < 0.05 and log2(drug-resistant/sensitive) > {+/-} 1). These 42 genes formed multiple tandem gene clusters and are known to be involved in virulence, pathogenicity and defence response to invading phages. A separate comparison showed a significantly high copy number of phage genes and a recently reported druggable target Rv1525 in pre- and extensively drug-resistant (Pre-XDR, XDR) compared to drug-sensitive clinical Mtb isolates. ConclusionThe identified gene sets in Mtb clinical isolates may be useful targets for lineage-specific therapeutics and diagnostics development.

genomics↗

bioRxiv2024

The conserved ATPase PCH-2 controls the number and distribution of crossovers by antagonizing crossover formation in C. elegans

Meiotic crossover recombination is essential for both accurate chromosome segregation and the generation of new haplotypes for natural selection to act upon. This requirement is known as crossover assurance and is one example of crossover control. While the conserved role of the ATPase, PCH-2, during meiotic prophase has been enigmatic, a universal phenotype when pch-2 or its orthologs are mutated is a change in the number and distribution of meiotic crossovers. Here, we show that PCH-2 controls the number and distribution of crossovers by antagonizing their formation. This antagonism produces different effects at different stages of meiotic prophase: early in meiotic prophase, PCH-2 prevents double strand breaks from becoming crossover-eligible intermediates, limiting crossover formation at sites of initial double strand break formation and homolog interactions. Later in meiotic prophase, PCH-2 winnows the number of crossover-eligible intermediates, contributing to the designation of crossovers and ultimately, crossover assurance. We also demonstrate that PCH-2 accomplishes this regulation through the meiotic HORMAD, HIM-3. Our data strongly support a model in which PCH-2s conserved role is to remodel meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors, coordinate meiotic recombination with synapsis, and contribute to the progressive implementation of meiotic recombination, guaranteeing crossover control.

cell biology↗

bioRxiv2022

Chromatin accessibility profiling in tissue sections by spatial ATAC

Current methods for epigenomic profiling are limited in the ability to obtain genome wide information with spatial resolution. Here we introduce spatial ATAC, a method that integrates transposase-accessible chromatin profiling in tissue sections with barcoded solid-phase capture to perform spatially resolved epigenomics. We show that spatial ATAC enables the discovery of the regulatory programs underlying spatial gene expression during mouse organogenesis, lineage differentiation and in human pathological samples.

genomics↗

bioRxiv2022

Lokatt: A hybrid DNA nanopore basecaller with an explicit duration hidden Markov model and a residual LSTM network

BackgroundBasecalling long DNA sequences is a crucial step in nanopore-based DNA sequencing protocols. In recent years, the CTC-RNN model has become the leading basecalling model, supplanting preceding hidden Markov models (HMMs) that relied on pre-segmenting ion current measurements. However, the CTC-RNN model operates independently of prior biological and physical insights. ResultsWe present a novel basecaller named Lokatt: explicit duration Markov model and residual-LSTM network. It leverages an explicit duration HMM (EDHMM) designed to model the nanopore sequencing processes. Trained on a newly generated library with methylation-free Ecoli samples and MinION R9.4.1 chemistry, the Lokatt basecaller achieves basecalling performances with a median single read identity score of 0.930, a genome coverage ratio of 99.750%, on par with existing state-of-the-art structure when trained on the same datasets. ConclusionOur research underlines the potential of incorporating prior knowledge into the basecalling processes, particularly through integrating HMMs and recurrent neural networks. The Lokatt basecaller showcases the efficacy of a hybrid approach, emphasizing its capacity to achieve high-quality basecalling performance while accommodating the nuances of nanopore sequencing. These outcomes pave the way for advanced basecalling methodologies, with potential implications for enhancing the accuracy and efficiency of nanopore-based DNA sequencing protocols. Supplementary informationSupplementary data are available online.

bioinformatics↗

bioRxiv2022

The conserved AAA-ATPase PCH-2 distributes its regulation of meiotic prophase events by remodeling multiple meiotic HORMADs in C. elegans

During meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination in C. elegans by remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs in C. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.

cell biology↗

bioRxiv2022

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

microbiology↗

bioRxiv2021

Low numbers of cytokine transcripts drive inflammatory skin diseases by initiating amplification cascades in localized epidermal clusters

Abundant polyclonal T cells infiltrate chronic inflammatory diseases and characterization of these cells is needed to distinguish disease-driving from bystander immune cells. Here, we investigated 52,000 human cutaneous transcriptomes of non-communicable inflammatory skin diseases (ncISD) using spatial transcriptomics. Despite the expected T cell infiltration, we observed only 1-10 pathogenic T cell cytokine per skin section. Cytokine expression was limited to lesional skin and presented in a disease-specific pattern. In fact, we identified responder signatures in direct proximity of cytokines, and showed that single cytokine transcripts initiate amplification cascades of thousands of specific responder transcripts forming localized epidermal clusters. Thus, within the abundant and polyclonal T cell infiltrates of ncISD, only a few T cells drive disease by initiating an inflammatory amplification cascade in their local microenvironment.

immunology↗

bioRxiv2021

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

microbiology↗

bioRxiv2021

Mad1s ability to interact with Mad2 is essential to regulate and monitor meiotic synapsis in C. elegans

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we show that mutations in some spindle checkpoint mutants affect PC movement early in meiotic prophase, consistent with a link between PC mobility and the regulation of synapsis. Further, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that abrogates Mad1s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is a major regulator of meiotic synapsis in C. elegans. AUTHOR SUMMARYSexual reproduction relies on production of gametes, such as eggs and sperm, which are produced during meiosis. During this specialized cell division, chromosomes replicate, pair with their homologs, undergo synapsis and finally undergo recombination, all of which are required for correct meiotic chromosome segregation. Chromosomes are highly mobile during these steps in meiosis but the specific role of this mobility is unclear. Here, we show that spindle assembly checkpoint proteins, Mad1 and Bub3, that regulate and monitor meiotic synapsis are implicated in chromosome movement, solidifying the functional link between chromosome mobility and synapsis. Moreover, we provide additional data that another spindle checkpoint effector, Mad2, and its regulation by Mad1, plays an important role in regulating meiotic synapsis.

cell biology↗

bioRxiv2020

Brilacidin, a COVID-19 Drug Candidate, Exhibits Potent In Vitro Antiviral Activity Against SARS-CoV-2

SummarySevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than one million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin--a de novo designed synthetic small molecule that captures the biological properties of HDPs--on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is primarily a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin has demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 and thus supports brilacidin as a promising COVID-19 drug candidate. HighlightsO_LIBrilacidin potently inhibits SARS-CoV-2 in an ACE2 positive human lung cell line. C_LIO_LIBrilacidin achieved a high Selectivity Index of 426 (CC50=241M/IC50=0.565M). C_LIO_LIBrilacidins main mechanism appears to disrupt viral integrity and impact viral entry. C_LIO_LIBrilacidin and remdesivir exhibit excellent synergistic activity against SARS-CoV-2. C_LI Significance StatementSARS-CoV-2, the emergent novel coronavirus, has led to the current global COVID-19 pandemic, characterized by extreme contagiousness and high mortality rates. There is an urgent need for effective therapeutic strategies to safely and effectively treat SARS-CoV-2 infection. We demonstrate that brilacidin, a synthetic small molecule with peptide-like properties, is capable of exerting potent in vitro antiviral activity against SARS-CoV-2, both as a standalone treatment and in combination with remdesivir, which is currently the only FDA-approved drug for the treatment of COVID-19.

microbiology↗

bioRxiv2020

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drugtreatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

microbiology↗

bioRxiv2019

PCH-2 collaborates with CMT-1 to proofread meiotic homolog interactions

The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds ATP but cannot hydrolyze it: pch-2E253Q. In vitro, this mutant binds its substrates but is unable to remodel them. This mutation results in non-homologous synapsis and loss of crossover assurance. Surprisingly, worms with a null mutation in PCH-2s adapter protein, CMT-1, the ortholog of p31comet, localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2E253Q mutants indicate that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.

cell biology↗

bioRxiv2019

Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3 untranslated region (3 UTR) of the virus. Three of the sites are \"canonical\" with all 8 seed sequence residues complimentary to miR-142-3p while one is \"non-canonical\" and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.\n\nAuthor SummaryEastern equine encephalitis virus (EEEV) is one of the most acutely virulent mosquito-borne viruses in the Americas. A major determinant of EEEV virulence is a mammalian microRNA (miRNA) that is primarily expressed in myeloid cells, miR-142-3p. Like miRNA suppression of host mRNA, miR-142-3p binds to the 3 untranslated region (UTR) of the EEEV genome only in myeloid cells suppressing virus replication and the induction of the innate immune response. In this study, we used point mutations in all four miR-142-3p binding sites in the EEEV 3 UTR to understand the mechanism behind this miRNA suppression. We observed that decreasing the number of miR-142-3p binding sites leads to virus escape and ultimately attenuation in vivo. Furthermore, another virus, western equine encephalitis virus, also encodes miR-142-3p binding sites that contribute to virulence in vivo. These results provide insight into the mechanism of how cell-specific miRNAs can mediate suppression of virus replication.

microbiology↗

bioRxiv2018

PCH-2TRIP13 regulates spindle checkpoint strength

Spindle checkpoint strength is dictated by three criteria: the number of unattached kinetochores, cell volume and cell fate. We show that the conserved AAA-ATPase, PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in C. elegans. When we manipulate embryos to decrease cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role in checkpoint strength is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2s localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This role may be specifically relevant in scenarios where maintaining genomic stability is particularly challenging, such as in oocytes and early embryos enlarged for developmental competence and germline cells that maintain immortality.

cell biology↗