Roles of Pneumovirus Proteins in Virus Replication, Path

肺病毒蛋白在病毒复制、路径中的作用

• 批准号: 6808226
• 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/6808226
• 关键词: RNA biosynthesis; cystic fibrosis; genetic transcription; interferons; laboratory mouse; microorganism culture; nucleic acid sequence; nucleocapsid; polymerase chain reaction; posttranslational modifications; protein structure function; recombinant virus; respiratory epithelium; respiratory syncytial virus; tissue /cell culture; transfection; virus RNA; virus envelope; virus genetics; virus protein; virus replication

Human respiratory syncytial virus (HRSV) is an enveloped RNA-containing virus of Family Paramyxoviridae, Order Mononegavirales, the nonsegmented negative strand RNA viruses. HRSV is the most important viral agent of pediatric respiratory tract disease worldwide but lacks an approved vaccine or effective antiviral therapy. We previously showed that its genome is a single negative strand of RNA of 15,222 nucleotides that encodes 11 proteins, and previous studies identified the functions of a number of these proteins. We also have extended our molecular studies to human metapneumovirus (HMPV), a recently-identified virus that has been found to be an etiologic agent of pediatric respiratory disease that previously had gone undetected. Last year we initiated study of HRSV infection in primary human airway epithelial (HAE) cells that are grown on a filter membrane at an air-liquid interface and form a differentiated pseudostratified polarized mucocilliary tissue that is nearly indistinguishable morphologically and functionally from ex vivo airway epithelium. This culture system is maintained at the University of North Carolina Cystic Fibrosis and Pulmonary Research and Treatment Center. Infection of these cultures with a recombinant HRSV that expresses the green fluorescent protein (GFP) showed that HRSV infection, replication and virus release was restricted to ciliated cells at the apical face, and the virus appeared to be spread by cilia beat. Remarkably, HRSV infection of HAE cultures did not result in overt cell destruction even over a period of several weeks, in contrast to the rapid and extensive cell destruction by an influenza virus control. Thus, while HRSV is highly cytopathic in monolayer cell culture, it is much less cytopathic in highly organized, polarized multicellular tissue that resembles the epithelium of the human airway. This suggests that host immunity, rather than direct viral cytopathogenesis, is responsible for the damage that occurs to the ciliated epithelial cells in the airways during HRSV infection in vivo (and much more so than for influenza virus). However, more careful analysis indicated that, over time, HRSV-infected cells underwent a change in shape and became rounder, likely reflecting a stage leading to cell death. There was a modest increase in the release of intracellular enzyme markers, suggesting that some cell destruction did occur even though the apical surface remained intact. The observation that the appearance of the tissue did not change with time suggests that presumptively destroyed cells might be replaced with new cells. The observation that HRSV is specific for ciliated cells will help guide the search for cellular proteins involved in HRSV infection and attachment, and this in vitro model will be useful for studying the response of the epithelium to viral infection, such as changes in gene expression and the release of cytokines and chemokines. We also have extended this study to human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 1 (HPIV1) and HMPV, including biologically-derived virus as well as derivatives that are derived from cDNA and express GFP. We also are studying gene deletion mutants, such as recombinant HRSV (or HMPV) derivatives that express GFP but lack the SH glycoprotein, the G glycoprotein, or both SH and G. This work is the most advanced in the case of HPIV3. Like HRSV, HPIV3 was found to infect only the apical surface and is shed only from the apical surface. Infection is almost completely inhibited by prior neuraminidase treatment of the cells whereas that of HRSV is modestly enhanced (likely due to the effect of clearing the cell surface). Thus, their modes of attachment indeed are distinct. HPIV3 infection also was specific to ciliated cells. However, HPIV3 appeared to infect all ciliated cells (which can be stained with a beta tubulin-specific antibody) whereas HRSV infection was found to be specific to a subset of ciliated cells (ones which stained with a keratan sulfate-specific antibody). The basis for this is unknown. Whereas infection with HRSV led to cell rounding and likely cell death, infection with HPIV3 did not cause any apparent shape change and thus appeared to be even less cytopathic. We are continuing to explore the gross features (and eventually finer details) of infection of this model system with these common respiratory tract viruses. This will provide an ideal model for investigating the functions and effects of pneumovirus proteins in cells of the respiratory epithelium. Cystic fibrosis (CF) is a disease associated with a single defective gene, that encoding the cystic fibrosis transmembrane conductance regulator (CFTR). It thus has been an attractive candidate for gene therapy. It is thought that the ciliated cell of the apical surface of the respiratory lumen represents the necessary target for CF gene transfer. Remarkably, despite more than a decade of work, a vector system that can efficiently deliver a trans gene to this cell type had not been identified. The tropism of HRSV and HPIV3 described above, together with their relative lack of cytopathology, were exactly appropriate for use as vectors to deliver CFTR. We inserted the 4.5 kb cDNA encoding CFTR between the HN and L genes of HPIV3. Remarkably, this yielded infectious virus that replicated in vitro with an efficiency comparable to that of wild type HPIV3. This virus also retained the ability to efficiently infect the apical cells of the HAE cultures. Abundant expression of CFTR in the ciliated cells was confirmed, and functional assays confirmed that it was active in ion transport. Importantly, infection of HAE cultures derived from CF patients confirmed the ability to reverse the airway dehydration and mucociliary dysfunction characteristic of CF disease, thus validating the concept and target of CF genetherapy. This provides the first appropriate model system for studying the correction of the CF defect by gene transfer, and provides a starting point for investigating the quantitative and functional aspects of CF gene therapy and for developing new vectors and packaging systems. We also studied interferon induction by wild type and gene-deletion HRSV. Wild-type HRSV is a poor inducer of type I interferon (IFN) alpha and beta. However, recombinant HRSV lacking the NS1 and NS2 genes (deltaNS1/2) induced high levels of IFN alpha and beta in human pulmonary epithelial cells (A549) as well as in macrophages derived from primary human peripheral blood monocytes. Results with NS1 and NS2 single- and double-gene deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role. This pattern of inhibition by HRSV NS1 and NS2 also extended to the newly described antiviral cytokines IFN lambda 1, 2 and 3. This identifies the basis for the attenuation phenotype of the NS1 and NS2 deletion mutations, which have been included in vaccine candidate viruses now in clinical trials. In addition, type I IFN stimulates aspects of innate and adaptive immunity, and the use of a vaccine virus that does not suppress the IFN response likely will increase the immunogenicity of a live attenuated HRSV vaccine.