RESUMO
BACKGROUND: The Alphapapillomavirus 9 (α-9 HPV) is a member of the Alphapapillomavirus genus and Papillomaviridae family. These viruses are almost all carcinogenic HPV, which is closely related to 75% of invasive cervical cancer worldwide, and has a high prevalence in Sichuan. The carcinogenic function is mainly realized by its E6 oncoprotein. METHODS: Cell samples were collected by cervical scraped for HPV detecting and typing. HPV-16, HPV-31, HPV-33, HPV-52, HPV-58 5 α-9 genus HPV subtype positive samples were selected, their E6 gene was sequenced and analyzed. The positive selection sites of HPV E6 genes were estimated by PAML 4.8 server. The secondary and tertiary structure of E6 protein were predicted by PSIPred and Swiss-model. The T-cell antigen epitopes of E6 protein were predicted by IEDB. RESULTS: α-9 HPV has a high prevalence in Sichuan, China. From 2012 to 2017, 18,067 cell cervical samples were collected, and 3135 were detected with α-9 HPV infection. Among which, 250 cases HPV-16 E6, 96 cases HPV-31 E6, 216 cases HPV-33 E6, 288 cases HPV-52 E6 and 405 cases HPV-58 E6 were successfully amplified, 17, 6, 6, 13, and 4 non-synonymous nucleotide mutations were respectively detected in HPV-16, 31, 33, 52, and 58 E6, 7 positive selection sites of α-9 HPV E6 were selected out (D32E of HPV-16 E6, K35N, K93N and R145I of HPV-33 E6, K93R of HPV-52 E6, K93N and R145K of HPV-58 E6). The structure and antigen epitopes of E6 protein with amino acid substitution differ from those of wild-type E6 protein, especially for the mutation located in the E6 positive selection site. CONCLUSIONS: HPV E6 nucleotide non-synonymous mutation in the positive selection site influence the protein structure and decrease the antigen epitopes affinity of the E6 protein overall, making it more difficult for the HPV-infected cells to be detected by the immune system, and enhancing the HPV adaptability to the environment. Mutations influence the validity of HPV clinical diagnostic probes, the polymorphism analysis of α-9 HPV E6 enrich the data of HR-risk HPV in Sichuan China, and the detection probes designed with the polymorphism data in mind can improve the efficiency of clinical detection; Mutations influence epitopes affinity, the association of E6 polymorphism and epitope affinity can improve the design of therapeutic vaccine with good immunity and high generality antigen epitope; The above study all provide a good theoretical basis for the prevention and treatment of HPV-related diseases.
Assuntos
Alphapapillomavirus , Proteínas Oncogênicas Virais , Proteínas Repressoras , Alphapapillomavirus/genética , China/epidemiologia , Epitopos de Linfócito T/genética , Feminino , Papillomavirus Humano 16 , Humanos , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus , Filogenia , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/química , Proteínas Repressoras/genética , Neoplasias do Colo do ÚteroRESUMO
BACKGROUND: Human papillomavirus type 39 associated with genital intraepithelial neoplasia and invasive cancers, has a high prevalence in Southwest China. HPV E6, E7 are two main papillomavirus oncoproteins, closely relate to the function of HPV immortalization, cell transformation, and carcinogenesis. L1 is the major capsid protein, can reflect the replication status of the virus in cells and the progression of cervical lesions. The purpose of this study is to reveal the prevalence of HPV 39 and the genetic polymorphisms of HPV39 based on E6, E7 and L1 gene in southwest China. METHODS: Cell samples were collected by cervical scraped for HPV detecting and typing, and HPV39 positive samples were selected out. Important E6, E7 and L1 genes of HPV39 were sequenced and analyzed for the study of HPV39 genetic polymorphisms. Phylogenetic trees were constructed by Maximum-likelihood and Kimura 2-parameters methods in Molecular Evolutionary Genetics Analysis version 6.0. The selection pressures of E6, E7 and L1 genes were estimated by Datamonkey web server. The secondary and three-dimensional structure of HPV39 E6, E7 proteins were created by sopma server and SWISS-MODEL software. RESULTS: 344 HPV39 positive samples were selected from 5718 HPV positive cell samples. Among HPV39 E6-E7 sequences, 20 single nucleotide mutations were detected, including 10 non-synonymous and 10 synonymous mutations; 26 single nucleotide mutations were detected in HPV39 L1 sequences, including 7 non-synonymous and 19 synonymous mutations respectively. 11 novel variants of HPV39 E6-E7 (5 in E6 and 6 in E7) and 14 novel variants of HPV39 L1 were identified in this study. A-branch was the most frequent HPV39 lineage in southwest China during our investigation. Selective pressure analysis showed that codon sites 26, 87, 151 in E6 and 75, 180, 222, 272, 284, 346, 356 in L1 were positively selected sites, as well as codon sites 45, 138, 309, 381 were negative selection sites in L1 gene, E7 has neither positive selection sites nor negative selection sites. A certain degree of secondary and three-dimensional structure dislocation was existed due to the non-synonymous mutations. CONCLUSIONS: Amino acid substitution affected the secondary and three-dimensional structure of HPV39, and resulting in the differences of carcinogenic potential and biological functions as well as the immune response due to the antigen epitopes difference, the antigen epitopes with stronger adaptability in Southwest will be screened out based on the above research results for the later vaccine development. And gene polymorphism of HPV39 in Southwest China may improve the effectiveness of clinical test and vaccine design, specifically for women in Southwest China.
Assuntos
Alphapapillomavirus , Genes Virais , Variação Genética , Infecções por Papillomavirus , Alphapapillomavirus/genética , China , Análise Mutacional de DNA , Epitopos , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Filogenia , Desenvolvimento de VacinasRESUMO
BACKGROUND: Variations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China. METHODS: LCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database. RESULTS: 50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387-7391 (TTATG) and 7698-7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287-7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY. CONCLUSION: This study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.
Assuntos
Alphapapillomavirus , Proteínas Oncogênicas Virais , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Filogenia , Alphapapillomavirus/genética , Teorema de Bayes , China , Epitopos de Linfócito B , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Fatores de Transcrição , Neoplasias do Colo do Útero/virologia , Desenvolvimento de VacinasRESUMO
BACKGROUND: Human papillomavirus (HPV) E6 and E7 oncoproteins play a crucial role in HPV-related diseases, such as cervical cancer, and can be used as ideal targets for therapeutic vaccines. Human leukocyte antigen (HLA) participates in the immune response to block HPV infection and invasion by its target/recognition function. HPV-33 and HPV-58 are highly prevalent among Chinese women. Therefore, it is of great significance to study the E6 and E7 region-specific gene polymorphisms of HPV-33 and HPV-58 in Southwest China and to identify ideal epitopes for vaccine design. Both HPV-33 and HPV-58 belong to α-9 genus HPV and are highly homologous, so their correlations are included in our research. METHODS: To study the E6 and E7 variations and polymorphisms of HPV-33 and HPV-58 in Southwest China, we collected samples, extracted and sequenced DNA, and identified variants. Nucleotide sequences were translated into amino acids by Mega 6.0 software. The physical/chemical properties, amino acid-conserved sequences and secondary structure of protein sequences were analysed by the Protparam server, ConSurf server and PSIPRED software. The T and B cell epitopes of the E6/E7 reference and variant sequences in HPV-33 and HPV-58 were predicted by the Immune Epitope Database (IEDB) analysis server and the ABCpred server, respectively. RESULTS: Five and seven optimal HLA-I restricted T cell epitopes were selected from HPV-33 and HPV-58 E6, respectively, and these optimal epitopes are mainly located in 41-58EVYDFAFADLTVVYREGN of HPV-33 E6 and 40-60SEVYDFVFADLRIVYRDGNPF of HPV-58 E6. Six optimal HLA-I-restricted T cell epitopes were selected from HPV-33 and HPV-58 E7, and these epitopes are mainly located in 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7. CONCLUSIONS: HPV-33/HPV-58 E6/E7 gene polymorphisms and T/B cell epitopes of their reference and variant sequences were studied, and candidate epitopes were selected by bioinformatics techniques for therapeutic vaccine design for people in Southwest China. This study was the first to investigate the correlation of epitopes between HPV-33 and HPV-58. After experimental validation, these selected epitopes will be employed to induce a wide range of immune responses in heterogeneous HLA populations.
Assuntos
Epitopos/imunologia , Variação Genética , Papillomaviridae/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Epitopos/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Infecções por Papillomavirus/epidemiologia , Filogenia , Neoplasias do Colo do Útero/virologiaRESUMO
CONTEXT: In the replication of SARS-CoV-2, the main protease (Mpro/3CLpro) is significant. It is conserved in a number of novel coronavirus variations, and no known human proteases share its cleavage sites. Therefore, 3CLpro is an ideal target. In the report, we screened five potential inhibitors (1543, 2308, 3717, 5606, and 9000) of SARS-CoV-2 Mpro through a workflow. The calculation of MM-GBSA binding free energy showed that three of the five potential inhibitors (1543, 2308, 5606) had similar inhibitor effects to X77 against Mpro of SARS-CoV-2. In conclusion, the manuscript lays the groundwork for the design of Mpro inhibitors. METHODS: In the virtual screening phase, we used structure-based virtual screening (Qvina2.1) and ligand-based virtual screening (AncPhore). In the molecular dynamic simulation part, we used the Amber14SB + GAFF force field to perform molecular dynamic simulation of the complex for 100 ns (Gromacs2021.5) and performed MM-GBSA binding free energy calculation according to the simulation trajectory.
Assuntos
Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Simulação de Dinâmica Molecular , SARS-CoV-2 , Humanos , Endopeptidases , Simulação de Acoplamento Molecular , Farmacóforo , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologiaRESUMO
INTRODUCTION: Cervical cancer is a common malignancy among woman, strong molecular epidemiological data show that high risk HPV infection is the main cause of cervical cancer. MATERIAL AND METHODS: Samples were collected from Sichuan women's and children's hospital based on the relevant guidelines and regulations, HPV DNA was extracted and evaluated by Human Papillomavirus Genotyping Kit for 21 types, according to the manufacturer's guidelines to analyze the epidemic age, mixed infection types, variation trend of HPV types in Sichuan from 2014 to 2021; Results: Out of 51174 samples11165 (21.82 %) HPV positive samples were detected, all belonging to alpha family, 53.32 % HPV positive samples and 61.51 % high-risk (HR) HPV positive samples are alpha-9 genus; The three commonest HR were HPV-52, HPV-16, HPV-58, and the low-risk (LR) HPV were HPV-81, HPV-6, HPV-11; Single infection was absolutely predominant and the age group with the highest HPV detection rate was 26-30 years old. During 2014-2021, HPV-16, HPV-6 and HPV-11 decline, while HPV-58 and HPV-52 increased; Conclusions: The most prevalent age group of HPV in this region was 26-30 years old. The detection rate of HPV-52 increased in the region, overtaking HPV-16 as the commonest type of HPV. α-9 genus HPV with strong pathogenicity is the commonest HR HPV. HPV prevalence systematic comparison in certain areas and continuous time can accurately and intuitively understand its distribution changes, achieve analysis of the epidemic trend, and provide guidance for the prevention, treatment and scientific research of HPV in Sichuan.
Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Criança , Feminino , Humanos , Adulto , Neoplasias do Colo do Útero/diagnóstico , Genótipo , Papillomaviridae/genética , Papillomavirus Humano 16 , Prevalência , China/epidemiologiaRESUMO
HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.
Assuntos
Alphapapillomavirus/genética , Mutação , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/epidemiologia , Alphapapillomavirus/química , Alphapapillomavirus/classificação , Alphapapillomavirus/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Linfócitos B/imunologia , Linfócitos B/virologia , Sítios de Ligação , Colo do Útero/imunologia , Colo do Útero/virologia , China/epidemiologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Genótipo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Modelos Moleculares , Tipagem Molecular , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Filogenia , Prevalência , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Artocarpus nanchuanensis (Moraceae), which is naturally distributed in China, is a representative and extremely endangered tree species. In this study, we obtained a high-quality chromosome-scale genome assembly and annotation information for A. nanchuanensis using integrated approaches, including Illumina, Nanopore sequencing platform, and Hi-C. A total of 128.71 Gb of raw Nanopore reads were generated from 20-kb libraries, and 123.38 Gb of clean reads were obtained after filtration with 160.34× coverage depth and a 17.48-kb average read length. The final assembled A. nanchuanensis genome was 769.44 Mb with a 2.09 Mb contig N50, and 99.62% (766.50 Mb) of the assembled data was assigned to 28 pseudochromosomes. In total, 39,596 genes (95.10%, 39,596/41,636) were successfully annotated, and 129 metabolic pathways were detected. Plants disease resistance/insect resistance genes, plant-pathogen interaction metabolic pathways, and abundant biosynthesis pathways of vitamins, flavonoid, and gingerol were detected. Unigene reveals the basis of species-specific functions, and gene family in contraction and expansion generally implies strong functional differences in the evolution. Compared with other related species, a total of 512 unigenes, 309 gene families in contraction, and 559 gene families in expansion were detected in A. nanchuanensis. This A. nanchuanensis genome information provides an important resource to expand our understanding of the unique biological processes, nutritional and medicinal benefits, and evolutionary relationship of this species. The study of gene function and metabolic pathway in A. nanchuanensis may reveal the theoretical basis of a special trait in A. nanchuanensis and promote the study and utilization of its rare medicinal value.
Assuntos
Artocarpus , Moraceae , Artocarpus/genética , Cromossomos , Frutas , Anotação de Sequência Molecular , Moraceae/genética , Filogenia , Árvores/genéticaRESUMO
Idiopathic asthenozoospermia, a common factor in male infertility, is characterized by altered sperm motility function in fresh ejaculate. Although the ß-defensin 126 (DEFB126) protein is associated with asthenozoospermia, DEFB126 gene polymorphisms have not been extensively studied. Therefore, the association between DEFB126 gene polymorphisms and asthenozoospermia requires further investigation. Screening was performed by semen analysis, karyotype analysis, and Y microdeletion detection, and 102 fertile men and 106 men with asthenozoospermia in Chengdu, China, were selected for DEFB126 gene sequence analyses. Seven nucleotide mutations and two nucleotide deletions in the DEFB126 gene were detected. rs11467417 (317-318 del/del), rs11467497 (163-166 wt/del), c.152T>C, and c.227A>G were significantly different between the control and asthenozoospermia groups, likely representing high-risk genetic factors for asthenozoospermia among males. DEFB126 expression was not observed in sperm with rs11467497 homozygous deletion and was unstable in sperm with rs11467417 homozygous deletion. The rs11467497 four-nucleotide deletion leads to truncation of DEFB126 at the carboxy-terminus, and the rs11467417 binucleotide deletion produces a non-stop messenger RNA (mRNA). The above deletions may be responsible for male hypofertility and infertility by reducing DEFB126 affinity to sperm surfaces. Based on in silico analysis, the amino acids 51M and 76K are located in the highly conserved domain; c.152T>C (M51T) and c.227A>G (K76R) are predicted to be damaging and capable of changing alternative splice, structural and posttranslational modification sites of the RNA, as well as the secondary structure, structural stability, and hydrophobicity of the protein, suggesting that these mutations are associated with asthenozoospermia.
Assuntos
Astenozoospermia , beta-Defensinas , Masculino , Humanos , Astenozoospermia/genética , Astenozoospermia/metabolismo , Motilidade dos Espermatozoides/genética , Homozigoto , Polimorfismo de Nucleotídeo Único , Sêmen , Deleção de Sequência/genética , Espermatozoides/metabolismo , Nucleotídeos/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
BACKGROUND: After the first epidemic wave from February through May 2013, the influenza A (H7N9) virus emerged and has followed a second epidemic wave since June 2013. As of June 27, 2014, the outbreak of H7N9 had caused 450 confirmed cases of human infection, with 165 deaths included. The case-fatality rate of all confirmed cases is about 36%, making the H7N9 virus a significant threat to people's health. At present, neuraminidase inhibitors are the only licensed antiviral medications available to treat H7N9 infections in humans. Oseltamivir is the most commonly used inhibitor, and it is also a front-line drug for the threatening H7N9. Unfortunately, it has been reported that patients treated with oseltamivir can induce R294K (Arg294Lys) substitution in the H7N9 virus, which is a rare mutation and can reduce the antiviral efficacy of inhibitors. Even worse, deaths caused by such mutation after oseltamivir treatment have already been reported, indicating that the need to find substitutive neuraminidase inhibitors for currently available drugs to treat drug-resistant H7N9 is really pressing. MATERIALS AND METHODS: First, the structure of H7N9 containing the R294K substitution was downloaded from the Protein Data Bank, and structural information of approved drugs was downloaded from the ZINC (ZINC Is Not Commercial) database. Taking oseltamivir carboxylate as a reference drug, we then filtered these molecules through virtual screening to find out potential inhibitors targeting the mutated H7N9 virus. For further evaluation, we carried out a 14 ns molecular dynamic simulation for each H7N9-drug complex and calculated the binding energy for each candidate drug. RESULTS: We found five inhibitors that could be candidate drugs for treating the mutated H7N9 virus. Docking poses showed these drugs could bind to the virus effectively, with the contribution of hydrogen bonds and hydrophobic interactions. With regard to the molecular dynamic simulations, receptor-ligand complexes formed by these candidate drugs were more stable than the one formed by oseltamivir carboxylate. The binding energy of oseltamivir carboxylate was -122.4 kJ/mol, while those for these potential inhibitors were -417.5, -404.7, -372.2, -304.3, and -289.9 kJ/mol, much better than the reference drug. CONCLUSION: Given the current and future threat of the mutated H7N9 virus, it is urgent that potent drugs and effective antiviral therapeutics be found. Our study therefore is able to complement currently available drugs for influenza A infectors and helps to prevent the ongoing threat of H7N9 virus.