In Vitro Models of Paramyxovirus Infection

我们正在与北卡罗来纳州大学囊性纤维化/肺研究和治疗中心合作，研究人呼吸道合胞病毒（HRSV）、人偏肺病毒（HMPV）和人副流感病毒1、2和3（HPIV 1、2和3）在人气道上皮（HAE）体外模型中的感染。该体外模型由原代气道上皮细胞组成，所述原代气道上皮细胞在空气-液体界面处的滤膜上生长并形成分化的假复层极化粘液纤毛组织，所述分化的假复层极化粘液纤毛组织在形态上和功能上与离体气道上皮几乎不可区分。 本报告侧重于HPIV 3。我们构建了表达增强型绿色荧光蛋白（GFP）的重组HPIV 3，并使用该病毒rgPIV 3感染HAE培养物。HAE的顶面对rgPIV 3感染高度敏感，而当病毒应用于基底外侧表面时，仅偶尔感染细胞。感染只涉及纤毛细胞。几乎没有病毒介导的细胞病理学证据，特别是没有细胞融合或病毒扩散超出管腔柱状细胞的证据。这表明HPIV 3本身不是一种高度致细胞病变的病毒，并意味着体内发生的细胞破坏可能是由于宿主免疫机制而不是直接的病毒介导的损伤。我们已经用表达GFP的HRSV（rgRSV）以及用野生型HRSV获得了类似的结果。 rgPIV 3对纤毛细胞的感染通过用对N-乙酰神经氨酸-α 2，6-半乳糖（α 2，6-唾液酸）特异性的神经氨酸酶处理顶端表面而完全消除，而α 2，3-和α 2，8-键的裂解几乎没有影响。这提供了rgPIV 3 HN利用α 2，6-连接的唾液酸残基启动感染的证据，这是先前对人流感病毒描述的特异性。荧光标记凝集素的探测表明，唾液酸存在于微绒毛上，但在纤毛上检测不到。rgRSV的感染不受HAE的神经氨酸酶处理的影响或适度增强，这与其受体不同于唾液酸一致。 HPIV 3 F糖蛋白仅被运输到纤毛细胞的顶端表面，这也是子代病毒的释放位点。有趣的是，F糖蛋白主要定位于纤毛轴结构的膜，这表明子代病毒可能从这些细胞结构中出芽。因此，HPIV 3出芽的位点可能不同于附着和进入的位点。F糖蛋白向顶端表面的极化运输也可能限制其与邻近细胞的相互作用，并可能导致细胞间融合的缺乏。 来自囊性纤维化（CF）患者的HAE对rgPIV 3感染或细胞病变效应并不更敏感，但由于纤毛搏动减少导致管腔分泌物运动受损，因此病毒传播有限。 rgPIV 3与rgRSV感染的结局之间存在细微差异。具体而言，rgRSV感染导致细胞变得更圆，这可能反映了导致细胞死亡的阶段。我们正在研究这些病毒在诱导程序性细胞死亡方面是否不同。当在较长时间（13天）内检查rgPIV 3感染的培养物时，HAE培养物的形态显示含粘蛋白细胞的化生和粘蛋白产生的增加。我们将继续探索这些常见呼吸道病毒对该模型系统的感染，以表征病毒感染和细胞病变机制以及宿主细胞反应。 囊性纤维化（CF）是与编码囊性纤维化跨膜传导调节因子（CFTR）的单个缺陷基因相关的疾病。因此，它已成为一个有吸引力的候选基因治疗。据认为，呼吸腔顶面的纤毛细胞代表CF基因转移的必要靶标。值得注意的是，尽管经过了十多年的研究，一种能够有效地将转基因传递到这种细胞类型的载体系统还没有被发现。上述HRSV和HPIV 3的嗜性以及它们相对缺乏细胞病理学，完全适合用作递送CFTR的载体。我们将编码CFTR的4.5kb cDNA插入HPIV 3的HN和L基因之间。值得注意的是，这产生了感染性病毒，其在体外复制的效率与野生型HPIV 3相当。该病毒还保留了有效感染HAE培养物的顶端细胞的能力。证实了CFTR在纤毛细胞中的丰富表达，并且功能测定证实了其在离子转运中具有活性。重要的是，来自CF患者的HAE培养物的感染证实了逆转CF疾病的气道脱水和粘膜纤毛功能障碍特征的能力，从而验证了CF基因治疗的概念和靶点。这提供了第一个适当的模型系统，用于研究通过基因转移校正CF缺陷，并提供了一个起点，用于研究CF基因治疗的定量和功能方面，并用于开发新的载体和包装系统。

We are studying infection by human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and the human parainfluenza viruses 1, 2 and 3 (HPIV1, 2 and 3) in an in vitro model of human airway epithelium (HAE), in collaboration with the University of North Carolina Cystic Fibrosis/Pulmonary Research and Treatment Center. This in vitro model consists of primary airway epithelium 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. The present report focuses on HPIV3. We constructed a recombinant HPIV3 that expresses enhanced green fluorescent protein (GFP) and used this virus, rgPIV3 to infect HAE cultures. The apical surface of HAE was highly susceptible to rgPIV3 infection, whereas only occasional cells were infected when virus was applied to the basolateral surface. Infection involved exclusively ciliated cells. There was little evidence of virus-mediated cytopathology and, in particular, no evidence of cell fusion or spread of the virus beyond the luminal columnar cells. This indicates that HPIV3 is not inherently a highly cytopathic virus, and implies that the cell destruction that occurs in vivo might be due to host immune mechanisms rather than direct virus-mediated damage. We have obtained similar results with HRSV that expresses GFP (rgRSV) as well as with wild-type HRSV. Infection of ciliated cells by rgPIV3 was completely abolished by treatment of the apical surface with a neuraminidase specific for N-acetylneuraminic acid-a2,6-galactose (a2,6-sialic acid), whereas cleavage of a2,3- and a2,8-linkages had little effect. This provided evidence that rgPIV3 HN utilizes a2,6-linked sialic acid residues for initiating infection, a specificity previously described for human influenza viruses. Probing with fluorescently-labeled lectins indicated that the sialic acid was present on microvilli but was not detectable on the cilia. Infection by rgRSV was unaffected or modestly enhanced by neuraminidase treatment of the HAE, consistent with its receptor being distinct from sialic acid. The HPIV3 F glycoprotein was trafficked exclusively to the apical surface of ciliated cells, which also was the site of release of progeny virus. Interestingly, F glycoprotein was localized mainly to the membranes of the cilial shaft structures, suggesting that progeny viruses may bud from these cellular structures. Thus, the site of HPIV3 budding might be different from the site of attachment and entry. The polarized trafficking of F glycoprotein to the apical surface also likely restricts its interaction with neighboring cells and could account for the lack of cell-to-cell fusion. HAE derived from cystic fibrosis (CF) patients were not more susceptible to rgPIV3 infection or cytopathic effect, but did exhibit limited spread of virus due to impaired movement of lumenal secretions due to a reduced cilial beat. There was a subtle difference between the outcome of infection by rgPIV3 versus rgRSV. Specifically, rgRSV infection caused the cells to become rounder, which might reflect a stage leading to cell death. We are investigating whether these viruses differ with regard to induction of programmed cell death, among other things. When rgPIV3-infected cultures were examined over a longer period of time (13 days), the morphology of the HAE culture exhibited a metaplasia of mucin-containing cells and an increase in mucin production. We are continuing to explore infection of this model system with these common respiratory tract viruses to characterize viral infection and cytopathic mechanisms and the host cell response. 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 gene therapy. 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.