Functional Analyses of the Histone-like A104R Protein of African Swine Fever Virus and of a Homologous Pseudogene Product Found in Soft Tick Genomes

Figures

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  Figure 1. Schematic maps of A104R mutants of ASFV. (A) Map of the AK1033 genome with the inverted terminal repeats (IR), left and right variable regions (LVR and RVR), and the central conserved region (CCR). The A104R gene region is enlarged and the viral ORFs are indicated by arrows. Nucleotide (nt) positions refer to the published genome sequence [29] (GenBank# OZ005801). (B) Corresponding genome regions of the deletion and substitution mutants AKΔA104R-GFP, AKA104Rtick-GFP, including the ORFs of green fluorescent protein (GFP), a truncated version of the human cell surface protein CD4, the synthetic A104Rtick gene, ASFV promoters (Pp30, Pp72), and LoxP sites. Only the first 6 nt of the original A104R ORF were retained. (C) AKΔA104R-loxP was generated through Cre/Lox-mediated excision of the transgene insertion from AKΔA104R-GFP. Revertant virus A104RR was generated by reintroduction of the authentic A104R gene region into AKΔA104R-GFP via homologous recombination.

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  Figure 2. Amino acid sequence alignment of pA104R of indicated ASFV isolates and of the A104R-like pseudogene product of O. moubata (ASFLI-A104R). Non-conserved residues are highlighted in red. Arginine-69, which is part of the DNA-binding domain, is conserved in all ASFV isolates (highlighted in green), including the tick derived ASFLI-A104R. The frameshift-repaired part of ASFLI-A104R is highlighted in blue. Asterisks (*) indicate predicted proteins which are N-terminally elongated by 23 additional amino acids. GenBank accession numbers of the compared sequences are indicated on the left.

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  Figure 3. Genome and protein analyses of A104R deletion and substitution mutants of AK1033. (A) PCR products of the A104R gene region of WT ASFV and indicated virus mutants were separated on a 0.7% agarose gel. Fragment sizes (kbp) of a DNA ladder (Thermo Fisher Scientific) and calculated PCR product sizes (bp) are shown on the left and right, respectively. (B) Western blot analysis of non-infected WSL cells and cells infected with the AK1033 variants (MOI = 2, 2 d p.i.). Proteins were separated by SDS-PAGE and probed with antibodies against ASFV pA104R, p30, and p72 or the host cell protein α-tubulin. Molecular masses of marker proteins (Thermo Fisher Scientific) are indicated on the left.

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  Figure 4. In vitro growth of A104R deletion and substitution mutants of AK1033 is moderately affected in WSL cells but severely impaired in porcine PBMCs. (A) WSL cells or (B) GM-CSF differentiated and activated porcine PBMCs were infected with WT and mutant viruses at an MOI of 0.03 and harvested at the indicated times p.i. Progeny virus titers (PFU/mL) were determined through plaque assays on WSL cells. The mean results of 4 (WSL cells) and 2 (PBMC) experiments, each, and standard deviations are shown. Significant differences (one-way ANOVA, Dunnett’s multiple comparisons test) to WT titers at 72 and 120 h are indicated by correspondingly colored asterisks above the growth curves of AKΔA104R-GFP, AKΔA104R-loxP, and AKA104Rtick-GFP. *** p < 0.001 and **** p < 0.0001. No significant differences were observed between wild-type and revertant viruses and between the three deletion/substitution mutants, respectively.

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  Figure 5. Deletion or substitution of A104R leads to reduced plaque sizes. (A) WSL cells infected with WT and mutant ASFV were incubated under semi-solid medium and fixed after 96 h. Virus plaques were visualized through IFA staining of the ASFV proteins p72 and pA104R (scale bars = 0.5 mm). (B) Sizes of ≥50 plaques per virus and experiment were determined via IFA staining of p72. Shown are the mean relative percentage area normalized to WT virus plaques in four independent experiments. Error bars represent standard deviations (n = 4). Significant differences (one-way ANOVA, Tukey’s multiple comparisons test) to WT plaques are indicated above the bars. * p < 0.05, ** p < 0.01, and **** p < 0.0001. No significant plaque size differences were observed between wild-type and revertant viruses and between the three deletion/substitution mutants, respectively.

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  Figure 6. Viral DNA replication kinetics of wild-type ASFV and A104R mutants are similar. WSL cells were infected with the indicated viruses at an MOI of 3, and total DNA was prepared from samples harvested (0, 2, 4, 8, 16 and 32 h p.i). ASFV DNA was quantified through duplex qPCR detection of the viral B646L gene normalized to the host cell β-actin gene. Viral genome copies were calculated using serial dilutions of plasmid-cloned B646L. Error bars represent standard deviation (n = 2).

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  Figure 7. Viral early CP204L (p30) and late B646L (p72) gene transcription of A104R-deleted ASFV is not impaired. WSL cells were infected at an MOI of 3 with the investigated ASFV variants, and samples were harvested at the indicated times post-infection. Total RNA was extracted, and relative amounts of (A) CP204L (p30) and (B) B646L (p72) transcripts were determined through RT-qPCR and normalized to host cell β-actin mRNA. Error bars represent standard deviation (n = 2).

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  Figure 8. DNA-binding activity of pA104R WT and pA104Rtick. Clarified cell lysates were prepared 2 d after transfection of RK13 cells with expression plasmids for ASFV (WT) or ASFLI tick genome element-derived pA104R, or mock transfection, and incubated with or without (Ø) biotinylated double-stranded oligonucleotides (DBtest-1F/R, -2F/R) in streptavidin-coated test strips. After washing, bound pA104R was quantified using a colorimetric antibody binding assay. The measured absorptions (A450 nm) were normalized to those of the respective negative controls (Ø), which were set to one. Shown are the mean results of three experiments with standard deviations.

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  Figure 9. pA104R exhibits complex interactions with other viral and host cell proteins. Network of host and virus proteins interacting with pA104R in ASFV-infected WSL cells. Each node represents a protein (circles, host proteins; diamonds, ASFV proteins). Proteins are clustered by the enriched GO terms indicated above the blue-green boxes and grouped into protein complexes according to the EBI Complex Portal, as indicated by the yellow highlights. Each edge is colored according to the type of treatment (blue, without benzonase; red, with benzonase). Edge thickness is proportional to the log10 iBAQ value. Physical interactions among host proteins were curated from the STRING database.