Abstract
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.
Highlights
Astroviruses are non-enveloped, positive-sense, single-stranded RNA viruses
In order to generate DNA-launched infectious clones of porcine astrovirus (PAstV), the T7 promoter in pMDF123 was replaced with a CMV promoter and an hepatitis delta virus ribozyme (HDVr) element was inserted immediately after the viral tail region of pMDF123 in order to produce the exact 30 end sequence of the viral genome (Figure 1a)
We found that Flag-tagged hypervariable region (HVR) protein co-localized with capsid protein (Cap) and ORF2b proteins in the cytoplasm, indicating that interactions between these viral proteins are closely connected
Summary
The astrovirus genome is approximately 6.2 to 7.9 kb in length and contains 50 untranslated regions (UTR), four open reading frames (ORF1a, ORF1b, ORF2, and ORF2b), a 30 UTR, and a 30 poly (A) tail [1,2,3]. ORF1a is situated at the 50 end of the genome and encodes a nonstructural polyprotein, nsP1a, which can be proteolytically cleaved into serine proteases;. ORF1b is expressed by a programmed ribosomal frameshifting mechanism and encodes an RNAdependent RNA polymerase (RdRp). ORF2 is located at the 30 end of the genome and encodes a capsid protein (Cap) translated from a sub-genomic RNA (sgRNA) [6]. The newly identified ORF2b overlaps with the 50 end of the ORF2. The ORF2b protein may have a certain amount of permeabilization activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
