O’nyong-nyong virus adaptive mutations in non-structural protein 1 and 3 enhance RNA replication and overcome FHL1 requirement

Figures

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  Figure 1.. Lunet cells are non-permissive for ONNV infection. (A) Schematic of ONNV (RRC013 SG mCherry), CHIKV (37997 E2 mCherry), and VEEV (TC-83 SG GFP) infectious clones with fluorescent marker (FM). (B) Growth kinetics of ONNV, CHIKV, and VEEV in Lunet and HEK293 cells (MOI 0.1, n = 2, detection limit 1.78 × 102 TCID50/ml indicated as dotted line). (C) Fluorescence microscopy of transfected ONNV, CHIKV, and VEEV infectious clones in Lunet and HEK293 cells at 24 h post transfection. (D,E) Fluorescence microscopy of ONNV, CHIKV, and mock-infected Lunet and HEK293 cells (MOI 0.1) at 24 h post infection. Cell nuclei stained with Hoechst, and virus infection visualized by (C) virus-encoded fluorescent marker (GFP recoloured), (D) anti-nsP1, and (E) anti-dsRNA immunofluorescent staining.

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  Figure 2.. The absence of host dependency factors hinders ONNV infection in Lunet cells. (A) Schematic overview of the PEG1500-mediated cell fusion of HEK293-Tet-On and Lunet-Tre-ZsGreen co-cultures, and subsequent ONNV infection. (B) Fluorescence microscopy of ONNV-infected Lunet and HEK293 co-cultures and heterokaryons (MOI 0.1) at 24 h post infection. Cell nuclei stained with Hoechst, heterokaryon visualized by ZsGreen, and virus infection by mCherry. (C) The percentage of infected heterokaryons/total heterokaryons and (D) total number of infected cells, upon ONNV and CHIKV infection of Lunet-HEK293 heterokaryons in co-cultures (MOI 0.1 or 1, 24 h post infection). (E) The total number of ONNV and CHIKV infected cells in HEK293 monocultures (MOI 0.1 or 1, 24 h post infection). Bars represent the mean and standard deviation of three independent experiments with each six biological replicates. Ratios and significance between groups indicated above graph (two-way ANOVA, ****P < 0.0001). (F) Ratios of CHIKV:ONNV infected cells in the infected heterokaryons, Lunet and HEK293 co-cultures, and HEK293 mono-cultures. Bars represent the mean and standard deviation of three independent experiments…

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  Figure 3.. ONNV-CHIKV chimaeras and laboratory ONNV strains replicate in Lunet cells. (A) Schematic of ONNV, CHIKV, and ONNV-CHIKV chimaeras (asterix indicates opal stop codon, triangle indicates subgenomic promoter). (B) Growth kinetics of ONNV, CHIKV, and chimaeras in Lunet and HEK293 cells (MOI 0.1, n = 2, detection limit 1.78 × 102 TCID50/ml indicated as dotted line). (C) Growth kinetics of multiple ONNV strains in Lunet, HEK293 and HEK293 ΔFHL1 cells (MOI 0.1, n = 2, detection limit 1.78 × 102 TCID50/ml indicated as dotted line). (D,E) Multiple sequence alignment of Illumina-sequenced ONNV strains with difference to the ONNV RRC013 genome highlighted for (D) the non-structural genes and (E) structural genes.

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  Figure 4.. Serial passaging of ONNV in mammalian and Aedes cells results in Lunet cell adaptation. (A) Schematic representation of the serial passaging assay with passaging of ONNV on cells and verification of adaptive mutants by virus-encoded mCherry expression in Lunet cells. After five passages, mutants were isolated from Lunet cells. (B) Detection of ONNV mutants that express mCherry in Lunet cells after serial passaging in monocultures of HEK293, Vero, Lunet, C6/36 or MSQ43 cells, co-culture of HEK293 + Lunet cells or Vero + Lunet cells, or alternating passaging between MSQ43 and HEK293 cells. Figure represents a triplicate experiment with an initial MOI of 0.1. (C) Growth kinetics of isolated ONNV mutants in Lunet cells with indication of the passaging cell line (MOI 0.1, n = 2, detection limit 1.78 × 102 TCID50/ml indicated as dotted line).

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  Figure 5.. Single ONNV nsP1 and nsP3 mutations result in infection of Lunet and A549 cells. (A) Missense mutations identified by Illumina RNA sequencing of three Lunet-adapted ONNV mutants: sequence 1 was derived from ONNV isolate passaged in HEK293 monoculture, sequence 2 from HEK293 + Lunet co-culture, and sequence 3 from Vero monoculture. (B) Verification of nsP mutations in the 18 isolated ONNV mutants by Sanger sequencing (nsP1 314 (17xM, 1xV), nsP1 386 (11x I, 6xT, 1xL), nsP3 117 (15xG, 3xR), nsP3 563 (14x*, 2xR, 2xL), with asterisks referring to the opal stop codon). (C) Schematic representation of the site-directed mutagenesis generated ONNV mutants. (D) Growth kinetics of ONNV mutants in Lunet (human hepatoma), HEK293 (human embryonic kidney), HEK293 ΔFHL1, human fibroblasts, C6/36 (Aedes albopictus), MSQ43 (Anopheles stephensi), and A549 (human lung epithelial) cells (MOI 0.1, n = 2, detection limit 1.78 × 102 TCID50/ml indicated as dotted line).

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  Figure 6.. ONNV nsP mutations enhance RNA replication. (A,B) Fluorescence microscopy of ONNV WT and mutants in (A) HEK293 and (B) Lunet cells (MOI 0.1). Cell nuclei stained with Hoechst, and virus infection visualized by anti-dsRNA immunofluorescent staining. (C,D) Negative- and positive-strand RNA quantification in (C) HEK293 and (D) Lunet cells upon ONNV WT and mutant infections (MOI 0.1) based on strand specific qRT-PCR. RNA levels were normalized to GAPDH and presented relative to ONNV positive-strand RNA at 4 hpi. Bars represent the mean and standard deviation (n = 4, two-way ANOVA, ****P < 0.0001, ***P < 0.0005, *P < 0.05). (E,F) Flow cytometry quantification of subgenomic encoded mCherry expression following transfection of ONNV WT, ONNV mutants, and a replication deficient ONNV (ONNV nsP4 GAA, negative control) infectious clone. (E) Mean fluorescence intensity (MFI) in HEK293 at 12hpt and (F) Lunet cells at 24hpt. Bars represent the mean and standard deviation (n = 3). Asterisks indicate statistically significant differences compared with ONNV WT (one-way ANOVA, ****P < 0.0001, ***P < 0.0005, **P < 0.001, and *P < 0.05). (G) Growth kinetics of ONNV infection in Lunet cells at MOI…