Reverse genetics as a tool to study molecular determinants of cell culture-adaptation and persistence in wild-type canine distemper virus


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Thèse: thèse de doctorat.
Reverse genetics as a tool to study molecular determinants of cell culture-adaptation and persistence in wild-type canine distemper virus
Plattet P.
Wittek R.
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Université de Lausanne, Faculté de biologie et médecine
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REROID:R003739655; 30 cm ill.; Old school value: Université de Lausanne
The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system of dogs. A75/17-CDV, which infects cell lines very inefficiently, was successfully adapted to grow in Vero cells and designated A75/17-V. Both genomes were sequenced, and the comparison revealed 7 differences located in the M, P/V/C and L genes. The mutations resulted in amino acid changes in the P, V, M and L proteins. Interestingly, the phenotype of infection of A75/17-V in Vero cells is mainly characterized by a cell-to-cell dissemination with no obvious syncytia formation.
Since both CDV strains contain identical glycoproteins but differ in their RNA polymerase genes, we first investigated whether the inability of A75/17-CDV to infect Vero cells was linked to a restricted RNA synthesis. Towards this end, the A75/17-V N, P and L genes, which have been demonstrated to be necessary and sufficient to promote paramyxovirus replication, were cloned into the eukaryotic expression vector pTM-1. Their functionality was then assessed by showing their ability to rescue a bi-cistronic minireplicon construct, containing the eGFP and CAT reporter genes flanked by the two A75/17-V promoter regions. The A75/17-V P and L genes were then mutated to generate the wild-type A75/17-CDV P and L proteins. Surprisingly, the polymerase of A75/17-V was found to be significantly less active in Vero cells than that of A75/17- CDV. This result suggested that restricted wild-type CDV infection in Vero cells was likely due to an impediment in cell entry, despite the fact that the wild-type and Vero- adapted virus harbor the same glycoprotein genes.
To investigate the molecular determinants of A75/17-V involved in the persistent infection in Vero cells, a second rescue system was established based on a molecularly cloned full-length A75/17-V genome. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein was inserted at the 3' proximal position in the A75/17-V cDNA clone. In this system, we used stably expressing T7 RNA polymerase cells (Bsr-T7), which allowed us to avoid the use of recombinant vaccinia virus to deliver the polymerase. By transfecting the relevant cDNA clone and the N, P and L expression plasmids in Bsr-T7 cells, and followed by a subculture with Vero cells, we indeed succeeded in recovering a persistent, GFP-expressing CDV. The virus was then characterized in vitro, and our results showed that the cloned virus behaved similarly to the parental A75/17-V.
The Onderstepoort CDV vaccine strain (OP-CDV), which is genetically more distant to wild-type isolate, induces a cytopathic effect characterized by extensive cell-to-cell fusion. To investigate the roles of wild-type F and H proteins in producing the persistent phenotype of infection, the surface glycoproteins of OP-CDV and A75/17-CDV were first compared in a transient-expression system using Vero cells. In addition, the role of the cellular receptor SLAM (CD150) in fusion efficiency was tested in a Vero cell line expressing the dog SLAM molecule. We demonstrate that not only the wild-type H protein, but also the wild-type F protein is major determinant of a low fusion activity. These results were confirmed by studying recombinant A75/17-V viruses bearing OP-CDV surface glycoproteins. Moreover, we showed that the presence of SLAM modulates the fusion activity, demonstrating a crucial role of host cellular factors in determining cytolytic versus persistent infection. Furthermore, detailed analysis of the F protein showed that the unusually long signal peptide plays a major role in modulating the fusogenic activity of the protein.
Finally, in a natural course of infection of dogs with CDV, infection of canine footpads can result in the so-called hard pad disease, characterized by proliferation of footpad keratinocytes. Subsequently, primary dog keratinocytes (PDK) have been shown to be permissive to support A75/17-CDV infection, which were characterized by a cell-to-cell spread with no obvious syncytium formation. Preliminary results showed that by passaging the wild virus only three times in these cells, an efficient and rapid dissemination was observed. This phenotype is associated with an increase of infectious virus release, without modulating the persistent infection. By sequencing both genomes, three mutations were found in the P, M and H genes, which all result in amino acid changes, suggesting an important role of the wild-type M protein in the unefficient infectious virus production.
Création de la notice
19/11/2007 13:40
Dernière modification de la notice
29/06/2021 11:16
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