Biology subjects

Mondal, A.

Publications and source records attributed to Mondal, A..

43 records · Page 3Linked to original sources

bioRxiv2021

A species-specific signature residue in the PB2 subunit of the bat influenza virus polymerase restricts viral RNA synthesis.

Bat influenza A viruses (H17N10 and H18N11) are genetically distant from conventional influenza A viruses and replicates poorly in non-bat hosts species. However, the reason behind the lower replication fitness of these viruses are yet to be elucidated. In this work, we have identified species-specific signature residues, present in viral PB2 protein, which is a major determinant of polymerase fitness in human, avian and bat cell lines. Through extensive sequence and structural comparison between the bat and non-bat influenza virus RNA polymerases, we have identified a previously uncharacterized PB2-282 residue, which is serine in bat virus PB2 protein but harbours highly conserved glutamic acid in conventional influenza A viruses. Introduction of these bat specific signatures in the polymerase of a human adapted strain of influenza A/H1N1 virus drastically reduces its polymerase activity and replication efficiency in cell lines of human, bat and canine origin. In contrast, introduction of the human specific signatures in bat virus PB2 (H17N10), significantly enhances its function in the context of a chimeric RNA polymerase. Interestingly, the PB2-282 resides within an evolutionary conserved "S-E-S" motif present across different genera of influenza viruses but is replaced with a "S-S-T" motif in bat influenza viruses, indicating that this E to S transition may serve as a species-specific adaptation signature that modulates the activity of bat virus polymerase in other host species. ImportanceRecent isolation of influenza A like viruses (H17N10 and H18N11) in bats raised concerns about their potential of zoonotic transmission in human. Here we present species-specific signature residues present in the bat influenza virus polymerase, which may act as critical modulators of bat virus propagation in non-bat host species. We utilize bioinformatics based comparative analysis followed by functional screening in order to identify the PB2-282nd position, which harbors a highly conserved glutamic acid in conventional influenza A viruses, but contains an unusual serine in case of bat influenza viruses. Human adapted polymerase, harboring bat specific signature (PB2-S282) performs poorly, while bat PB2 protein harboring human specific signature (PB2-E282) shows increased fitness in human cells. Together, our data identifies novel species-specific signatures present within the influenza virus polymerase that may serve as a key factor in the adaptation of influenza viruses from bat to non-bat host species and vice versa.

microbiology↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

Structure Specific Neuro-toxicity of α-Synuclein Oligomer

Parkinsons disease (PD) is linked to -synuclein (aS) aggregation and deposition of amyloid in the substantia nigra region of the brain tissues. Recent reports suggested that oligomeric assembly structure could be neurotoxic to neuronal cells. In the current investigation we produced two distinct classes of aS oligomers and link the protein conformation state and stability to neuronal cell toxicity. Natural oligomers (NO) enriched with alpha-helical folds are produced in storage of aS at below -20{degrees}C for 7 days. Induced oligomer (IO), often observed in the aggregation pathway of aS were made incubating the protein solution at 37{degrees}C. Natural oligomers remained stable and did not transform into {beta}-sheet rich amyloid fiber and exhibited higher toxicity (80% cell death) compared to induced oligomers. Natural oligomers were ovular shape and the size ranged between 4-5.5 nm. It maintained significant number ([~] 60%) of residues in -helical conformational space. However, initiation of hydrophobic zipping with beta sheet conformation was evidenced in induced oligomer (IO) and a lesser number residues (45%) remained with preference to -helical secondary structure. Hydrophobic collapse leads the transformation of IO into thermodynamically most stable {beta}-sheet rich amyloid fibril. Molten globule like secondary structure stabilized by H-bonding in natural oligomers caused enhanced stability and cellular toxicity compared to induced oligomer. Thus off-pathway/natural oligomers could be plausible reason of neuronal cell death and possible cause of Parkinsons disease.

biophysics↗

bioRxiv2020

Development of a copper-graphene nanocomposite based transparent coating with antiviral activity against influenza virus

Respiratory infections by RNA viruses are one of the major burdens upon global health and economy. Viruses like influenza or coronaviruses can be transmitted through respiratory droplets or contaminated surfaces. An effective antiviral coating can decrease the viability of the virus particles in the outside environment significantly, hence reducing their transmission rate. In this work, we have screened a series of nanoparticles and their composites for antiviral activity using Nano Luciferase based highly sensitive influenza A reporter virus. Using this screening system, we have identified copper-graphene (Cu-Gr) nanocomposite shows strong antiviral activity. Extensive material and biological characterization of the nanocomposite suggested a unique metal oxide embedded graphene sheet architecture that can inactivate the virion particles only within 30 minutes of pre-incubation and subsequently interferes with the entry of these virion particles into the host cell. This ultimately results in reduced viral gene expression, replication and production of progeny virus particles, slowing down the overall pace of progression of infection. Using PVA as a capping agent, we have been able to generate a Cu-Gr nanocomposite based highly transparent coating that retains its original antiviral activity in the solid form.

microbiology↗

bioRxiv2020

Mechanism of Dynamic Binding of Replication Protein A to ssDNA

Replication protein A (RPA) serves as hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with ssDNA intermediates before handing over them to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the sizes of ssDNA intermediates. We further estimated the association kinetics and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive bulge formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. Our analysis explains how the short-lived, long ssDNA intermediates are processed quickly in vivo. The study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication. Significance StatementDespite ssDNA be the common intermediate to all pathways involving RPA, how does the latter function differently in the DNA processing events such as DNA repair, replication, and recombination just based on the length of ssDNA intermediates remains unknown. The major hindrance is the difficulty in capturing the transient interactions between the molecules. Even attempts to crystallize RPA complexes with 32nt and 62nt ssDNA have yielded a resolved structure of only 25nt ssDNA wrapped with RPA. Here, we used a state-of-the-art coarse-grained protein-ssDNA model to unravel the detailed mechanism of binding of RPA to ssDNA. Our study illustrates the molecular origin of variations in RPA action during various DNA processing events depending on the length of ssDNA intermediates.

biophysics↗

bioRxiv2020

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. We previously showed that phosphorylation regulates genome replication by controlling assembly of the replication machinery (Mondal, et al. 2017). However, it remained unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

microbiology↗

bioRxiv2019

Microbially induced calcite precipitation using Bacillus velezensis with guar gum

The present study was performed to explore the efficiency of microbially induced calcite precipitation (MICP) via locally isolated bacterial strains. Strains were isolated from soil and were screened for urease activity as well as microbial precipitation. Among all screened isolates, a carbonate precipitating soil bacterium was subjected for 16S rRNA gene sequencing. This strain was identified as Bacillus velezensis. The MICP characteristics of this strain were explored under three different media compositions and significant amount of precipitation in all cases was observed. Highest amount of precipitation was seen with guar as a biopolymer additive medium under experimented conditions. Activity of isolated strain with reference to pH profile, and ammonia concentration and total reducing sugar was further explored under media supplemented with four concentrations of guar (0.25%, 0.5%, 0.75% and 1% w/v). Microstructural analysis of microbial precipitation was performed with the help of scanning electron microscopy (SEM) and X-ray powder diffraction (XRD) analysis, which confirmed the presence of calcium carbonate in different phases. The strain was subjected to bio-cementation and locally available sand was successfully consolidated. XRD results confirmed the presence of calcium carbonate on consolidated samples.

microbiology↗