The Spike Protein of Transmissible Gastroenteritis Coronavirus Controls the Tropism of Pseudorecombinant Virions Engineered Using Synthetic Minigenomes
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The minimum sequence required for the replication and packaging of transmissible gastroenteritis virus (TGEV)-derived minigenomes has been determined. To this end, cDNAs encoding defective RNAs have been cloned and used to express heterologous spike proteins, to determine the influence of the peplomer protein in the control of TGEV tropism.
A TGEV defective interfering RNA of 9.7 kb (DI-C) was isolated, and a cDNA complementary to DI-C RNA was cloned under the control of T7 promoter. In vitro transcribed DI-C RNA was replicated in trans upon transfection of helper virus-infected cells. A collection of DI-C deletion mutants (TGEV minigenomes) was generated and tested for their ability to be replicated and packaged. The size of the smallest minigenome replicated in trans was 3.3 kb. The rescue system was used to express the spike protein of an enteric TGEV isolate (Cll) using as helper virus a TGEV strain (C8) that replicates very little in the gut. A mixture of two pseudorecombinant viruses containing either the helper virus genome or the minigenome was obtained. These pseudorecombinants display in the surface the S proteins from the enteric and the attenuated virus, and showed 104-fold increase in their gut replication levels as compared to the helper isolate (C8). In addition, the pseudorecombinant virus increased its enteric pathogenicity as compared to the C8 isolate.
KeywordsInfectious Bronchitis Virus Hepatitis Delta Virus Helper Virus Mouse Hepatitis Virus Spike Protein
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