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.
Showing the most recent 10 out of 19 publications
