The smallest open reading frame (ORF) encoded protein ORF3 of hepatitis E virus (HEV), recently, has been demonstrated to perform multiple functions besides accessory roles. ORF3 could act as a target for vaccine against HEV infections. The IDR (intrinsically disordered region); IDP (ID protein)/IDPR (ID protein region), plays critical role in various regulatory functions of viruses. The dark proteome of HEV-ORF3 protein including its structure and function was systematically examined by computer predictors to explicate its role in viral pathogenesis and drug resistance beyond its functions as accessory viral protein. Amino acid distribution showed ORF3 enrichment with disorder-promoting residues (Ala, Pro, Ser, Gly) while deficiency in order-promoting residues (Asn, Ile, Phe, Tyr and Trp). Initial investigation revealed ORF3 as IDP (entirely disordered protein) or IDPR (proteins consisting of IDRs with structured globular domains). Structural examination revealed preponderance of disordered regions interpreting ORF3 as moderately/highly disordered protein. Further disorder predictors categorized ORF3 as highly disordered protein/IDP. Identified sites and associated-crucial molecular functions revealed ORF3 involvement in diverse biological processes, substantiating them as targets of regulation. As ORF3 functions are yet to completely explored, thus, data on its disorderness could help in elucidating its disorder related functions.
BACKGROUND: Hepatitis E virus (HEV) is the cause of a liver disease hepatitis E. The translation product of HEV ORF2 has recently been demonstrated as a protein involved in multiple functions besides performing its major role of a viral capsid. As intrinsically disordered regions (IDRs) are linked to various essential roles in the virus's life cycle, we analyzed the disorder pattern distribution of the retrieved ORF2 protein sequences by employing different online predictors. Our findings might provide some clues on the disorder-based functions of ORF2 protein that possibly help us in understanding its behavior other than as a HEV capsid protein. RESULTS: The modeled three dimensional (3D) structures of ORF2 showed the predominance of random coils or unstructured regions in addition to major secondary structure components (alpha helix and beta strand). After initial scrutinization, the predictors VLXT and VSL2 predicted ORF2 as a highly disordered protein while the predictors VL3 and DISOPRED3 predicted ORF2 as a moderately disordered protein, thus categorizing HEV-ORF2 into IDP (intrinsically disordered protein) or IDPR (intrinsically disordered protein region) respectively. Thus, our initial predicted disorderness in ORF2 protein 3D structures was in excellent agreement with their predicted disorder distribution patterns (evaluated through different predictors). The abundance of MoRFs (disorder-based protein binding sites) in ORF2 was observed that signified their interaction with binding partners which might further assist in viral infection. As IDPs/IDPRs are targets of regulation, we carried out the phosphorylation analysis to reveal the presence of post-translationally modified sites. Prevalence of several disordered-based phosphorylation sites further signified the involvement of ORF2 in diverse and significant biological processes. Furthermore, ORF2 structure-associated functions revealed its involvement in several crucial functions and biological processes like binding and catalytic activities. CONCLUSIONS: The results predicted ORF2 as a protein with multiple functions besides its role as a capsid protein. Moreover, the occurrence of IDPR/IDP in ORF2 protein suggests that its disordered region might serve as novel drug targets via functioning as potential interacting domains. Our data collectively might provide significant implication in HEV vaccine search as disorderness in viral proteins is related to mechanisms involved in immune evasion.
Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus's pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.
Chronic kidney disease (CKD) is defined as a persistent abnormality in the structure and function of kidneys and leads to high morbidity and mortality in individuals across the world. Globally, approximately 8%-16% of the population is affected by CKD. Proper screening, staging, diagnosis, and the appropriate management of CKD by primary care clinicians are essential in preventing the adverse outcomes associated with CKD worldwide. In light of this, the identification of biomarkers for the appropriate management of CKD is urgently required. Growing evidence has suggested the role of mRNAs and microRNAs in CKD, however, the gene expression profile of CKD is presently uncertain. The present study aimed to identify diagnostic biomarkers and therapeutic targets for patients with CKD. The human microarray profile datasets, consisting of normal samples and treated samples were analyzed thoroughly to unveil the differentially expressed genes (DEGs). After selection, the interrelationship among DEGs was carried out to identify the overlapping DEGs, which were visualized using the Cytoscape program. Furthermore, the PPI network was constructed from the String database using the selected DEGs. Then, from the PPI network, significant modules and sub-networks were extracted by applying the different centralities methods (closeness, betweenness, stress, etc.) using MCODE, Cytohubba, and Centiserver. After sub-network analysis we identified six overlapped hub genes (RPS5, RPL37A, RPLP0, CXCL8, HLA-A, and ANXA1). Additionally, the enrichment analysis was undertaken on hub genes to determine their significant functions. Furthermore, these six genes were used to find their associated miRNAs and targeted drugs. Finally, two genes CXCL8 and HLA-A were common for Ribavirin drug (the gene-drug interaction), after docking studies HLA-A was selected for further investigation. To conclude our findings, we can say that the identified hub genes and their related miRNAs can serve as potential diagnostic biomarkers and therapeutic targets for CKD treatment strategies.
Lung cancer is the major cause of cancer-associated deaths across the world in both men and women. Lung cancer consists of two major clinicopathological categories, i.e., small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Lack of diagnosis of NSCLC at an early stage in addition to poor prognosis results in ineffective treatment, thus, biomarkers for appropriate diagnosis and exact prognosis of NSCLC need urgent attention. The proposed study aimed to reveal essential microRNAs (miRNAs) involved in the carcinogenesis of NSCLC that probably could act as potential biomarkers. The NSCLC-associated expression datasets revealed 12 differentially expressed miRNAs (DEMs). MiRNA-mRNA network identified key miRNAs and their associated genes, for which functional enrichment analysis was applied. Further, survival and validation analysis for key genes was performed and consequently transcription factors (TFs) were predicted. We obtained twelve miRNAs as common DEMs after assessment of all datasets. Further, four key miRNAs and nine key genes were extracted from significant modules based on the centrality approach. The key genes and miRNAs reported in our study might provide some information for potential biomarkers profitable to increased prognosis and diagnosis of lung cancer.
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , MicroRNAs , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Male , MicroRNAs/genetics , MicroRNAs/metabolism
Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein's function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/ß structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.
Background: Hepatitis E virus (HEV) is a member of the family Hepeviridae and causes acute HEV infections resulting in thousands of deaths worldwide. The zoonotic nature of HEV in addition to its tendency from human to human transmission has led scientists across the globe to work on its different aspects. HEV also accounts for about 30% mortality rates in case of pregnant women. The genome of HEV is organized into three open reading frames (ORFs): ORF1 ORF2 and ORF3. A reading frame encoded protein ORF4 has recently been discovered which is exclusive to GT 1 isolates of HEV. The ORF4 is suggested to play crucial role in pregnancy-associated pathology and enhanced replication. Though studies have documented the ORF4's importance, the genetic features of ORF4 protein genes in terms of compositional patterns have not been elucidated. As codon usage performs critical role in establishment of the host-pathogen relationship, therefore, the present study reports the codon usage analysis (based on nucleotide sequences of HEV ORF4 available in the public database) in three hosts along with the factors influencing the codon usage patterns of the protein genes of ORF4 of HEV. Results: The nucleotide composition analysis indicated that ORF4 protein genes showed overrepresentation of C nucleotide and while A nucleotide was the least-represented, with random distribution of G and T(U) nucleotides. The relative synonymous codon usage (RSCU) analysis revealed biasness toward C/G-ended codons (over U/A) in all three natural HEV-hosts (human, rat and ferret). It was observed that all the ORF4 genes were richly endowed with GC content. Further, our results showed the occurrence of both coincidence and antagonistic codon usage patterns among HEV-hosts. The findings further emphasized that both mutational and selection forces influenced the codon usage patterns of ORF4 protein genes. Conclusions: To the best of our knowledge, this is first bioinformatics study evaluating codon usage patterns in HEV ORF4 protein genes. The findings from this study are expected to increase our understanding toward significant factors involved in evolutionary changes of ORF4. Supplementary Information: The online version contains supplementary material available at 10.1186/s43088-022-00244-w.
BACKGROUND: Hepatitis E virus (HEV) is a positive-sense RNA virus belonging to the family Hepeviridae. The genome of HEV is organized into three open-reading frames (ORFs): ORF1, ORF2, and ORF3. The ORF1 non-structural Y-domain region (YDR) has been demonstrated to play an important role in the HEV pathogenesis. The nucleotide composition, synonymous codon usage bias in conjunction with other factors influencing the viral YDR genes of HEV have not been studied. Codon usage represents a significant mechanism in establishing the host-pathogen relationship. The present study for the first time elucidates the detailed codon usage patterns of YDR among HEV and HEV-hosts (Human, Rabbit, Mongoose, Pig, Wild boar, Camel, Monkey). RESULTS: The overall nucleotide composition revealed the abundance of C and U nucleotides in YDR genomes. The relative synonymous codon usage (RSCU) analysis indicated biasness towards C and U over A and G ended codons in HEV across all hosts. Codon frequency comparative analyses among HEV-hosts showed both similarities and discrepancies in usage of preferred codons encoding amino acids, which revealed that HEV codon preference neither completely differed nor completely showed similarity with its hosts. Thus, our results clearly indicated that the synonymous codon usage of HEV is a mixture of the two types of codon usage: coincidence and antagonism. Mutation pressure from virus and natural selection from host seems to be accountable for shaping the codon usage patterns in YDR. The study emphasised that the influence of compositional constraints, codon usage biasness, mutational alongside the selective forces were reflected in the occurrence of YDR codon usage patterns. CONCLUSIONS: Our study is the first in its kind to have reported the analysis of codon usage patterns on a total of seven different natural HEV hosts. Therefore, knowledge of preferred codons obtained from our study will not only augment our understanding towards molecular evolution but is also envisaged to provide insight into the efficient viral expression, viral adaptation, and host effects on the HEV YDR codon usage.
BACKGROUND: Hepatitis E is a liver disease caused by the pathogen hepatitis E virus (HEV). The largest polyprotein open reading frame 1 (ORF1) contains a nonstructural Y-domain region (YDR) whose activity in HEV adaptation remains uncharted. The specific role of disordered regions in several nonstructural proteins has been demonstrated to participate in the multiplication and multiple regulatory functions of the viruses. Thus, intrinsic disorder of YDR including its structural and functional annotation was comprehensively studied by exploiting computational methodologies to delineate its role in viral adaptation. RESULTS: Based on our findings, it was evident that YDR contains significantly higher levels of ordered regions with less prevalence of disordered residues. Sequence-based analysis of YDR revealed it as a "dual personality" (DP) protein due to the presence of both structured and unstructured (intrinsically disordered) regions. The evolution of YDR was shaped by pressures that lead towards predominance of both disordered and regularly folded amino acids (Ala, Arg, Gly, Ile, Leu, Phe, Pro, Ser, Tyr, Val). Additionally, the predominance of characteristic DP residues (Thr, Arg, Gly, and Pro) further showed the order as well as disorder characteristic possessed by YDR. The intrinsic disorder propensity analysis of YDR revealed it as a moderately disordered protein. All the YDR sequences consisted of molecular recognition features (MoRFs), i.e., intrinsic disorder-based protein-protein interaction (PPI) sites, in addition to several nucleotide-binding sites. Thus, the presence of molecular recognition (PPI, RNA binding, and DNA binding) signifies the YDR's interaction with specific partners, host membranes leading to further viral infection. The presence of various disordered-based phosphorylation sites further signifies the role of YDR in various biological processes. Furthermore, functional annotation of YDR revealed it as a multifunctional-associated protein, due to its susceptibility in binding to a wide range of ligands and involvement in various catalytic activities. CONCLUSIONS: As DP are targets for regulation, thus, YDR contributes to cellular signaling processes through PPIs. As YDR is incompletely understood, therefore, our data on disorder-based function could help in better understanding its associated functions. Collectively, our novel data from this comprehensive investigation is the first attempt to delineate YDR role in the regulation and pathogenesis of HEV.
Hepatitis E virus (HEV) is a major causative agent of hepatitis E infections across the globe. Although the essentiality of HEV nonstructural polyprotein (pORF1) putative Y-domain (Yd) has been established in viral pathogenesis, its structural-functional role remains elusive. The current research discusses the novel exploration on Yd protein expression, purification, biophysical characterization and structure-based docking analysis. The codon optimized synthetic gene and optimized expression parameters i.e., 5 h induction with 0.25 mM IPTG at 37 °C, resulted in efficient production of Yd protein (~40 kDa) in E. coli BL21(DE3) cells. Majority of the recombinant Yd (rYd) protein expressed as inclusion bodies was solubilized in 0.5% N-lauroylsarcosine and purified using Ni-NTA chromatography. Circular dichroism (CD) and UV visible absorption spectroscopic studies on Yd revealed both secondary and tertiary structure stability in alkaline range (pH 8.0-10.0), suggesting correlation with its physiological activity. Thus, loss in structure at low pH perhaps play crucial role in cytoplasmic-membrane interaction. The biophysical data were in good agreement with insilico structural analyses, which suggested mixed α/ß fold, non-random and basic nature of Yd protein. Furthermore, due to Yd protein essentiality in HEV replication and pathogenesis, it was considered as a template for docking and drug-likeness analyses. The 3D modeling of Yd protein and structure-based screening and drug-likeness of inhibitory compounds, including established antiviral drugs led to the identification of top nine promising candidates. Nonetheless, in vitro studies on the predicted interaction of Yd with intracellular-membrane towards establishing replication-complexes as well as validations of the proposed therapeutic agents are warranted.
Hepatitis E virus/chemistry , Recombinant Proteins/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Catalytic Domain , Escherichia coli/genetics , Hepatitis E virus/genetics , Humans , Inclusion Bodies/chemistry , Molecular Docking Simulation , Protein Conformation , Recombinant Proteins/genetics , Solubility , Viral Proteins/genetics
Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host-pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host-pathogen interaction.
Hepatitis E virus (HEV) is the main cause of acute hepatitis worldwide. HEV accounts for up to 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (G1) HEV. The contributing factors in adverse cases during pregnancy in women due to HEV infection is still debated. The mechanism underlying the pathogenesis of viral infection is attributed to different genomic component of HEV, i.e., open reading frames (ORFs): ORF1, ORF2, ORF3 and ORF4. Recently, ORF4 has been discovered in enhancing the replication of GI isolates of HEV through regulation of an IRES-like RNA element. However, its characterization through computational methodologies remains unexplored. In this novel study, we provide comprehensive overview of ORF4 protein's genetic and molecular characteristics through analyzing its sequence and different structural levels. A total of three different datasets (Human, Rat and Ferret) of ORF4 genomes were built and comparatively analyzed. Several non-synonymous mutations in conjunction with higher entropy values were observed in rat and ferret datasets, however, limited variation was observed in human ORF4 genomes. Higher transition to tranversion ratio was observed in the ORF4 genomes. Studies have reported the association of intrinsic disordered proteins (IDP) with drug discovery due to its role in several signaling and regulatory processes through protein-protein interactions (PPIs). As PPIs are potent drug target sources, thus the ORF4 protein was explored by analyzing its polypeptide structure in order to shed light on its intrinsic disorder. Pressures that lead towards preponderance of disordered-promoting amino acid residues shaped the evolution of ORF4. The intrinsic disorder propensity analysis revealed ORF4 protein (Human) as a highly disordered protein (IDP). Predominance of coils and lack of secondary structure further substantiated our findings suggesting its involvement in binding to ligand molecules. Thus, ORF4 contributes to cellular signaling processes through protein-protein interactions, as IDPs are targets for regulation to accelerate the process of drug designing strategies against HEV infections.
Chikungunya fever is an arboviral infection caused by the Chikungunya virus (CHIKV) and is transmitted by Aedes mosquito. The envelope protein (E2) of Chikungunya virus is involved in attachment of virion with the host cell. The present study was conceptualized to determine the structure of E2 protein of CHIKV and to identify the potential viral entry inhibitors. The secondary and tertiary structure of E2 protein was determined using bioinformatics tools. The mutational analysis of the E2 protein suggested that mutations may stabilize or de-stabilize the structure which may affect the structure-function relationship. In silico screening of various compounds from different databases identified two lead molecules i.e. phenothiazine and bafilomycin. Molecular docking and MD simulation studies of the E2 protein and compound complexes was carried out. This analysis revealed that bafilomycin has high docking score and thus high binding affinity with E2 protein suggesting stable protein-ligand interaction. Further, MD simulations suggested that both the compounds were stabilizing E2 protein. Thus, bafilomycin and phenothiazine may be considered as the lead compounds in terms of potential entry inhibitor for CHIKV. Further, these results should be confirmed by comprehensive cell culture, cytotoxic assays and animal experiments. Certain derivatives of phenothiazines can also be explored in future studies for entry inhibitors against CHIKV. The present investigation thus provides insight into protein structural dynamics of the envelope protein of CHIKV. In addition the study also provides information on the dynamics of interaction of E2 protein with entry inhibitors that will contribute towards structure based drug design.
