Cross-utilization of viral polymerase: parainfluenza virus hijacks the RdRp of porcine sapelovirus to facilitate its replication during co-infection.

Figures

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  Fig 1. Co-infection of PSV and PIV5 promotes diarrhea in suckling piglets. (A) Detection rates of PSV and PIV5 in diarrheal samples collected from 2016 to 2024. (B) PIV5 and PSV are frequently co-detected in diarrheal piglets. (C) Co-detection rate of PSV and PIV5 in samples negative for enteric coronaviruses and rotavirus. (D) Schematic diagram of the five-group animal experiment design. Eighteen 7-day-old piglets were randomly divided into six groups and orally inoculated with different viruses as indicated. (E and F) Daily viral shedding of PSV (E) and PIV5 (F) from anal swabs. (G) Clinical symptoms in piglets challenged with both PSV and PIV5 or either virus alone. (H) H&E staining of ileum sections from challenged piglets. (I and J) Viral titers of PSV (I) and PIV5 (J) in digestive tissues.

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  Fig 2. PSV co-infection significantly enhances PIV5 pathogenicity in vivo. (A and B) Growth curves of PIV5 (A) and PSV (B) during co-infection in PK-15 cells. (C and D) Growth curves of PIV5 (C) and PSV (D) during co-infection in Vero cells. (E and F) Growth curves of PIV5 (E) and PSV (F) in PK-15 cells under different infection sequences.

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  Fig 3. PSV and PIV5 co-infection occurs intracellularly in vitro and in vivo. (A and B) Confocal microscopy of PK-15 (A) and IPEC-J2 (B) cells infected with PSV, PIV5, or both. All cells were fixed at 36 hpi and stained with anti-PSV-VP1 mAb and anti-PIV5-NP. (C) STORM imaging of PK-15 cells infected with PSV, PIV5, or both. All cells were fixed at 36 hpi and stained with anti-PSV-VP1 mAb and anti-PIV5-NP. (D) Transmission electron microscopy of PK-15 cells co-infected with PSV and PIV5. Red arrows indicate PIV5 virions (150–200 nm); blue arrows indicate PSV virions (~20 nm). (E) Immunofluorescence assay of ileum tissue collected from piglets co-infected with PSV and PIV5. White arrows indicate cells positive for both viruses.

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  Fig 4. PSV 3D protein promotes PIV5 replication. (A) Screening of all PSV genes for effects on PIV5 replication via plasmid transfection in PK-15 cells. Cells were transfected with plasmids carrying the indicated genes (2 μg per sample) and infected with PIV5 (MOI of 0.01) after 12 h. The supernatant was collected at 48 hpi for viral loading determination. (B) Growth curve of PIV5 in PK-15 cells transfected with increasing doses of PSV 3D. Cells were transfected with plasmids carrying PSV 3D and infected with PIV5 (MOI of 0.01) after 12 h. The supernatant was collected for viral loading determination. (C) Western blot analysis of PIV5 M protein levels in PSV 3D-transfected cells at 60 hpi with relative intensity indicated. *P < 0.05, **P < 0.01.

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  Fig 5. PSV 3D protein directly interacts with PIV5 nucleoprotein. (A) Co-immunoprecipitation showing interaction between PSV 3D and PIV5 NP. HEK293T cells were transfected with the indicated plasmids and lysed at 48 hpt. Cell lysate was immunoprecipitated with anti-GFP or anti-Flag antibodies and subjected to Western blot analysis. (B) Endogenous immunoprecipitation confirming PSV 3D–PIV5 NP interaction. PK-15 cells co-infected with PSV and PIV5 were lysed at 36 hpi. The cell lysate was immunoprecipitated with anti-PIV5-NP antibodies and subjected to Western blot analysis. (C) Confocal microscopy showing co-localization of PSV 3D and PIV5 NP. (D and E) Co-immunoprecipitation showing interaction of PIV5 NP with the N-terminal domain of PSV 3D (D) and the core domain of PIV5 NP (E). HEK293T cells were transfected with plasmids carrying the truncated variants and lysed at 48 hpt. The cell lysate was immunoprecipitated with anti-GFP or anti-Flag antibodies and subjected to Western blot analysis. (F) AlphaFold3-based structural prediction of the interactions among PSV 3D, PIV5 NP, and PIV5 L protein.

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  Fig 6. PSV 3D binds to PIV5 genomic RNA. (A) RNA immunoprecipitation confirms binding of PIV5 RNA to PSV 3D. PK-15 cells were transfected with GFP-PSV-VP1 or GFP-PSV 3D, and then infected with PIV5 of MOI 0.01. The cells were then UV cross-linked at 36 hpi and subjected to RNA immunoprecipitation analysis. The affinity of PSV-VP1/PSV 3D to PIV5 genomic RNA was assessed by RT-qPCR. (B) RNA pull-down assay showing interaction between PIV5 RNA and PSV 3D. The vRNA was extracted from purified PIV5 virions and biotinylated, which was further incubated with cell lysate containing GFP-3D or not. The beads were finally washed and detected by Western blotting. **P < 0.01.

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  Fig 7. PSV 3D enhances activity of the PIV5 mini-genome system. (A) Schematic of PIV5 mini-genome construction. (B) Luciferase assay showing mini-genome activity. HEK293T-T7 cells were co-transfected with pBR-PIV5-luciferase (0.3 μg), pCAGGs-PIV5-P (0.3 μg), and phRL-TK (0.05 μg), with pCAGGs-PIV5-L or pCAGGS-PSV 3D. After 36 h, the cells were lysed and assayed for Fluc and renilla activity using a dual-luciferase reporter assay system. (C) Mini-genome activity driven by 3D proteins from different PSV strains. HEK293T-T7 cells were co-transfected with pBR-PIV5-Luciferase (0.3 μg), pCAGGs-PIV5-P (0.3 μg) and phRL-TK (0.05 μg), and pCAGGS-PSV 3D. After 36 h, the cells were lysed and assayed for Fluc and renilla activity using a dual-luciferase reporter assay system. (D and E) qRT-PCR quantification of PIV5 plus-strand (D) and minus-strand (E) RNAs. PK-15 cells of different groups were transfected with PSV 3D/infected with PSV/mock-treated, and further infected with PIV5. The total RNA was extracted at indicated time points and transcribed by RNase H with different primers against the plus- and minus-strand RNAs of PIV5. The number of PIV5 RNAs was quantified by qRT-PCR analysis. *P <…

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  Fig 8. PSV 3D supports transcription of full-length PIV5 RNA. (A) Schematic of the full-length PIV5 genome, infectious clone, and mutant constructs. (B) Western blot analysis of NP expression in cells transfected with PIV5 infectious clones or mutants. (C) IFA of NP expression in transfected cells. (D) TEM images of PIV5 virions. PK-15 cells infected with the rescued PIV5 (F1) were subject to TEM observation at 24 hpi. (E) Growth kinetics of rescued PIV5 compared with the parental strain. (F and G) qRT-PCR analysis of plus-strand (F) and minus-strand (G) PIV5 RNAs from infectious clone and mutants. Cells transfected with PIV5 infectious clones or mutants were subjected to RNA extraction. The total RNA was transcribed by RNase H with different primers against the plus- and minus-strand RNAs of PIV5 and quantified by qRT-PCR analysis.

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  Fig 9. 3D proteins of other enteric picornaviruses enhance PIV5 replication. (A and B) Co-immunoprecipitation confirms interactions of PIV5 NP with 3D proteins from PKV (A) and PTV (B). HEK293T cells were transfected with plasmids carrying different genes and lysed at 48 hpt. Cell lysate was immunoprecipitated with anti-GFP or anti-Flag antibodies and subjected to Western blot analysis. (C and D) Dose-dependent enhancement of PIV5 mini-genome activity by PKV 3D (C) and PTV 3D (D). HEK293T-T7 cells were co-transfected with pBR-PIV5-luciferase (0.3 μg), pCAGGs-PIV5-P (0.3 μg), and phRL-TK (0.05 μg) with pCAGGS-PKV-3D or pCAGGS-PTV-3D. After 36 h, the cells were lysed and assayed for Fluc and renilla activity using a dual-luciferase reporter assay system. (E–H) PKV 3D (E) and PTV 3D (F) promote PIV5 replication and increase both plus-strand (G) and minus-strand (H) RNA levels. PK-15 cells were transfected with plasmids carrying the 3D gene (1.0 μg) and infected with PIV5 12 h later. The supernatant was collected for viral growth curve description, while the total RNA was extracted for PIV5 RNA quantification by qRT-PCR analysis.

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  Fig 10. Schematic diagram summarizing the role of PSV 3D in facilitating PIV5 replication.