Why Does the E1219V Mutation Expand T-Rich PAM Recognition in Cas9 from Streptococcus pyogenes?

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.

E-Protein Protonation Titration-Induced Single-Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV.

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strains of the dengue virus (DENV) show different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely, DENV-2 and DENV-4. We have performed E-protein protonation titration-induced single-particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on the virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of the DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. We also highlighted here the role of the microenvironment around the titrable residues (in the 3D-folded structure of the protein) in altering the pKa. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations.

The CRISPR/Cas9 system is a popular genome-editing tool with immense therapeutic potential. It is a simple two-component system (Cas9 protein and RNA) that recognizes the DNA sequence on the basis of RNA:DNA complementarity, and the Cas9 protein catalyzes the double-stranded break in the DNA. In the past decade, near-atomic resolution structures at various stages of the CRISPR/Cas9 DNA editing pathway have been reported along with numerous experimental and computational studies. Such studies have boosted knowledge of the genome-editing mechanism. Despite such advancements, the application of CRISPR/Cas9 in therapeutics is still limited, primarily due to off-target effects. Several studies aim at engineering high-fidelity Cas9 to minimize the off-target effects. Molecular Dynamics (MD) simulations have been an excellent complement to the experimental studies for investigating the mechanism of CRISPR/Cas9 editing in terms of structure, thermodynamics, and kinetics. MD-based studies have uncovered several important molecular aspects of Cas9, such as nucleotide binding, catalytic mechanism, and off-target effects. In this Review, the contribution of MD simulation to understand the CRISPR/Cas9 mechanism has been discussed, preceded by an overview of the history, mechanism, and structural aspects of the CRISPR/Cas9 system. These studies are important for the rational design of highly specific Cas9 and will also be extremely promising for achieving more accurate genome editing in the future.

A comprehensive analysis of NAC gene family in Oryza sativa japonica: a structural and functional genomics approach.

NAC gene family regulates diverse aspects of plant growth and developmental processes. The NAC DNA binding domains together with cis-acting elements play inter-related roles in regulating gene expression. In this study, an in silico approach for genome wide analysis of NAC gene in Oryza sativa japonica lead to an identification of 11 NAC genes, distributed over 12 chromosomes. A detailed analysis of phylogenetic relationship, motifs, gene structure, duplication patterns, positive-selection pressure and cis-elements of 11 OsNAC genes were performed. Three pairs of NAC genes with a high degree of homology in terminal nodes were observed and were inferred to be paralogous pairs. One conserved NAC domain was analyzed in all the NAC proteins. Only one gene was studied to be intronless and the majority had 2 introns. Segmental gene duplication pattern was predominant in 11 NAC genes. Ka/Ks ratio of 3 pairs of segmentally duplicated gene was substantially lower than 1, suggesting that the OsNAC sequences are under strong purifying selection pressure. NAC74 and NAC71 gene showed the maximum responsiveness for several factors. The paralogous genes, NAC2 and NAC67 were found to have maximum mya values, respectively. They showed maximum difference amongst themselves in all the categories of responsiveness. Responsiveness towards abscisic acid was observed to be absent in NAC67, but present in NAC2, while responsiveness to meristem inducibility was observed to remain absent in NAC2 but present in NAC67. These results would provide a platform for the future identification and analysis of NAC genes in Oryza sativa japonica.Communicated by Ramaswamy H. Sarma.

Identification of a neutrophil-specific PIK3R1 mutation facilitates targeted treatment in a patient with Sweet syndrome.

BackgroundAcute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. The disease pathophysiology remains elusive, and current dogma suggests that Sweet syndrome is a process of reactivity to an unknown antigen. Corticosteroids and steroid-sparing agents remain frontline therapies, but refractory cases pose a clinical challenge.MethodsA 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole-genome sequencing, transcriptomic profiling, and cellular assays of the patient's skin and neutrophils were performed.ResultsWe identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration toward IL-1ß and neutrophil respiratory burst. Targeted treatment of the patient with an IL-1 receptor 1 antagonist resulted in a dramatic therapeutic response and enabled a tapering off of corticosteroids.ConclusionDysregulated PI3K/AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests that this syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogeneous disease that resulted in a rational and successful clinical intervention. Future patients will benefit from efforts to identify potential mutations. The ability to directly interrogate the diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for patients with clinically challenging disease.Funding SourcesBerstein Foundation, NIH, Veterans Affairs (VA) Administration, Moseley Foundation, and H.T. Leung Foundation.

Clinical aspects and presumed etiology of multisystem inflammatory syndrome in children (MIS-C): A review.

The COVID-19 outbreak sparked by SARS-CoV-2, begat significant rates of malady worldwide, where children with an abnormal post-COVID ailment called the Multisystem Inflammatory Syndrome (MIS-C), were reported by April 2020. Here we have reviewed the clinical characteristics of the pediatric patients and the prognosis currently being utilized. A vivid comparison of MIS-C with other clinical conditions has been done. We have addressed the probable etiology and fundamental machinery of the inflammatory reactions, which drive organ failure. The involvement of androgen receptors portrays the likelihood of asymptomatic illness in children below adolescence, contributing to the concept of antibody-dependent enhancement.

FIBCD1 Deficiency Decreases Disease Severity in a Murine Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold associated with the development of pulmonary diseases that include invasive pulmonary aspergillosis (IPA), an often fatal opportunistic infection. FIBCD1 is a transmembrane endocytic membrane receptor widely expressed on human epithelium. Although FIBCD1 was previously shown to bind chitin, modulate fungal colonization of the gut, and inhibit intestinal inflammation, the role of FIBCD1 in the context of lung fungal infection remains unknown. In this study, we observed that mortality, fungal burden, and tissue histopathology were decreased in the absence of FIBCD1 in murine IPA. Quantitative RT-PCR analyses demonstrated decreased inflammatory cytokines in the lungs of neutrophil-depleted FIBCD1-/- mice with IPA, when compared with wild-type controls. In contrast, inflammatory cytokines were increased in immune-competent FIBCD1-/- mice after fungal aspiration, suggesting that the presence of neutrophils is associated with cytokine modulation. In contrast to the clear IPA phenotype, FIBCD1-/- mice with systemic infection or bleomycin-induced lung injury exhibited similar morbidity and mortality when compared with their wild-type counterparts. Thus, our study identifies a detrimental role of FIBCD1 in IPA.

In silico screening and exploration into phenotypic alterations of deleterious oncogenic single nucleotide polymorphisms in HSPB1 gene.

A small heat shock protein, HSP27, encoded by HSPB1 gene strongly favors survival, proliferation and metastasis of cancer cells and its expression is dependent on post-translational modifications like phosphorylation. This study performed an extensive in silico screening of 20 deleterious non-synonymous SNPs in the coding region of HSPB1 gene, among which four were identified to be cancer associated. The SNP variant I181S introduced a new phosphorylation site in position 181, which might elevate the protein's activation potential. Emergence of other post-translational modifications was also observed in SNP variants: L144P and E130K.Significant conformational changes were observed in I181S, L144P and E130K SNP variants with respect to wild-type HSP27. These SNPs appear in one among 105 individuals, making them more susceptible towards cancer. This study would therefore, instigate development of novel biomarkers for cancer risk detection and would provide a detailed understanding towards varied cancer susceptibility of human population.

Development of new vaccine target against SARS-CoV2 using envelope (E) protein: An evolutionary, molecular modeling and docking based study.

COVID-19 is one of the fatal pandemic throughout the world. For cellular fusion, its antigenic peptides are presented by major histocompatibility complex (MHC) in humans. Therefore, exploration into residual interaction details of CoV2 with MHCs shall be a promising point for instigating the vaccine development. Envelope (E) protein, the smallest outer surface protein from SARS-CoV2 genome was found to possess the highest antigenicity and is therefore used to identify B-cell and T-cell epitopes. Four novel mutations (T55S, V56F, E69R and G70del) were observed in E-protein of SARS-CoV2 after evolutionary analysis. It showed a coil➔helix transition in the protein conformation. Antigenic variability of the epitopes was also checked to explore the novel mutations in the epitope region. It was found that the interactions were more when SARS-CoV2 E-protein interacted with MHC-I than with MHC-II through several ionic and H-bonds. Tyr42 and Tyr57 played a predominant role upon interaction with MHC-I. The higher ΔG values with lesser dissociation constant values also affirm the stronger and spontaneous interaction by SARS-CoV2 proteins with MHCs. On comparison with the consensus E-protein, SARS-CoV2 E-protein showed stronger interaction with the MHCs with lesser solvent accessibility. E-protein can therefore be targeted as a potential vaccine target against SARS-CoV2.

Volume and Location of the Defect as Predictors of Swallowing Outcome After Glossectomy: Correlation with a Classification.

This study aimed to characterize the swallowing outcomes after glossectomy and analyze factors affecting them. An attempt is made to propose a classification system and corroborate it to the results. This is a cross-sectional study to assess swallowing in carcinoma tongue patients treated surgically with or without reconstruction, followed by adjuvant therapy as indicated. One hundred and six patients were evaluated with videofluoroscopy (VFS). Volume defects were classified as I: less than one-third, II: one-third to half, III: half to two-thirds, IV: two-thirds to total glossectomy. Location was assigned as lateral, tip, and sulcus defects. Predictors were T stage, surgical approach, volume, location, and adjuvant radiotherapy. Chi-square and logistic regression were used for statistical analysis. Defects were Class I, II, III, and IV in 36, 42, 16, and 12 patients, respectively. Adjuvant radiotherapy was given in 40% of cases. Mean evaluation time was 14 months from treatment. On, Functional Oral Intake Scale (FOIS) score, as the Class of the defect increased, the percentage of patients with low scores (poor swallowing outcomes) showed an increasing trend (p < 0.001). Defect volume, T stage, approach, and radiotherapy correlated significantly with an abnormality of all VFS parameters (p < 0.001). On multivariate analysis, defect volume remained an independent predictor for oral parameters; radiotherapy emerged as the only independent predictor for pharyngeal parameters. The incremental volume of the defect is a significant independent predictor of swallowing. Based on this, we propose a classification for glossectomy.

Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses.

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-ß and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis.

Exploring Single Nucleotide Polymorphisms in ITGAV for Gastric, Pancreatic and Liver Malignancies: An Approach Towards the Discovery of Biomarker.

BACKGROUND: Integrin αV, encoded by ITGAV gene, is one of the most studied protein subunits, closely associated with liver, pancreatic and stomach cancer progression and metastasis via regulation of angiogenesis. The occurrence of Single Nucleotide Polymorphisms (SNPs) in cancer- associated proteins is a key determinant for varied susceptibility of an individual towards cancer. METHODOLOGY: The study investigated the deleterious effects of these cancer-associated SNPs on the protein's structure, stability and cancer causing potential using an in silico approach. Numerous computational tools were employed that identified the most deleterious cancer-associated SNPs and those to get actively involved in post-translational modifications. The impact of these SNPs on the protein structure, function and stability was also examined. Conclusion and Future Scope: A total 63 non-synonymous SNPs in ITGAV gene were observed to be associated in these three gastrointestinal cancers and among this, 63, 19 were the most deleterious ones. The structural and functional importance of residues altered by most damaging SNPs was analyzed through evolutionary conservation and solvent accessibility. The study also elucidated three-dimensional structures of the 19 most damaging mutants. The analysis of conformational variation identified 5 SNPs (D379Y, G188E, G513V, L950P, and R540L) in integrin αV, which influence the protein's structure. Three calcium binding sites were predicted at residues: D379, G384 and G408 and a peptide binding site at residue: R369 in integrin αV. Therefore, SNPs D379Y, G384C, G408R and R369W have the potential to alter the binding properties of the protein. Screening and characterization of deleterious SNPs could advance novel biomarker discovery and therapeutic development in the future.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti.

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as ß-actin, ef-1ß, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Structural impact due to PPQEE deletion in multiple cancer associated protein - Integrin αV: An In silico exploration.

A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin ß3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount ß-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.

Identification of novel therapeutic target and epitopes through proteome mining from essential hypothetical proteins in Salmonella strains: An In silico approach towards antivirulence therapy and vaccine development.

Salmonella strains are responsible for a huge mortality rate through foodborne ailment in the world that necessitated the discovery of novel drugs and vaccines. Essential hypothetical proteins (EHPs), whose structures and functions were previously unknown, could serve as potential therapeutic and vaccine targets. Antivirulence therapy shall emerge as a superior therapeutic approach that uses virulence factors as drug targets. This study annotated the biological functions of 96 out of total 106 essential hypothetical proteins in five strains of Salmonella and classified into nine important protein categories. 34 virulence factors were predicted among the EHPs, out of which, 11 were identified to be pathogen specific potential drug targets for antivirulence therapy. These targets were non-homologous to both human and gut microbiota proteome to avoid cross-reactivity with them. Seven identified targets had druggable property, while the rest four targets were novel targets. Four identified targets (DEG10320148, DEG10110027, DEG10110040 and DEG10110142) had antigenic properties and were further classified as: two membrane-bound Lipid-binding transmembrane proteins, a Zinc-binding membrane protein and an extracellular glycosylase. These targets could be potentially used for the development of subunit vaccines. The study further identified 11 highly conserved and exposed epitope sequences from these 4 vaccine targets. The three-dimensional structures of the vaccine targets were also elucidated along with highlighting the conformation of the epitopes. This study identified potential therapeutic targets for antivirulence therapy against Salmonella. It would therefore instigate in novel drug designing as well as provide important leads to new Salmonella vaccine development.

In Silico Drug Target Discovery Through Proteome Mining from M. tuberculosis: An Insight into Antivirulent Therapy.

AIM AND OBJECTIVE: One of the challenges to conventional therapies against Mycobacterium tuberculosis is the development of multi-drug resistant pathogenic strains. This study was undertaken to explore new therapeutic targets for the revolutionary antivirulence therapy utilizing the pathogen's essential hypothetical proteins, serving as virulence factors, which is the essential first step in novel drug designing. METHODS: Functional annotations of essential hypothetical proteins from Mycobacterium tuberculosis (H37Rv strain) were performed through domain annotation, Gene Ontology analysis, physicochemical characterization and prediction of subcellular localization. Virulence factors among the essential hypothetical proteins were predicted, among which pathogen-specific drug target candidates, non-homologous to human and gut microbiota, were identified. This was followed by druggability and spectrum analysis of the identified targets. RESULTS AND CONCLUSION: The study successfully assigned functions of 83 essential hypothetical proteins of Mycobacterium tuberculosis, among which 25 were identified as virulence factors. Out of 25, 12 virulence factors were observed as potential pathogen-specific drug target candidates. Nine potential targets had druggable properties and rest three were considered as novel targets. Exploration of these targets will provide new insights into future drug development. Characterization of subcellular localizations revealed that most of the predicted targets were cytoplasmic which could be ideal for intracellular drugs, while two drug targets were membranebound, ideal for vaccines. Spectrum analysis identified one broad-spectrum and 11 narrowspectrum targets. This study would, therefore, instigate designing novel therapeutics for antivirulence therapy, which have the potential to serve as revolutionary treatment instead of conventional antibiotic therapies to overcome the lethality of antibiotic-resistant strains.

Detailed Molecular Biochemistry for Novel Therapeutic Design Against Nipah and Hendra Virus: A Systematic Review.

BACKGROUND: Nipah virus (NiV) and Hendra virus (HeV) of genus Henipavirus are the deadliest zoonotic viruses, which cause severe respiratory ailments and fatal encephalitis in humans and other susceptible animals. The fatality rate for these infections had been alarmingly high with no approved treatment available to date. Viral attachment and fusion with host cell membrane is essential for viral entry and is the most essential event of viral infection. Viral attachment is mediated by interaction of Henipavirus attachment glycoprotein (G) with the host cell receptor: Ephrin B2/B3, while viral fusion and endocytosis are mediated by the combined action of both viral glycoprotein (G) and fusion protein (F). CONCLUSION: This review highlights the mechanism of viral attachment, fusion and also explains the basic mechanism and pathobiology of this infection in humans. The drugs and therapeutics used either experimentally or clinically against NiV and HeV infection have been documented and classified in detail. Some amino acid residues essential for the functionality of G and F proteins were also emphasized. Therapeutic designing to target and block these residues can serve as a promising approach in future drug development against NiV and HeV.