Biology subjects

Rani, P.

Publications and source records attributed to Rani, P..

8 recordsLinked to original sources

bioRxiv2026

A bifunctional RNase H protein supports DNA replication and transcription in nucleus-forming Chimalliviridae phages

Jumbo bacteriophages of the Chimalliviridae family replicate their genomes within a proteinaceous phage nucleus that spatially segregates viral DNA replication and transcription from translation. While the phage nucleus protects viral DNA from host defenses, it also imposes unique constraints on genome replication and gene expression. Here we identify the phage-encoded protein Rhc (RNase H-clamp) as a central mediator of genome transactions within the phage nucleus. Using the Escherichia coli infecting bacteriophage Goslar, we show that Rhc functions as an essential component of a non-canonical three-subunit DNA polymerase complex composed of DNAPN, DNAPC and Rhc. Rhc physically interacts with the DNAPC subunit, and both the catalytic activity of Rhc's N-terminal RNase H domain and its C-terminal sliding clamp-derived DNA binding domain are required for genome replication, nuclear maturation, and productive infection. Further, Goslar Rhc also supports transcription within the phage nucleus through a function that is separable from its DNA replication function. Rhc depletion results in reduced transcription of a subset of late genes, in a pattern that suggests a role for Rhc in resolving R-loops. Imaging and cryo-electron tomography reveal that loss of Rhc arrests phage development at a nascent nuclear stage, uncoupling nuclear assembly from maturation. Together, these findings identify Rhc as a multifunctional nuclear factor that mediates both DNA replication and transcription in nucleus-forming jumbo phages, revealing how viruses repurpose a conserved enzymatic activity to coordinate gene expression within a compartmentalized replication environment.

cell biology↗

bioRxiv2026

Pleiotropic antimalarial activities and immunomodulation exhibited by Himalayan Buransh (Rhododendron arboreum) in human and rodent malaria models

Emerging drug resistance against the malaria parasite is worrisome and necessitates the development of novel antimalarials. Himalayan Buransh (Rhododendron arboreum) is a well-known medicinal plant found in the northern states of India. In this study, we observed the pleiotropic antimalarial activities and immunomodulation exhibited by the aqueous extract of Buransh flower (AEBF). AEBF demonstrated significant IC50 values (16-29 {micro}g/ml) against the asexual stages of various P. falciparum strains (3D7, Dd2-chloroquine-resistant and C580Y-artemisinin resistant). The oral administration of AEBF (200 mg/kg) in mice, suppressed [~]80% P. berghei parasitemia, improved mean survival time (MST-23.5 days) and prevented splenomegaly. Notably, the combination of AEBF and artesunate not only cleared primary infection, but also conferred sustained immunity. This immunomodulatory effect, driven by protective IFN-{gamma} resulted in reduced parasitemia during a homologous challenge without the need for further treatment. It is important to highlight the malaria transmission blocking activity of AEBF, resulting in reduced sexual stage male gametocyte exflagellation. Furthermore, the virtual drug screening of selected bioactive constituents from Buransh flower demonstrated potent binding against multiple P. falciparum proteins, suggested a pleiotropic mode of action. Altogether, our results corroborated the first ever evidence of the multistage antimalarial potential of Buransh flower, supported by in vitro cell studies, in vivo rodent malaria model and in silico docking analyses. Based on our studys findings and the traditional use of Buransh juice as a medicinal beverage in Uttarakhand, India, we propose exploring it as an adjunct therapy for drug-resistant malaria, subject to further clinical validation.

pathology↗

bioRxiv2026

A DNA damage-activated kinase controls bacterial immune pathway expression

Bacteria encode myriad stress-response pathways that protect their hosts against both internal and external threats. A key question is how these pathways are regulated, especially anti-phage immune pathways that mediate host cell killing. Here, we identify two proteins termed CapK and CapS that are encoded upstream of diverse immune operons, and regulate their expression in response to DNA damage. CapK resembles bacterial anti-sigma factor kinases, and CapS resembles these proteins STAS domain antagonists. CapS is a DNA-binding transcriptional repressor, and phosphorylation of CapS by CapK results in dissociation of a CapS homodimer and de-repression of transcription. CapKs kinase activity is directly activated by single-stranded DNA generated as a by-product of DNA repair. Finally, we show that CapK and CapS-like proteins have been co-opted into an anti-phage toxin-antitoxin system with a VapC-like protein, where they similarly respond to DNA damage to activate VapCs nuclease activity. Overall, our results reveal how a kinase-substrate pair can regulate expression of an adjacent operon in response to DNA damage, and highlight the modularity of immune and other stress-response pathways.

microbiology↗

bioRxiv2023

microRNA-22 displaces ITAFs from the 5'UTR and inhibit the translation of Coxsackievirus B3 RNA

microRNAs play an essential role in gene regulation during virus infections and have major consequences on viral pathogenesis. During RNA virus infections, the host miRNAs can target both host mRNAs and the virus genomic RNA. Using the CVB3 virus as a model, we have investigated how a host miRNA can target viral genomic RNA and act as an antiviral factor limiting the growth of the virus. CVB3 is an RNA virus whose infection causes myocarditis and, eventually, dilated cardiomyopathy. We shortlisted miRNAs with a potential binding site in the CVB3 genomic RNA. Among these, miR-22 was picked for further studies as its binding site was putatively located in a region in the CVB3 5 UTR, important for recruiting ITAFs and ribosomes for IRES-mediated translation. Using mutational analysis and pull-down assays, we first confirmed the binding of miR-22 on the 5UTR. This binding negatively regulated the translation of CVB3 RNA. However, miR-22 binding-defective mutant of CVB3 RNA had no effect of miR-22 overexpression and could translate normally. Moreover, cells from which miR-22 was knocked out, showed a higher level of CVB3 infection as compared to the wild type. We have further demonstrated that the binding of miR-22 interferes with the recruitment of several ITAFs (La, PSF, and PTB) on viral mRNA. This abrogates the spatial structure necessary for ribosome recruitment on the CVB3 RNA, ultimately inhibiting its translation. Also, the level of miR-22 increases 4 hours post-infection, presumably after the synthesis of viral 2A protease, to regulate infection in the host cell more effectively. Along with the direct effect on viral RNA, the altered level of miR-22 affects the level of its cellular targets which might contribute to CVB3 infection. To identify the possible players, we obtained a list of miR-22 targets and performed pathway analysis. Several targets were shortlisted among the top hits and their levels upon CVB3 infection were checked. Protocadherin-1 (PCDH-1), a single-pass transmembrane protein, followed an expected trend, and its levels were significantly downregulated upon CVB3 infection in miR-22 dependent manner. miR-22 mediated suppression of PCDH1 levels during CVB3 infection points towards the possible role of miR-22 in either modulating antiviral signaling or in virus entry, in addition to regulating the viral translation.

molecular biology↗

bioRxiv2023

Human antigen R differentially modulates the translation of SARS-CoV-2 genomic and sub-genomic RNAs

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) viral RNA associates with different RNA-binding host proteins at each stage of its life cycle. We found sequence dependent binding of one such important protein, human antigen R (HuR) to SARS-CoV-2 5'UTR and studied its potential role in virus life cycle. The knockdown and knockout studies revealed importance of such binding in viral translation. We identified 5'-UTR mutations in SARS-CoV-2 variants of concern that altered the HuR-binding affinity. Interestingly, HuR enhanced non-structural protein translation through the genomic 5'-UTR, by promoting polypyrimidine tract-binding protein binding to the 5'-UTR. However, HuR suppressed the structural protein translation from sub genomic 5UTR. HuR knockout increased the sensitivity to remdesivir treatment by decreasing its half-maximal inhibitory concentration. An antisense oligonucleotide (whose binding site overlapped the HuR-binding site) reduced viral RNA levels in wild-type cells but not HuR-knockout cells. Our results indicate that HuR regulates the balance between SARS-CoV-2 structural and non-structural proteins and guides the infection of viral variants, implying that HuR can potentially be targeted for therapeutic interventions. Author SummaryViruses interacts with various host proteins throughout their life cycle. One significant protein is HuR, an RNA-binding protein that regulates RNA stability and translation. HuR binds to viral RNAs at the 5UTR or 3UTR, impacting their translation and replication. We identified conserved HuR binding sites in the SARS-CoV-2 5UTR across different beta coronaviruses. This binding enhanced the initiation of translation from the genomic 5 UTR, increasing the production of non-structural proteins essential for viral replication. Additionally, we discovered that another host protein, PTB, promotes HuR binding to the viral 5 UTR, facilitating its loading onto ribosomes. Conversely, HuR plays an antagonistic role concerning subgenomic RNAs (sgRNAs), which code for structural proteins, by regulating and limiting their levels. This dual regulation indicates that the virus exploits HuR for its benefit while the host employs it to control viral spread. Targeting HuR may help manipulate the SARS-CoV-2 life cycle. We found that HuR knockout increased sensitivity to the antiviral drug Remdesivir. Using an antisense oligonucleotide to block HuR binding effectively reduced viral RNA levels. Our findings highlight the critical role of HuR in regulating viral protein production and its potential as a therapeutic target.

microbiology↗

bioRxiv2022

A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models.

The COVID-19 pandemic has highlighted the need for novel antivirals for pandemic management and preparedness. Targeting host processes that are co-opted by viruses is an attractive strategy for developing antivirals with a high resistance barrier. Picolinic acid (PA) is a byproduct of tryptophan metabolism, endogenously produced in humans and other mammals. Here we report broad-spectrum antiviral effects of PA against enveloped viruses, including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Influenza A virus (IAV), Flaviviruses, Herpes Simplex Virus, and Human Parainfluenza Virus. We further demonstrate using animal models that PA is effective against SARS-CoV-2 and IAV, especially as an oral prophylactic. The mode of action studies revealed that PA inhibits viral entry of enveloped viruses, primarily by interfering with viral-cellular membrane fusion, inhibiting virus-mediated syncytia formation, and dysregulating cellular endocytosis. Overall, our data establish PA as a broad-spectrum antiviral agent, with promising preclinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

microbiology↗

bioRxiv2020

A multifunctional Type1A DNA/RNA topoisomerase with RNA hydrolysis activity from Mycobacterium smegmatis and Mycobacterium tuberculosis

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression, indicated the enzymes back-up support to RNases involved in rRNA processing. We demonstrate that the active site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity while retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically-related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival.

biochemistry↗

bioRxiv2019

Study on the influence of G82S RAGE polymorphism on RAGE-Amyloid interaction in AD pathology

Receptor for advanced glycation end products (RAGE) has been implicated in the pathophysiology of AD due to its ability to bind amyloid-beta and mediate inflammatory response. G82S RAGE polymorphism is associated with AD but the molecular mechanism for this association is not understood. Our previous in silico study indicated a higher binding affinity for mutated G82S RAGE, which could be caused due to changes in N linked glycosylation at residue N81. To confirm this hypothesis, in the present study molecular dynamics (MD) simulations were used to simulate the wild type (WT) and G82S glycosylated structures of RAGE to identify the global structural changes and to find the binding efficiency with A{beta}42 peptide. Binding pocket analysis of the MD trajectory showed that cavity/binding pocket in mutant G82S glycosylated RAGE variants is more exposed and accessible to external ligands compared to WT RAGE, which can enhance the affinity of RAGE for A{beta}. To validate the above concept, an in vitro binding study was carried using SHSY5Y cell line expressing recombinant WT and mutated RAGE variant individually to which HiLyte Fluor labeled A{beta}42 was incubated at different concentrations. Saturated binding kinetics method was adopted to determine the Kd values for A{beta}42 binding to RAGE. The Kd value for A{beta}42-WT and A{beta}42-mutant RAGE binding were 92{+/-}40 nM (95% CI-52 to 152nM; R2-0.92) and 45{+/-}20 nM (95% CI -29 to 64nM; R2-0.93), respectively. The Kd value of <100nM observed for both variants implicates RAGE as a high-affinity receptor for A{beta}42 and mutant RAGE has higher affinity compared to WT. The alteration in binding affinity is responsible for activation of the inflammatory pathway as implicated by enhanced expression of TNF and IL6 in mutant RAGE expressing cell line which gives a mechanistic view for the G82S RAGE association with AD.

neuroscience↗