AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in vari...AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.展开更多
目前,临床上治疗乙型肝炎病毒(Hepatitis B virus,HBV)导致的慢性乙肝药物存在应答率低、副作用大和耐药性等缺点,急需寻找抗病毒治疗的新靶点。HBV反转录过程中,反转录酶(P蛋白)和位于病毒前基因组RNA(Pregenome RNA,pgRNA)5′邻近的RN...目前,临床上治疗乙型肝炎病毒(Hepatitis B virus,HBV)导致的慢性乙肝药物存在应答率低、副作用大和耐药性等缺点,急需寻找抗病毒治疗的新靶点。HBV反转录过程中,反转录酶(P蛋白)和位于病毒前基因组RNA(Pregenome RNA,pgRNA)5′邻近的RNA包装信号(ε)的相互作用(又称P-ε相互作用)非常关键,该相互作用有宿主蛋白如热激蛋白的参与。P-ε复合物形成后,一方面引发反转录的启动,另一方面启动核衣壳的包装。目前封闭P-ε相互作用的策略主要有热激蛋白抑制剂、ε适配子和阻断P蛋白的化合物三个方面,其机制为针对性靶向P蛋白或者宿主蛋白,从而直接或间接地阻断P-ε相互作用。前期,我们首次体外筛选到干扰P-ε相互作用的适配子,体外实验及动物实验均证实,该类适配子强烈抑制HBV复制。总之,P-ε相互作用为抗乙肝研究的临床治疗提供了一个极具吸引力和应用前景的药物靶点,上述三种策略绕过或克服了目前临床上HBV对化疗药的耐药问题,具有较高的应用前景。展开更多
基金Supported by the Instituto de Salud Carlos III,No.PI15/00856the European Regional Development Fund(ERDF),No.PI15/00856
文摘AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.
文摘目前,临床上治疗乙型肝炎病毒(Hepatitis B virus,HBV)导致的慢性乙肝药物存在应答率低、副作用大和耐药性等缺点,急需寻找抗病毒治疗的新靶点。HBV反转录过程中,反转录酶(P蛋白)和位于病毒前基因组RNA(Pregenome RNA,pgRNA)5′邻近的RNA包装信号(ε)的相互作用(又称P-ε相互作用)非常关键,该相互作用有宿主蛋白如热激蛋白的参与。P-ε复合物形成后,一方面引发反转录的启动,另一方面启动核衣壳的包装。目前封闭P-ε相互作用的策略主要有热激蛋白抑制剂、ε适配子和阻断P蛋白的化合物三个方面,其机制为针对性靶向P蛋白或者宿主蛋白,从而直接或间接地阻断P-ε相互作用。前期,我们首次体外筛选到干扰P-ε相互作用的适配子,体外实验及动物实验均证实,该类适配子强烈抑制HBV复制。总之,P-ε相互作用为抗乙肝研究的临床治疗提供了一个极具吸引力和应用前景的药物靶点,上述三种策略绕过或克服了目前临床上HBV对化疗药的耐药问题,具有较高的应用前景。
