Search Results (10)

Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at >620 μM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and π-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS⁻ and 2-PyOH as competitive, and DCMQ/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a ΔT_m ≥ 1.5 °C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 μM, exhibiting two orders of magnitude higher potency than 2-MPO (400 μM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses. Full article

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5′ → 3′ direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6–4) pyrimidone (6–4PP) photolesion. The experimentally observed unwinding rate constants (k_obs) and processivities (P) were examined. Relative to undamaged DNA, the k_obs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2–5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest k_obs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions’ inhibitory effects than unwinding rate constants. Full article

The current investigation endeavors to identify differentially expressed alternatively spliced (DAS) genes that exhibit concordant expression with splicing factors (SFs) under diverse multifactorial abiotic stress combinations in Arabidopsis seedlings. SFs serve as the post-transcriptional mechanism governing the spatiotemporal dynamics of gene expression. The different stresses encompass variations in salt concentration, heat, intensive light, and their combinations. Clusters demonstrating consistent expression profiles were surveyed to pinpoint DAS/SF gene pairs exhibiting concordant expression. Through rigorous selection criteria, which incorporate alignment with documented gene functionalities and expression patterns observed in this study, four members of the serine/arginine-rich (SR) gene family were delineated as SFs concordantly expressed with six DAS genes. These regulated SF genes encompass cactin, SR1-like, SR30, and SC35-like. The identified concordantly expressed DAS genes encode diverse proteins such as the 26.5 kDa heat shock protein, chaperone protein DnaJ, potassium channel GORK, calcium-binding EF hand family protein, DEAD-box RNA helicase, and 1-aminocyclopropane-1-carboxylate synthase 6. Among the concordantly expressed DAS/SF gene pairs, SR30/DEAD-box RNA helicase, and SC35-like/1-aminocyclopropane-1-carboxylate synthase 6 emerge as promising candidates, necessitating further examinations to ascertain whether these SFs orchestrate splicing of the respective DAS genes. This study contributes to a deeper comprehension of the varied responses of the splicing machinery to abiotic stresses. Leveraging these DAS/SF associations shows promise for elucidating avenues for augmenting breeding programs aimed at fortifying cultivated plants against heat and intensive light stresses. Full article

Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5′–3′ polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication–transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC₅₀ = 1.25 μM) and B3 (IC₅₀ = 0.98 μM) as potential lead antiviral compounds for further targeted drug research. Full article

Helicases, motor proteins present in both prokaryotes and eukaryotes, play a direct role in various steps of RNA metabolism. Specifically, SF2 RNA helicases, a subset of the DEAD-box family, are essential players in plant developmental processes and responses to biotic and abiotic stresses. Despite this, information on this family in the physic nut (Jatropha curcas L.) remains limited, spanning from structural patterns to stress responses. We identified 79 genes encoding DEAD-box RNA helicases (JcDHX) in the J. curcas genome. These genes were further categorized into three subfamilies: DEAD (42 genes), DEAH (30 genes), and DExH/D (seven genes). Characterization of the encoded proteins revealed a remarkable diversity, with observed patterns in domains, motifs, and exon–intron structures suggesting that the DEAH and DExH/D subfamilies in J. curcas likely contribute to the overall versatility of the family. Three-dimensional modeling of the candidates showed characteristic hallmarks, highlighting the expected functional performance of these enzymes. The promoter regions of the JcDHX genes revealed potential cis-elements such as Dof-type, BBR-BPC, and AP2-ERF, indicating their potential involvement in the response to abiotic stresses. Analysis of RNA-Seq data from the roots of physic nut accessions exposed to 150 mM of NaCl for 3 h showed most of the JcDHX candidates repressed. The protein–protein interaction network indicated that JcDHX proteins occupy central positions, connecting events associated with RNA metabolism. Quantitative PCR analysis validated the expression of nine DEAD-box RNA helicase transcripts, showing significant associations with key components of the stress response, including RNA turnover, ribosome biogenesis, DNA repair, clathrin-mediated vesicular transport, phosphatidyl 3,5-inositol synthesis, and mitochondrial translation. Furthermore, the induced expression of one transcript (JcDHX44) was confirmed, suggesting that it is a potential candidate for future functional analyses to better understand its role in salinity stress tolerance. This study represents the first global report on the DEAD-box family of RNA helicases in physic nuts and displays structural characteristics compatible with their functions, likely serving as a critical component of the plant’s response pathways. Full article

Rapeseed (Brassica napus) is one of the most important vegetable oil crops worldwide. Abiotic stresses such as salinity are great challenges for its growth and productivity. DEAD-box RNA helicase 6 (RH6) is a subfamily member of superfamily 2 (SF2), which plays crucial roles in plant growth and development. However, no report is available on RH6 in regulating plant abiotic stress response. This study investigated the function and regulatory mechanism for BnRH6. BnRH6 was targeted to the nucleus and cytoplasmic processing body (P-body), constitutively expressed throughout the lifespan, and induced by salt stress. Transgenic overexpressing BnRH6 in Brassica and Arabidopsis displayed salt hypersensitivity, manifested by lagging seed germination (decreased to 55–85% of wild-type), growth stunt, leaf chlorosis, oxidative stress, and over-accumulation of Na ions with the K⁺/Na⁺ ratio being decreased by 18.3–28.6%. Given the undesirable quality of knockout Brassica plants, we utilized an Arabidopsis T-DNA insertion mutant rh6-1 to investigate downstream genes by transcriptomics. We constructed four libraries with three biological replicates to investigate global downstream genes by RNA sequencing. Genome-wide analysis of differentially expressed genes (DEGs) (2-fold, p < 0.05) showed that 41 genes were upregulated and 66 genes were downregulated in rh6-1 relative to wild-type under salt stress. Most of them are well-identified and involved in transcription factors, ABA-responsive genes, and detoxified components or antioxidants. Our research suggests that BnRH6 can regulate a group of salt-tolerance genes to negatively promote Brassica adaptation to salt stress. Full article

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements. Full article

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b–NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement. Full article

Helicases are enzymes that convert the chemical energy stored in ATP into mechanical work, allowing them to move along and manipulate nucleic acids. The helicase superfamily 1 (Sf1) is one of the largest subgroups of helicases and they are required for a range of cellular activities across all domains of life. Sf1 helicases can be further subdivided into two classes called the Sf1a and Sf1b helicases, which move in opposite directions on nucleic acids. The results of this movement can range from the separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. Here, we describe the characteristics of the Sf1a helicase Srs2 and the Sf1b helicase Pif1, both from the model organism Saccharomyces cerevisiae, focusing on the roles that they play in homologous recombination, a DNA repair pathway that is necessary for maintaining genome integrity. Full article

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions. Full article