DNAJC14-Independent Replication of the Atypical Porcine Pestivirus

Figures

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  FIG 1. Scheme of the genome organization of the synthetic APPV genomes used in this study. NTR at the 5′ and 3′ ends of the genome and the mature processing products of the polyproteins are indicated. The labeled bars represent the N-terminal autoprotease (Npro), capsid protein (Core), glycoproteins (Erns, E1, and E2), and nonstructural proteins (p7 and NS2, -3, -4A, -4B, -5A, and -5B). A cDNA copy of an APPV field strain was cloned in a plasmid backbone and amplified by PCR, and infectious RNA was transcribed, generating recAPPV-wt. A nonreplicative subgenome was generated using a truncated PCR template. This negative control is lacking the NS5B gene sequence and the 3′ NTR and was named recAPPV-wtΔNS5B. To generate a replicative APPV-DI, we replaced the genes encoding Npro to NS2 with a ubiquitin gene (Ubi*). Expression of mature NS3 in a complete genomic APPV was achieved by inserting Ubi* into NS5B gene sequences from the APPV-DI in frame with the truncated NS4 (NS4B*), resulting in APPV-Ubi*. The mutations H1237A and C1280A were introduced to inactivate the NS2 autoprotease.

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  FIG 2. Reactivity of MAb N6 anti-APPV NS3 against recombinant NS3H expressed in E. coli. Total protein of noninduced (n.i.) and induced (i.) cultures of E. coli transfected with a pet11a vector encoding His-APPV-NS3H were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane. The membranes were probed with MAb N6 anti-APPV NS3 (A) and MAb 10B6 anti-His (B). Both MAbs reacted with a single protein species with an apparent molecular weight of about 60 kDa (NS3H). The bands of a protein ladder are indicated on the left, in kilodaltons.

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  FIG 3. Reactivity of MAb N6 to APPV NS3 against recombinant GFP-NS3 expressed in 293T cells. A pcDNA3.1 plasmid encoding GFP-NS3 was transfected into 293T cells. Transfected and mock-transfected control cells were fixed, permeabilized, and probed with MAb N6 against APPV NS3. Binding of the MAb was visualized with a Cy3-labeled secondary antibody. A close correlation between Cy3 signals and GFP fluorescence was observed in the transfected cells, as demonstrated in the overlay picture with dominant yellow staining. No signals occurred in the mock-transfected cells. Cellular nuclei were localized with a DAPI stain. Bars, 100 μm.

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  FIG 4. Molecular clone of an APPV field strain. SK-6 cells were transfected with synthetic RNA of recAPPV-wtΔNS5B (negative control) and recAPPV-wt by electroporation. SK-6 cells persistently infected with APPV field strain AUT-2016_C (APPV-wt) served as a positive control. After an incubation time of 48 h, the cells were fixed, permeabilized, and probed with MAb N6 anti-APPV NS3. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Dividing antigen-positive cells with characteristic cytoplasmic NS3 staining appeared in recAPPV-wt-transfected cells, and all cells were antigen positive in the persistently APPV-wt-infected cell line. Bars, 100 μm. Note the weaker and more focal staining in the persistently infected cells.

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  FIG 5. DNAJC14 dependency of CSFV. Transfection of synthetic RNA of CSFV (strain Alfort-Tuebingen) in SK-6 cells, SK-6 DNAJC14 knockout cells (SK-6 ΔDNAJC14), and SK-6 ΔDNAJC14 cells with knock-in of active Jiv90 (+ Jiv90) or inactive Jiv90 W39A (+ Jiv90 W39A). Transfection was done side by side with APPV RNAs as a control experiment (Fig. 6). At 48 h postelectroporation, the cells were fixed, permeabilized, and probed with MAb 8.12.7 against CSFV NS3. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm. CSFV antigen was detectable after transfection of RNA into SK-6 and SK-6 ΔDNAJC14 + Jiv90 cells. Note the strong antigen expression in SK-6 ΔDNAJC14 + Jiv90 and the induction of cytopathic effects. As described earlier, (63) the SK-6 ΔDNAJC14 and SK-6 ΔDNAJC14 + Jiv90 W39A were resistant to CSFV infection and showed no antigen expression.

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  FIG 6. DNAJC14-independent replication of APPV. Transfection of synthetic RNA of recAPPV-wt in SK-6 cells, SK-6 ΔDNAJC14 cells, and SK-6 ΔDNAJC14 cells with knock-in of active Jiv90 or inactive Jiv90 W39A. At 48 h postelectroporation, the cells were fixed, permeabilized, and probed with MAb N6 against APPV NS3. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm. APPV antigen was detectable in all cell lines after transfection of recAPPV-wt RNA. Note that no CPE is visible in SK-6 ΔDNAJC14 + Jiv90 cells.

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  FIG 7. APPV NS3 is not detectable in mock-transfected cells (control experiment). SK-6 cells, SK-6 ΔDNAJC14 cells, and SK-6 ΔDNAJC14 cells with knock-in of active Jiv90 or inactive Jiv90 W39A were fixed, permeabilized, and probed with MAb N6 against APPV NS3. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm.

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  FIG 8. The recAPPV-wt genome replicates in SK-6 ΔDNAJC14 cells with knock-in of active Jiv90 without visible CPE. SK-6 ΔDNAJC14 cells with knock-in of active Jiv90 were transfected with CSFV (strain Alfort-Tuebingen) or recAPPV-wt or mock infected. An aliquot of the cells was stained at 24 h posttransfection using MAb 8.12.7 (CSFV), MAb N6 (APPV), or MAb 8.12.7 and N6 (Mock) to verify the success of the transfection. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm. Another aliquot of the cells was passaged and documented in a bright-field image at day 10 posttransfection. Note that the cellular monolayer is completely destroyed in CSFV-infected SK-6 ΔDNAJ-C14 + Jiv90 cells, while no obvious changes are visible in recAPPV-wt transfections in comparison with the mock infection control.

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  FIG 9. Alignment of pestiviral NS2 sequences. The NS2 sequences of APPVs (strain AUT-2016_C and 000515), BVDV-1 (strain NCP7), CSFV (strain Alfort/Tuebingen), and HCV (strain BC) were aligned using the program CLC Workbench with default settings. The proposed active-site residues of the NS2 autoprotease C1280 and H1237 are highlighted in red. Amino acids are labeled with polarity colors to display homologies between the sequences. A consensus sequence is displayed using the “majority” setting and an X as symbol for ambiguous residues. Note the poor homologies and similarities between the sequences, which are almost exclusively restricted to the C-terminal protease domain.

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  FIG 10. The single-amino-acid changes C1280A and H1237A completely abolish the replication ability of recAPPV-wt, while APPV-Ubi* is not affected. SK-6 cells were transfected with synthetic RNA of recAPPV-wtC1280A and APPV-Ubi*C1280A (top rows) as well as recAPPV-wtH1237A and APPV-Ubi*H1237A (bottom rows) by electroporation. The cells were fixed, permeabilized, and probed with MAb N6 against APPV NS3 after an incubation of 48 h. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm. No antigen-positive cells appeared in the recAPPV-wtC1280A and recAPPV-wtH1237A images, while APPV-Ubi*C1280A and APPV-Ubi*H1237A were not affected by the mutation. The experiment indicates a functional relevance of these residues for NS2-3 cleavage and the importance of NS3 release for APPV.

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  FIG 11. APPV genomes expressing mature NS3 independently of the NS2 autoprotease. SK-6 cells were transfected with synthetic RNA of recAPPV-wt, APPV-DI, and APPV-Ubi* by electroporation. The cells were fixed, permeabilized, and probed with MAb N6 anti-APPV NS3 after an incubation of 48 h. A Cy3-labeled goat anti-mouse IgG was used for detection and a DAPI counterstain for nucleus localization. Bars, 100 μm. Very intense staining of dividing cells was observed for subgenome and the genome with duplicated NS3 sequences, while a weaker staining is visible in the wild-type genome. Note that no CPE is visible in antigen-positive cells transfected with APPV genomes expressing mature NS3 independently of NS2 autoprotease.

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  FIG 12. APPV-DI is not cytopathogenic. SK-6 cells were transfected with synthetic RNA of APPV-DI by electroporation. Aliquots of the transfected cells were fixed at 48 h posttransfection as well as after passages 1 and 2 of the cells. After permeabilization, the cells were probed with MAb N6 against APPV NS3 and a Cy3-labeled goat anti-mouse IgG. A DAPI counterstain was used for nucleus localization. Bars, 500 μm. A smaller magnification had to be chosen to show the formation of foci and to show their decreasing number. Note that cells transfected with the DI continue to divide and form multicellular foci.