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. 2021 Oct 21;13(11):2119.
doi: 10.3390/v13112119.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

Yanjie Du  1 , Teng Liu  1 , Yifeng Qin  1 , Qinting Dong  1 , Ying Chen  1 , Kang Ouyang  1 , Zuzhang Wei  1 , Weijian Huang  1
Affiliations

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

Yanjie Du et al. Viruses. .

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.

Keywords: DNA-launched infectious clones; HVR; PASTV-GX1; astrovirus; iLOV; tag; transposons.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) Strategy for the construction of a full-length DNA-launched infectious clone of PAstV. The T7 promoter of pMD123 was replaced with a cytomegalovirus (CMV) promotor using SpeI and NgoMIV. The hepatitis delta virus ribozyme was inserted into the 3′ terminal end of the PAstV-GX1 genome with XhoI and SacII. The restriction enzyme sites above the full-length cDNA clones are indicated by arrows. (b) Cytopathic effects were observed in PK-15 cells infected with rescued viruses (magnification 10×). (c) IFA analysis of the Cap protein expression in PK-15 cells. PK-15 cells infected with rescued and parental viruses were fixed at approximately 48 h.p.i and subjected to immunofluorescence using a polyclonal antibody against capsid protein. The PK-15 cells were stained with goat anti-mouse IgG H&L (CoraLite488) (magnification 20×). (d) Growth curves of the recombinant and parental viruses. PK-15 cells were infected with recovered and parental viruses at a multiplicity of infection of 0.01. The viral titers were determined as TCID50.
Figure 2
Figure 2
Identification of 15 bp insertion sites in the ORF1a coding region of PAstV. Schematic diagram showing the transposon-based random insertion. MuA transposase-mediated random insertion was concentrated in a specific region of the PAstV genome, an HpaI-to-HindIII fragment containing half of the C-terminal of the coiled-coil structure, as well as the predicted genome-linked protein, VPg, and the hypervariable region.
Figure 3
Figure 3
Recovery and characteristics of recombinant viruses harboring epitope tags. (a) IFA analysis of the Cap expression in PK-15 cells. Recombinant virus-infected PK-15 cells were incubated with a mouse PcAb against capsid protein and stained with goat anti-mouse IgG H&L (CoraLite594). (b) IFA analysis of the tags expression in PK-15 cells. The PK-15 cells were infected with recombinant virus harboring epitope tags and incubated with mouse mAbs against the His, Flag, or HA-tag. The PK-15 cell was stained with goat anti-mouse IgG H&L (CoraLite488) (magnification 20×). (c) Growth curves of the recombinant and parental viruses. PK-15 cells were infected with recombinant virus harboring epitope tags at a multiplicity of infection of 0.01. Cell supernatants were collected at 6, 12, 24, 36, 48, and 60 h.p.i and the virus titers were determined by their TCID50 in PK-15 cells.
Figure 4
Figure 4
Flag-tagged ORF1a protein partially co-localizes with Cap and ORF2b proteins. (a) The PK-15 cells were infected with recombinant virus harboring Flag tag and parental viruses and subjected to immunofluorescence using a rabbit mAb against the Flag (red) and mouse mAb against capsid protein (green). (b) The PK-15 cells were subjected to immunofluorescence using a mouse mAb against the Flag (green) and a rabbit PcAb against the ORF2b protein (red). PK-15 cells were then stained with either goat anti-mouse IgG H&L (CoraLite488) or goat anti-rabbit IgG H&L (CoraLite594). The cell nuclei were counterstained with DAPI (blue) (magnification 20×).
Figure 5
Figure 5
Schematic diagram showing the site of insertion of iLOV.
Figure 6
Figure 6
Characterization of a recombinant PAstV with insertion of the iLOV gene into the HVR. (a) Cytopathic effects were observed in PK-15 cells infected with recombinant reporter virus. (b) IFA analysis of the capsid protein expression in PK-15 cells. The PK-15 cells were infected with recombinant virus harboring iLOV and subjected to immunofluorescence using mouse PcAb against capsid protein. PK-15 cells were stained with goat anti-mouse IgG H&L (CoraLite594) (magnification 20×). (c) Expression of auto-fluorescent protein in cells infected with reporter virus. PK-15 cell monolayers were infected with recombinant virus harboring iLOV at a multiplicity of infection of 0.01. Green fluorescence produced from iLOV protein was directly visualized using a fluorescent microscope at 6, 12, 24, and 36 h.p.i (magnification 20×). (d) Growth curves of the recombinant reporter virus. PK-15 cells were infected with recombinant virus harboring iLOV at a multiplicity of infection of 0.01. Cell supernatants were collected at 6, 12, 24, 36, 48, and 60 h.p.i and the virus titers were determined by their TCID50 in PK-15 cells.
Figure 7
Figure 7
(a) Analysis of the stability of reporter genes in the genomes of recombinant viruses in cells. Expression of reporter auto-fluorescence during serial passages of reporter viruses. PK-15 cells were infected with P1, P5, P10, and P15 viruses. At 24 h.p.i, live cells were imaged with a fluorescent microscope. (b) Agarose gel pictures showing the DNA fragments covering the reporter gene as generated by RT-PCR using the RNA extracted from cells infected with P3, P6, P9, P12, and P15 viruses. (c) The boundaries of the nucleotide deletions detected in the reporter gene of passaged viruses. Internal deletions were found in the genomes of passaged viruses. The numbers below the boxes indicate the nucleotide boundaries of the inserts and the internal deletions. The deleted regions are indicated by the dotted lines.
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