Papillomavirus Infectious Entry

乳头瘤病毒传染性进入

• 批准号: 7965815
• 负责人: Christopher Buck
• 金额: $ 10.7万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965815
• 关键词: Affinity Chromatography; Antibodies; Antigens; Anus; Attention; Benign; Binding; Biological; Cancer Etiology; Cell Culture Techniques; Cells; Cellular biology; Cervical; Cervical Cancer Screening; Chemicals; Client; Crosslinker; Culture Media; Cyclophilins; Cyclosporine; DNA Viruses; Developed Countries; Developing Countries; Development; Epithelial; Family; Gene Transfer; Genital system; Goals; Human Papillomavirus; Immunity; In Vitro; Infection; Licensing; Life Cycle Stages; Malignant neoplasm of cervix uteri; Mechanics; Methods; Molecular; Molecular Biology; Molecular Chaperones; Mucous Membrane; Nature; Neoplasms; Oncogenic Viruses; Papillomavirus; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Pliability; Process; Production; Proteins; Proteomics; Public Health; Risk; Serum; Skin; Skin Cancer; Staging; Structure; Symptoms; Technology; Throat Cancer; Tissues; Tropism; Vaccines; Viral; Viral Genome; Virion; Virus; Woman; Work; base; cyclophilin B; disulfide bond; flexibility; gene transfer vector; genetic vaccine; improved; in vivo; killings; member; pressure; protein crosslink; tool; transmission process; tumorigenic; vector

Human papillomaviruses (HPVs) are a group of non-enveloped DNA viruses that have evolved to exploit a specialized biological niche in epithelial tissues such as the skin or the genital mucosa. Although most HPV types are associated with clinically inapparent or benign symptoms, such as common skin warts, about a dozen sexually-transmitted HPV types cause nearly all cases of cervical cancer, as well as a substantial fraction of anal, genital and throat cancers. HPV-induced cervical cancer kills nearly 300,000 women per year worldwide, mostly in developing countries where access to cervical cancer screening is limited. A recently-licensed vaccine targeting two cancer-causing HPV types has helped focus public attention on the substantial risk these viruses pose to public health. Work in the Tumor Virus Molecular Biology Section is focused on the HPV virion. The virions of non-enveloped viruses are dynamic structures that must undergo a range of conformational changes during different stages of the virus life cycle. Virions must be flexible enough to allow encapsidation of the viral genome, yet stable enough to withstand environmental insults encountered during transmission between hosts. Virions must also become pliable enough to release the viral genome upon infection of a host cell. Although each of these steps in the virus life cycle requires discrete structural motifs that are typically well-conserved among members of a virus family, immunological pressure tends to select for virions in which conserved functional motifs are occluded from immunological recognition. Using HPV vector technologies and a range of proteomics tools, we seek to characterize conserved functional motifs of the virion, as well as their cellular binding targets. Work in the lab is aimed at elucidating the mechanics of virion assembly and infectious entry, as well as the basic cell biology that underpins these phases of the viral life cycle. However, our work also has translational goals. High-titer HPV-based gene transfer vectors (also known as HPV pseudoviruses) are beginning to show promise as genetic vaccine vehicles. Improved understanding of the assembly, infectivity and in vivo tropism of HPV vectors should improve their practicality for in vivo gene transfer applications. Antibodies targeting conserved virion structures have the potential to confer sterilizing immunity against a wide variety of HPV types. Thus, another translational goal of our work is to facilitate the ongoing development of improved vaccines that might confer protection against a broader range of tumorigenic HPV types than the currently available vaccine. During FY09 we completed our goal of using tandem affinity purification (TAP) strategy for identifying cellular proteins that the HPV virion contacts during the infectious entry process. In brief, the TAP strategy involved purification of cell-bound HPV virions baited with chemical crosslinkers. We utilized mass spectrometric analysis to identify cellular proteins crosslinked to the virion. The approach revealed two classes of host protein that facilitate the infectious entry of HPVs into host cells. Once candidate factor is a cellular protease. In FY10 we will work toward understanding what protein clients the protease acts on to facilitate HPV infectious entry. Our TAP analysis also revealed that a chaperone protein that is a member of the cyclophilin family is required for HPV infectious entry. Cyclophilin-inhibitory drugs, such as cyclosporin A, were found to inhibit HPV infectivity in vitro. Although the initial candidate protein identified in our TAP screen was cyclophilin B, further analysis suggests that a cyclophilin other than cyclophilin B is required to support HPV infectious entry. The unidentified cyclophilin appears not to be produced endogenously by the cell, but is rather provided by serum in the cell culture medium. In a separate line of work, we completed our goal of elucidating the pattern of disulfide bonds that stabilize the HPV virion. This line of work led to improved methods for production of fully mature HPV virions.

人乳头瘤病毒（HPV）是一组非发育的DNA病毒，这些病毒已进化为在上皮组织（例如皮肤或生殖器粘膜）中利用专门的生物生物裂市场。尽管大多数HPV类型都与临床上不可或缺的症状或良性症状（例如常见的皮肤疣相关），但大约十几种性传播的HPV类型几乎会导致所有宫颈癌病例，以及很大一部分肛门，生殖器，生殖器和喉咙癌。 HPV诱导的宫颈癌每年在全球范围内杀死近30万名女性，主要是在宫颈癌筛查的发展中国家受到限制的发展中国家。最近批准的两种引起癌症HPV类型的疫苗有助于将公众关注这些病毒对公共卫生的重大风险。肿瘤病毒分子生物学部分的工作集中在HPV病毒素上。非发育病毒的病毒体是动态结构，必须在病毒生命周期的不同阶段进行一系列构象变化。病毒体必须足够灵活，以允许封装病毒基因组，但足够稳定，可以承受宿主之间传播期间遇到的环境侮辱。病毒体还必须变得足够柔韧，以在感染宿主细胞后释放病毒基因组。尽管病毒生命周期中的每个步骤都需要在病毒家族成员中保存良好的离散结构基序，但免疫压力倾向于选择从免疫学识别中遮住保守功能基序的病毒体。使用HPV载体技术和一系列蛋白质组学工具，我们试图表征病毒素的保守功能基序及其细胞结合靶标。实验室中的工作旨在阐明病毒体组装和传染性进入的机制，以及基于病毒生命周期这些阶段的基本细胞生物学。但是，我们的工作也有翻译目标。 基于HPV的高细胞基因转移载体（也称为HPV伪病毒）开始表现为遗传疫苗车辆的希望。对组装，感染性和HPV载体的体内倾向的理解应提高其体内基因转移应用的实用性。靶向保守病毒体结构的抗体有可能对各种HPV类型进行灭菌。因此，我们工作的另一个翻译目标是促进不断发展的改进疫苗，这些疫苗可能与当前可用的疫苗相比，可以保护针对更广泛的肿瘤性HPV类型。在09财年期间，我们完成了使用串联亲和力纯化（TAP）策略来识别传染性进入过程中HPV病毒素接触的细胞蛋白的目标。简而言之，TAP策略涉及用化学交联诱导的细胞结合的HPV病毒体纯化。我们利用质谱分析来鉴定与病毒粒子交联的细胞蛋白。该方法揭示了两类的宿主蛋白，可促进HPV进入宿主细胞的传染性。一旦候选因子是细胞蛋白酶。在FY10中，我们将致力于了解蛋白质客户的蛋白质客户的作用，以促进HPV传染性进入。我们的TAP分析还表明，HPV传染性进入需要一种是环磷脂家族成员的伴侣蛋白。 发现环糖蛋白抑制性药物（例如环孢菌素A）在体外抑制HPV感染性。尽管在我们的水龙头筛选中鉴定出的最初的候选蛋白是环粒蛋白B，但进一步的分析表明，除环蛋白B以外的其他细胞周期蛋白需要支持HPV传染性进入。未鉴定的环蛋白似乎不是由细胞内源性产生的，而是由细胞培养基中的血清提供。在另一项工作中，我们完成了阐明稳定HPV病毒体的二硫键模式的目标。这项工作导致改进了生产完全成熟的HPV病毒体的方法。