Development of a reporter HBoV1 strain for antiviral drug screening and life cycle studies

Figures

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  Fig. 1. Insertion sites for the HiBiT coding sequence in the HBoV1 genome. A Diagram showing the pHBoV1 genome structure. Black arrows and numbers indicate HiBiT tag insertion sites. The HiBiT tag (yellow) and GSSG linker (purple) sequences are indicated. B Prediction of the accessibility of NS1, NS1-70, and VP3 after the insertion of the HiBiT tag using Alphafold2. Accessibility (from strong to weak): red ​> ​yellow ​> ​green ​> ​blue.

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  Fig. 2. Analysis of the replication capacity of the HBoV1 recombinant clones. A HEK293T cells overexpressing LgBiT were separately transfected with different pHBoV1-HiBiT plasmids (pHBoV1, pHBoV1-HiBiTNS1, pHBoV1-HiBiTNS1-70, pHBoV1-HiBiTVP3, pHBoV1-HiBiTVP3-206, or pHBoV1-HiBiTVP3-460); after 72 ​h, HiBiT-dependent luciferase activity was measured. B HEK293T cells were separately transfected with different pHBoV1-HiBiT plasmids; after 72 ​h, the intracellular DNA replication level was quantified by qPCR [compared to wild type (WT)]. C HEK293T cells were separately transfected with different pHBoV1-HiBiT plasmids; after 48 ​h, the protein levels of NS1, HiBiT, VPs, and NP1 were quantified by western blotting. D HEK293T cells were transfected with pHBoV1, pHBoV1-HiBiTNS1, pHBoV1-HiBiTNS1-70, pHBoV1-HiBiTVP3, pHBoV1-HiBiTVP3-206, or pHBoV1-HiBiTVP3-460; subsequently, Hirt-extracted DNA was digested with the DpnI restriction endonuclease for 12 ​h and separated using 1.2% agarose gel electrophoresis. dRF (double replicative form) DNA and mRF (monomer replicative form) DNA, which was resistant to digestion by DpnI, was detected in the transfected cells by Southern blotting. Data are…

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  Fig. 3. Characterization of recombinant HBoV1 carrying the HiBiT tag in the NS1 protein. A HEK293T cells were transfected with pHBoV1 or pHBoV1-HiBiTNS1 for 72 ​h, probed with the DNA-binding dye DAPI (blue), anti-HiBiT antibody (green) and anti-NS1 antibody (red) in an immunofluorescence analysis, and then subjected to confocal microscopy. Scale bars, 10 ​μm. B HEK293T cells were separately transfected with vector, pHBoV1, or pHBoV1-HiBiTNS1 plasmids; after 48 ​h, the supernatant was collected with benzonase digestion or not for 30 ​min, then the benzonase resistant or free DNA in the supernatants was quantified by qPCR. C–E HEK293T cells were transfected with pHBoV1 or pHBoV1-HiBiTNS1 and the intracellular DNA copy number (C); HiBiT activity (D); and NS1, VPs, NP1, and HiBiT protein levels (E) were quantified at the indicated time points. F HEK293T cells were transfected with pHBoV1 or pHBoV1-HiBiTNS1 for 72 ​h, after which HBoV1 and HBoV1-HiBiTNS1 virions were subjected to negative-stain scanning electron microscopy. Scale bars, 50 ​nm. G Transmission electron microscopy of immunogold labeling in HBoV1 and HBoV1-HiBiTNS1 virions. Anti-VP antibodies were used as the primary antibodies.…

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  Fig. 4. Evaluation of antiviral drugs using recombinant HBoV1-HiBiTNS1.A HEK293T cells were transfected with pHBoV1-HiBiTNS1 in the presence of cidofovir (200 ​μM), favipiravir (50 ​μM), ivermectin (10 ​μM), nevirapine (50 ​μM), remdesivir (10 ​μM), ribavirin (50 ​μM), sofosbuvir (50 ​μM), or zidovudin (50 ​μM). At 72 ​h post-transfection, the Nano-Glo luciferase assay was performed. B, C HEK293T cells were transfected with pHBoV1-HiBiTNS1 in the presence of increasing (2-fold) concentrations of ivermectin. At 72 ​h post-transfection, luciferase activity was measured using the Nano-Glo luciferase assay (B), while the intracellular DNA copy number was quantified by qPCR (C). The EC50 and CC50 values were determined by fitting the dose-response curves with four-parameter logistic regression in GraphPad Prism. ∗, P ​< ​0.05, ∗∗∗, P ​< ​0.001; ns, not significant.

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  Supplementary Fig. S1. Analysis of the insertion sites for the HiBiT coding sequence in the VP3 protein. A Bar chart showing the average solvent surface area of each atom in each amino acid calculated according to the structure of HBoV1 VP3 [Protein Data Bank (PDB) 7L0Y]. B Secondary structure analysis of the encoded pHBoV1 VP3 protein sequence; α-helical regions (blue), β-fold regions (red), β-corner regions (green), and random curl regions (purple) are indicated. C Multiple sequence alignment of the VP3 sequences of HBoV1–4; highly conserved regions (red), relatively conserved regions (yellow), and variable regions (no color) are indicated. The arrows indicate the HiBiT tag insertion sites in the VP3 protein.

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  Supplementary Fig. S2. Evaluation of the HEK293T-LgBiT cell line. A The expression of LgBiT in the HEK293T-LgBiT stable cell line was quantified by Western blotting. B HEK293T-LgBiT cells were transfected with the empty vector control (VC) or HiBiT expression plasmids for 72 ​h, after which HiBiT-dependent luciferase (NanoLuc) activity was measured.

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  Supplementary Fig. S3. Detection of NS1, NS1 C-terminus, NP1, VP1 N-terminus, and VP3 transcript levels. A–E HEK293T cells were separately transfected with different pHBoV1-HiBiT plasmids for 48 ​h, after which the NS1, NS1 C-terminus, NP1, VP1 N-terminus, and VP3 transcript levels were evaluated by qRT-PCR. F–H HEK293T cells were separately transfected with HBoV1-WT and HBoV1-HiBiTNS1 plasmids (F), or HBoV1-HiBiTVP3 plasmids (G), or HBoV1-HiBiTVP3-206 and HBoV1-HiBiTVP3-460 plasmids (H) for 48 ​h. The cells were subsequently treated with MG132 (20 ​μM), chloroquine (CQ; 50 ​μM) or 3-MA (60 ​μM) and incubated for 4 ​h, and the indicated protein levels were detected by western blotting.

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  Supplementary Fig. S4. Characteristics of the HBoV1-GFPNS1 recombinants. A HEK293T cells were transfected with the pHBoV1-GFPNS1 plasmid and EGFP fluorescence was detected at 48 ​h post-transfection. Scale bar, 10 ​μm. B HEK293T cells were transfected with pHBoV1 or pHBoV1-GFPNS1 for 48 ​h; subsequently, NS, NP1, and VP protein levels were detected by western blotting at the indicated time points. C HEK293T cells were transfected with pHBoV1, pHBoV1-GFPNS1, or empty vector control (VC); then, Hirt-extracted DNA was digested with DpnI restriction endonuclease for 12 ​h and separated using 1.2 ​% agarose gel electrophoresis. dRF (double replicative form) DNA and mRF (monomer replicative form) DNA, which is resistant to digestion by DpnI, was detected in transfected cells by Southern blotting. D Absolute quantification of the HBoV1 and HBoV1-GFP genomes after virus purification. Viral DNA copy number was quantified by qPCR using the NS1 primer.