Biology subjects

Anantpadma, M.

Publications and source records attributed to Anantpadma, M..

5 recordsLinked to original sources

bioRxiv2025

Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

Pooled CRISPR knockout (KO) screens using live viruses are a proven and valuable approach for identifying essential host factors acting across viral life cycles. Here we describe the development of a pooled genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically identify host factors essential for viral replication. Virus replicons are non-infectious, therefore enabling the study of highly virulent viruses under standard biosafety level 2 containment. We developed a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line to perform a pooled genome-wide FACS-based CRISPR KO screen. This benchmark DENV-2 replicon screen successfully identified host genes previously known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components) and confirmed two additional genes (DOHH and ZFP36L2) involved in replication that have not been recovered in prior live virus screens. We applied this replicon screening approach to two highly divergent viruses: chikungunya virus (CHIKV), a positive-sense RNA virus that replicates at the plasma membrane, and Ebola virus (EBOV), a negative-sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified two genes known to be required for replication (G3BP1 and G3BP2) and several additional, novel genes (CLEC4G, CSDE1, GOLGA7, HNF1A, and PCBD1). We verified two of them (CSDE1 and GOLGA7) in live CHIKV replication assays. A distinct set of genes (EHMT1, EHMT2, and USP7) were recovered in the EBOV replicon screen and were further confirmed using independent transient transfection assays. Thus, viral replicon-based screens provide a useful approach that can be extended to viruses of diverse taxa to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed medical countermeasures.

genomics↗

bioRxiv2024

Recombinant Pichinde reporter virus as a safe and suitable surrogate for high-throughput antiviral screening against highly pathogenic arenaviruses

Several arenaviruses, such as the Old World (OW) Lassa virus (LASV) and the New World (NW) Junin virus (JUNV), can cause severe and lethal viral hemorrhagic fevers in humans. Currently, no vaccines or specific antiviral therapies are FDA-approved for treating arenavirus infections. One major challenge for the development of new therapeutic candidates against these highly pathogenic viruses is that they are BSL-3/4 pathogens that need to be handled in high biocontainment laboratories. In this work, a recombinant non-pathogenic New World arenavirus, Pichinde virus (rPICV), was used for the development of a high-throughput screening (HTS) assay in the BSL-2 laboratory for the screening and identification of small molecule inhibitors against arenaviruses. The rPICV is a replication-competent virus expressing the firefly luciferase reporter gene in the infected cells proportionally to the infection rate. rPICV infection was optimized for an automated HTS in 384-well format with robust Z' scores, high signal-to-background ratios, and low intrinsic variance. Screening an established library allowed for the identification of five top hit compounds, which included ribavirin, a known inhibitor of arenaviral RNA synthesis, showing good potency and selectivity in inhibiting rPICV replication. The antiviral activity of the top hit compounds was further validated against another recombinant arenavirus, the OW lymphocytic choriomeningitis virus (rLCMV) and against laboratory strains of LASV (Josiah) and JUNV (Romero). The use of rPICV in the HTS-based antiviral assay under BSL-2 condition has proven to be safe and suitable for the identification of broad-spectrum small molecule inhibitors against highly pathogenic arenaviruses.

microbiology↗

bioRxiv2021

Multidose evaluation of 6,710 drug repurposing library identifies potent SARS-CoV-2 infection inhibitors In Vitro and In Vivo.

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

microbiology↗

bioRxiv2020

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs. In these interactions the largely alpha-helical carboxy-terminal domain of the EBOV VP30 engages with the motif such that the prolines adopt non-canonical orientations, as compared to other proline-rich motifs. Affinity tag-purification mass spectrometry identified additional PPxPxY-containing host proteins, including hnRNP L, hnRNPUL1 and PEG10, as VP30 interactors. Of these, hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV replication, whereas hnRNPUL1 enhances. Further, double knockdown studies support additive effects of RBBP6 and hnRNP L. Binding studies demonstrate variable capacity of PPxPxY motifs to bind VP30 and the extended motif PxPPPPxY is demonstrated to confer optimal binding and to inhibit RNA synthesis, with the fifth proline and the tyrosine being most critical. Competition binding and hydrogen-deuterium exchange studies demonstrate that each protein binds a similar interface on VP30 and impacts VP30 phosphorylation. VP30 therefore represents a novel proline recognition domain that allows multiple host proteins to target a single viral protein-protein interface to modulate viral transcription.

microbiology↗

bioRxiv2020

Pyronaridine Tetraphosphate Efficacy Against Ebola Virus Infection in Guinea Pig

The recent outbreaks of the Ebola virus (EBOV) in Africa have brought global visibility to the shortage of available therapeutic options to treat patients infected with this or closely related viruses. We have recently computationally identified three molecules which have all demonstrated statistically significant efficacy in the mouse model of infection with mouse adapted Ebola virus (ma-EBOV). One of these molecules is the antimalarial pyronaridine tetraphosphate (IC50 range of 0.82-1.30 {micro}M against three strains of EBOV and IC50 range of 1.01-2.72 {micro}M against two strains of Marburg virus (MARV)) which is an approved drug in the European Union and used in combination with artesunate. To date, no small molecule drugs have shown statistically significant efficacy in the guinea pig model of EBOV infection. Pharmacokinetics and range-finding studies in guinea pigs directed us to a single 300mg/kg or 600mg/kg oral dose of pyronaridine 1hr after infection. Pyronaridine resulted in statistically significant survival of 40% at 300mg/kg and protected from a lethal challenge with EBOV. In comparison, oral favipiravir (300 mg/kg dosed once a day) had 43.5 % survival. The in vitro metabolism and metabolite identification of pyronaridine and another of our EBOV active molecules, tilorone, which suggests significant species differences which may account for the efficacy or lack thereof, respectively in guinea pig. In summary, our studies with pyronaridine demonstrates its utility for repurposing as an antiviral against EBOV and MARV, providing justification for future testing in non-human primates. ImportanceThere is currently no antiviral small molecule drug approved for treating Ebola Virus infection. We have previously used machine learning models to identify new uses for approved drugs and demonstrated their activity against the Ebola virus in vitro and in vivo. We now describe the pharmacokinetic properties of the antimalarial pyronaridine in the guinea pig. In addition, we show that this drug is effective against multiple strains of EBOV and MARV in vitro and in the guinea pig model of Ebola virus infection. These combined efforts indicate the need to further test this molecule in larger animal efficacy studies prior to clinical use in humans. These findings also may be useful for repurposing this drug for use against other viruses in future.

pharmacology and toxicology↗