Viruses, Vol. 13, Pages 2119: Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

Viruses, Vol. 13, Pages 2119: Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus Viruses doi: 10.3390/v13112119 Authors: Yanjie Du Teng Liu Yifeng Qin Qinting Dong Ying Chen Kang Ouyang Zuzhang Wei Weijian Huang A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter vi...
Source: Viruses - Category: Virology Authors: Tags: Article Source Type: research