Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion.

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Whole genome characterization and diagnostics of prunus necrotic ringspot virus (PNRSV) infecting apricot in India.

Prunus necrotic ringspot virus (PNRSV) is a pathogen that infects Prunus species worldwide, causing major economic losses. Using one and two-step RT-PCR and multiplex RT-PCR, the whole genome of the PNRSV-infecting apricot was obtained and described in this study. Computational approaches were used to investigate the participation of several regulatory motifs and domains of the Replicase1, Replicase2, MP, and CP. A single degenerated reverse and three forward oligo primers were used to amplify PNRSV's tripartite genome. The size of RNA1 was 3.332 kb, RNA2 was 2.591 kb, and RNA3 was 1.952 kb, according to the sequencing analysis. The Sequence Demarcation Tool analysis determined a percentage pair-wise identity ranging between 91 and 99% for RNA1 and 2, and 87-98% for RNA3. Interestingly, the phylogenetic analysis revealed that closely related RNA1, RNA2, and RNA3 sequences of PNRSV strains from various geographical regions of the world are classified into distinct clades or groups. This is the first report on the characterization of the whole genome of PNRSV from India, which provides the cornerstone for further studies on the molecular evolution of this virus. This study will assist in molecular diagnostics and management of the diseases caused by PNRSV.

In silico analysis reveals hypoxia-induced miR-210-3p specifically targets SARS-CoV-2 RNA.

Human coronaviruses (HCoVs) until the emergence of SARS in 2003 were associated with mild cold and upper respiratory tract infections. The ongoing pandemic caused by SARS-CoV-2 has enhanced the potential for infection and transmission as compared to other known members of this family. MicroRNAs (miRNA) are 21-25 nucleotides long non-coding RNA that bind to 3' UTR of genes and regulate almost every aspect of cellular function. Several human miRNAs have been known to target viral genomes, mostly to downregulate their expression and sometimes to upregulate also. In some cases, host miRNAs could be sequestered by the viral genome to create a condition for favourable virus existence. The ongoing SARS CoV-2 pandemic is unique based on its transmissibility and severity and we hypothesised that there could be a unique mechanism for its pathogenesis. In this study, we exploited in silico approach to identify human respiratory system-specific miRNAs targeting the viral genome of three highly pathogenic HCoVs (SARS-CoV-2 Wuhan strain, SARS-CoV, and MERS-CoV) and three low pathogenic HCoVs (OC43, NL63, and HKU1). We identified ten common microRNAs that target all HCoVs studied here. In addition, we identified unique miRNAs which targeted specifically one particular HCoV. miR-210-3p was the single unique lung-specific miRNA, which was found to target the NSP3, NSP4, and NSP13 genes of SARS-CoV-2. Further miR-210-NSP3, miR-210-NSP4, and miR-210-NSP13 SARS-CoV-2 duplexes were docked with the hAGO2 protein (PDB ID 4F3T) which showed Z-score values of -1.9, -1.7, and -1.6, respectively. The role of miR-210-3p as master hypoxia regulator and inflammation regulation may be important for SARS-CoV-2 pathogenesis. Overall, this analysis advocates that miR-210-3p be investigated experimentally in SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Therapeutic Potential of Medicinal Plants against Dengue Infection: A Mechanistic Viewpoint.

Dengue is a tropical disease caused by the Dengue virus (DENV), a positive-sense, single stranded RNA virus of the family Flaviviridae, which is transmitted by Aedes mosquitoes. The occurrence of dengue has grown dramatically around the globe in recent decades, and it is rapidly becoming a global burden. Furthermore, all four DENV serotypes cocirculate and create a problematic hyperendemic situation. Characteristic symptoms range from being asymptomatic, dengue fever to life-threatening complications such as hemorrhagic fever and shock. Apart from the inherent virulence of the virus strain, a dysregulated host immune response makes the condition worse. Currently, there is no highly recommended vaccine or therapeutic agent against dengue. With the advent of virus strains resistant to antiviral agents, there is a constant need for new therapies to be developed. Since time immemorial, human civilization has utilized plants in traditional medicine to treat various diseases, including infectious viral diseases. With the advancement in molecular biology, cell biology techniques, and bioinformatics, recent studies have tried to provide scientific evidence and determine the mechanism of anti-dengue activity of various plant extracts and plant-derived agents. The current Review consolidates the studies on the last 20 years of in vitro and in vivo experiments on the ethnomedicinal plants used against the dengue virus. Several active phytoconstituents like quercetin, castanospermine, α-mangostin, schisandrin-A, hirsutin have been found to be promising to inhibition of all the four DENV serotypes. However, novel therapeutics need to be reassessed in relevant cells using high-throughput techniques. Further, in vivo dose optimization for the immunomodulatory and antiviral activity should be examined on a vast sample size. Such a Review should help take the knowledge forward, validate it, and use medicinal plants in different combinations targeting multiple stages of virus infection for more effective multipronged therapy against dengue infection.

Decrypting the interaction pattern of Piperlongumine with calf thymus DNA and dodecamer d(CGCGAATTCGCG)2 B-DNA: Biophysical and molecular docking analysis.

The mechanisms of interaction of DNA with pharmacological molecules are critical to understanding their therapeutic actions on physiological systems. Piperlongumine is widely studied for its anticancer potential. Multi-spectrometry, calorimetry and in silico studies were employed to study the interaction of piperlongumine and calf thymus DNA. UV-Vis spectroscopy illustrated a hyperchromic pattern in spectra of the calf thymus DNA-piperlongumine complex, while fluorescent quenching was observed in emission spectral studies. Competitive displacement assay demonstrated higher displacement and binding constant for DNA-rhodamine B complex by piperlongumine than DNA-methylene blue complex. Differential scanning calorimetry presented non-significant changes in melting temperature and molecular docking presented the precise interaction site of piperlongumine with calf thymus DNA at minor groove. Further, piperlongumine treatment did not result in pBluescript KS plasmid DNA cleavage as revealed from the DNA topology assay. All these experiments confirmed the binding of piperlongumine with DNA through minor groove binding mode.

Multi-Targeted Molecular Docking, Pharmacokinetics, and Drug-Likeness Evaluation of Okra-Derived Ligand Abscisic Acid Targeting Signaling Proteins Involved in the Development of Diabetes.

Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.

A bioinformatics approach to investigate serum and hematopoietic cell-specific therapeutic microRNAs targeting the 3' UTRs of all four Dengue virus serotypes.

Hyperendemic circulation of all four Dengue virus (DENV) serotypes is a severe global public health problem, so any vaccine or therapeutics should be able to target all four of them. Cells of hemopoietic origin are believed to be primary sites of DENV replication. This study aimed to identify potential host miRNAs that target 3' UTR of all four DENV serotypes, thereby directly regulating viral gene expression or indirectly modulating the host system at different virus infection steps. We used four prediction algorithms viz. miRanda, RNA22, RNAhybrid and StarMir for predicting miRNA, targeting 3'UTR of all four DENV serotypes. Statistically, the most significant miRNA targets were screened based on their Log10 P-value (> 0.0001) of Gene Ontology (GO) term and Kyoto Encyclopaedia of Gene and Genome (KEGG) pathway enrichment analysis. The intersection test of at least three prediction tools identified a total of 30 miRNAs, which could bind to 3'UTR of all four DENV serotypes. Of the 30, eight miRNAs were of hematopoietic cell origin. GO term enrichment and KEGG analysis showed four hemopoietic origin miRNAs target genes of the biological processes mainly involved in the innate immune response, mRNA 3'-end processing, antigen processing and presentation and nuclear-transcribed mRNA catabolic process.

Differential binding of SARS-CoV-2 Spike protein variants to its cognate receptor hACE2 using molecular modeling based binding analysis.

The current emergence of novel coronavirus, SARS-CoV-2 and its ceaseless expansion worldwide has posed a global health emergency that has adversely affected the humans. With the entire world striving to understand the newly emerged virus, differences in morbidity and infection rate of SARS-CoV-2 have been observed across varied geographic areas, which have been ascribed to viral mutation and evolution over time. The homotrimeric Spike (S) glycoprotein on the viral envelope surface is responsible for binding, priming, and initiating infection in the host. Our phylogeny analysis of 1947 sequences of S proteins indicated there is a change in amino acid (aa) from aspartate (Group-A) to glycine (Group-B) at position 614, near the receptor- binding domain (RBD; aa positions 331-524). The two variants are reported to be in circulation, disproportionately across the world, with Group-A dominant in Asia and Group-B in North America. The trimeric, monomeric, and RBD of S protein of both the variant groups (A & B) were modeled using the Swiss-Model server and were docked with the human receptor angiotensin-converting enzyme 2 (hACE2) employing the PatchDock server and visualized in PyMol. Group-A S protein's RBD bound imperceptibly to the two binding clefts of the hACE2 protein, on the other hand, Group-B S protein's RBD perfectly interacted inside the binding clefts of hACE2, with higher number of hydrogen and hydrophobic interactions. This implies that the S protein's amino acid at position 614 near the core RBD influences its interaction with the cognate hACE2 receptor, which may induce its infectivity that should be explored further with molecular and biochemical studies.