FUNCTIONS OF THE PROTEINS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS

人呼吸道合胞病毒蛋白质的功能

• 批准号: 6098950
• 负责人: PETER LEON COLLINS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/6098950
• 关键词: DNA directed RNA polymerase; RNA biosynthesis; genetic transcription; microorganism culture; microorganism growth; nucleocapsid; phosphoproteins; plasmids; posttranslational modifications; protein structure function; replicase; respiratory syncytial virus; tissue /cell culture; transfection; virus RNA; virus envelope; virus genetics; virus protein; virus replication

Human respiratory syncytial virus (RSV), an enveloped RNA- containing virus of the paramyxovirus family, is the most important viral agent of pediatric respiratory tract disease. Its genome is a single negative strand of RNA of 15,222 nucleotides that encodes ten major mRNAs. The viral genes are transcribed in a sequential fashion in which transcript abundance decreases with increasing distance of its gene from the promoter (polar transcription). The purpose of this project is to identify the functions of the viral proteins and to reconstruct events in the viral growth cycle under conditions where they can be more readily studied. Knowledge of the functions of the proteins is important for the design of live- attenuated recombinant vaccine viruses. We previously developed an intracellular transcription and replication system for RSV based on components expressed from transfected plasmid-borne cDNAs (see accompanying report). This involves transfecting tissue culture cells with plasmids which individually encode a helper-dependent minireplicon analog of negative-sense genomic or positive-sense antigenomic RNA as well as whatever mix of RSV proteins is desired. Three proteins, the nucleocapsid N protein, phosphoprotein P and large polymerase subunit L protein, constitute the RSV replicase whereas, unexpectedly, the transcriptase requires in addition expression of the M2 mRNA. The M2 mRNA contains two overlapping translational open reading frames (ORFs), and the upstream one (M2-1) was shown to encode a processivity factor. This is a completely novel finding for the negative strand RNA viruses, and among RNA viruses such a factor has only been described for human immunodeficiency virus 1, namely the Tat protein. Additional experiments indicated that the presence of M2-1 is not required for correct initiation, termination or polyadenylation. However, in addition to its processivity activity, the M2-1 protein was found to reduce the efficiency of transcriptional termnation at the gene-end (GE) signal and thus increases the amount of transcriptional readthrough. The effect of M2-1 on RSV sequential transcription is being examined using minigenomes which contain authentic groups of viral genes. Both activities, namely processivity within genes and partial readthrough of GE signals, has the effect of reducing the gradient of transciptional polarity. Transcription and the first step of RNA replication each involve the synthesis of positive-sense RNA off the genomic template. It is generally assumed that the two processes are interrelated and are regulated by the availability of N protein to direct encapsidation of the antigenome. In the absence of sufficient N protein, transcription would predominate. An alternative model is that the M2-1 transcription factor might have a regulatory effect by favoring transcription over replication. The reconstituted transcription/replication system provided the first opportunity to directly test these models. A series of tightly controlled experiments showed that the levels of transcription and RNA replication are insensitive to changes in the levels of N or of the other nucleocapsid-associated proteins. The available data suggests that these processes are independent rather than tightly interrelated. Expression of the second ORF of the M2 mRNA (M2-2) inhibits RNA replication and transcription, providing functional evidence that this represents an eleventh RSV gene and is a novel negative regulatory factor. We presently are working to detect this protein in infected cells and to determine the mechanism by which it is expressed from its overlapped ORF. The nonstructural protein NS1 also was found to be a potent inhibitor of RSV RNA synthesis. Like the M2-2 protein, NS1 strongly inhibited transcription and both steps of RNA replication. We found that it is possible to reconstitute virion morphogenesis by coexpression of appropriate envelope components with the above-mentioned transcription and replication system. Morphogenesis was assayed by the passage of a minireplicon to fresh cells. This showed that the matrix protein M, attachment glycoprotein G and fusion glycoprotein F protein are important for the formation of transmissible virus-like particles. The small hydrophobic SH protein, both ORFs of the M2 mRNA, and the nonstructural NS1 and NS2 proteins are completely dispensable for particle formation. Interestingly, coexpression of the M2-1 transcription factor reduced the efficiency of virion packaging, suggesting that a nucleocapsid which is engaged in fully processive transcription might be refractory to packaging. Consistent with this, the further coexpression of the M2-2 inhibitory factor increased the efficiency of packaging and countered the effects of M2-1. Thus, M2-2 might be a packaging factor.

人呼吸综合病毒（RSV）， paramyxovirus家族的包裹的RNA - 含有RNA的病毒是 小儿呼吸道疾病的最重要病毒剂。 它的基因组是15,222的RNA的单个负链 编码十个主要mRNA的核苷酸。病毒基因是 以连续的方式转录的转录。 随着其基因与启动子的距离的增加而减小 （极性转录）。该项目的目的是确定 病毒蛋白的功能并重建病毒中的事件 在可能更容易的条件下增长周期 研究。了解蛋白质功能对 重组疫苗病毒的生命衰减设计。我们 以前开发了细胞内转录和复制 根据转染表达的组件的RSV系统 质粒传播的cDNA（请参阅随附的报告）。这涉及 用质粒转染组织培养细胞，这些细胞单独 编码负态的辅助辅助型微型类似物类似物 基因组或阳性抗原组RNA以及其他 需要RSV蛋白的混合。三个蛋白质，核素n 蛋白质，磷蛋白P和大型聚合酶亚基L蛋白， 构成RSV复制酶，而意外的是 转录酶还需要表达M2 mRNA。这 M2 mRNA包含两个重叠的翻译开放阅读 帧（ORF）和上游一个（M2-1）显示用于编码 一个加工性因子。这是一个完全新颖的发现 阴性链RNA病毒，在RNA病毒中，这是一个因素 仅描述了人类免疫缺陷病毒1， 即TAT蛋白。其他实验表明 正确启动，终止不需要M2-1的存在 或聚腺苷酸化。但是，除了其加工性活动外， 发现M2-1蛋白可降低 基因末端（GE）信号的转录术语，因此 增加了转录读取的量。效果 RSV顺序转录的M2-1正在使用 含有真实的病毒基因组的微型素。两个都 活动，即基因内的加工性和部分读取 GE信号的效果是降低 跨性极性。转录和RNA的第一步 复制每个都涉及从阳性RNA的合成 基因组模板。通常认为这两个过程 是相互关联的，并受到N蛋白的可用性的调节 抗原组的直接封装。在没有足够的情况下 N蛋白，转录将占主导地位。另一种模型是 M2-1转录因子可能具有调节作用 偏爱转录而不是复制。重构 转录/复制系统提供了第一个机会 直接测试这些模型。一系列严格控制的实验 表明转录和RNA复制水平是 对N或其他水平的变化不敏感 核蛋白酶相关蛋白。可用数据表明 这些过程是独立的，而不是紧密相关的。 M2 mRNA（M2-2）的第二个ORF的表达抑制 RNA复制和转录，提供功能证据 这代表了第十一RSV基因，是一种新颖的负面 调节因素。我们目前正在努力检测这种蛋白质 感染细胞并确定其所在的机制 从其重叠的ORF表示。非结构蛋白NS1 还发现是RSV RNA合成的有效抑制剂。喜欢 M2-2蛋白NS1强烈抑制转录，两者都 RNA复制的步骤。我们发现有可能 通过合适的共表达重新构建病毒体形态发生 具有上述转录的包络组件和 复制系统。通过通过 米雷泼剂到新鲜细胞。这表明基质蛋白M， 附着糖蛋白G和融合糖蛋白F蛋白是 对于形成可传染性病毒样颗粒的重要性。这 小的疏水性SH蛋白，M2 mRNA的两个ORF和 非结构性NS1和NS2蛋白完全可分配 用于粒子形成。有趣的是，M2-1的共表达 转录因子降低了病毒粒子包装的效率， 提示有一个从事全程处理的核蛋白 转录可能是对包装的难治性。与此一致 M2-2抑制因子的进一步共表达增加了 包装的效率并抵消了M2-1的影响。因此， M2-2可能是包装因素。