Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus

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 r...

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Autores principales:	Yanjie Du, Teng Liu, Yifeng Qin, Qinting Dong, Ying Chen, Kang Ouyang, Zuzhang Wei, Weijian Huang
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	astrovirus PASTV-GX1 DNA-launched infectious clones HVR transposons tag Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/edb297d387a147518bdb7a8d392c06f9
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spelling	oai:doaj.org-article:edb297d387a147518bdb7a8d392c06f92021-11-25T19:12:24ZInsertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus10.3390/v131121191999-4915https://doaj.org/article/edb297d387a147518bdb7a8d392c06f92021-10-01T00:00:00Zhttps://www.mdpi.com/1999-4915/13/11/2119https://doaj.org/toc/1999-4915A 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 virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.Yanjie DuTeng LiuYifeng QinQinting DongYing ChenKang OuyangZuzhang WeiWeijian HuangMDPI AGarticleastrovirusPASTV-GX1DNA-launched infectious clonesHVRtransposonstagMicrobiologyQR1-502ENViruses, Vol 13, Iss 2119, p 2119 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	astrovirus PASTV-GX1 DNA-launched infectious clones HVR transposons tag Microbiology QR1-502
spellingShingle	astrovirus PASTV-GX1 DNA-launched infectious clones HVR transposons tag Microbiology QR1-502 Yanjie Du Teng Liu Yifeng Qin Qinting Dong Ying Chen Kang Ouyang Zuzhang Wei Weijian Huang Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
description	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 virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.
format	article
author	Yanjie Du Teng Liu Yifeng Qin Qinting Dong Ying Chen Kang Ouyang Zuzhang Wei Weijian Huang
author_facet	Yanjie Du Teng Liu Yifeng Qin Qinting Dong Ying Chen Kang Ouyang Zuzhang Wei Weijian Huang
author_sort	Yanjie Du
title	Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
title_short	Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
title_full	Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
title_fullStr	Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
title_full_unstemmed	Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus
title_sort	insertion of exogenous genes within the <i>orf1a</i> coding region of porcine astrovirus
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/edb297d387a147518bdb7a8d392c06f9
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Insertion of Exogenous Genes within the <i>ORF1a</i> Coding Region of Porcine Astrovirus

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