Multiple directed mutagenesis reduces enzymatic activity and antibody recognition of the African Swine Fever Virus E2 ubiquitin-conjugating protein (ASFV-pI215L)

Figures

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  Figure 1.. Sequence alignment of ASFV-pI215L with eukaryotic and viral ubiquitin-conjugating enzymes highlights conservation of UBC domain and suggests ASFV-pI215L evolutionary proximity to Eukarya. (A) Multiple sequence alignment of Ba71 V ASFV-pI215L with eukaryotic ubiquitin-conjugating proteins of high homology: E2R1 from H. sapiens (Cdc34), E2G2 from H. sapiens, S. scrofa, P. africanus and O. turicata. Clustal alignment: red for charged residues (negative and acidic D, E, positive and alkaline R, H, K), yellow for small residues (S, T, P, G, A), white for polar uncharged residues (N, Q), green for nonpolar aliphatic residues (C, V, I, L, M) and blue for nonpolar aromatic residues (F, Y, W). The black box indicates the ubiquitin-conjugating active site and black triangles indicate residues targeted for single-point mutagenesis. (B) Phylogenetic tree of a curated selection of ubiquitin-conjugating proteins: NCBI BLASTP search of proteins homologous to Ba71 V ASFV-pI215L, restricted to eukaryotic organisms and ASFV hosts (H. sapiens, D. melanogaster, S. cerevisiae, S. scrofa, P. africanus, O. turicata, in red), or non-ASFV viruses (all from Megaviricetes class, in blue); ASFV-pI215L…

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  Figure 2.. ASFV-pI215L shows high sequence conservation across genetically diverse ASFV isolates. (A) Phylogenetic tree of ASFV-pI215L sequences encoded by 222 ASFV genomes retrieved from the NCBI database, showing country, year of collection and, in parentheses, p72 genotype. The collapsed branch encompassing all genotype II sequences includes 116 entries from Asian and European origin, while the branch for Italy genotype I includes 60 entries. Branch length, measured in the number of substitutions per site, is indicated in the scale bar, and values next to nodes indicate bootstrap value, denoting cluster consistency (%). Accession numbers for each entry can be consulted in Supplementary Table S3. (B) Alignment of ASFV-pI215L sequences from the following ASFV isolates: highly virulent genotype I L60 isolate, low virulent genotype I NH/P68 isolate, avirulent genotype I Ba71 V and highly virulent genotype II Georgia 2007/1 isolate. Identical residues are shown in dark blue and the yellow box indicates ubiquitin-conjugating active site C85. Above the sequence alignment: black triangles indicate residues considered for single-point mutagenesis; black lines mark regions predicted by SVMTriP as…

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  Figure 3.. Western blotting detection of the ubiquitin-conjugating activity of ASFV-pI215L mutants showing functional impairment with single – and multi-residue substitutions in key positions of the ASFV-pI215L sequence. Graphical representation of band intensities of ubiquitin-linked protein (E1-ubq, pI215L-ubq), after incubation of ubiquitin-activating enzyme (E1), ubiquitin and ASFV-pI215L mutants, normalized to the band intensity from ASFV-pI215LWT. Band detection of ubiquitinated proteins can be consulted in Supplementary Figure S1. (A) 2-minutes ubiquitination of ASFV-pI215L single-residue mutants (R4E, R4S, R11A, R11E, S87D, S87R, E120R, E120S, S125D, S125R, D130R, D130S). (B) 1-hour ubiquitination of single-residue mutants. (C) 2-minutes ubiquitination of ASFV-pI215L multi-residue mutants (11A, 11B, 11C, 130A, 130B, 130C, 11 + 130A). (D) 1-hour ubiquitination of multi-residue mutants.

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  Figure 4.. ELISA assessment of ASFV-pI215L immunogenicity after incubation of full-length recombinant ASFV-pI215L with sera collected from ASFV-infected pigs, showing late detection of IgG, early detection of IgM, and decrease of IgG recognition with analysis of multiple-residue mutations. (A) Absorbance detection of total IgM or total IgG after ASFV-pI215LWT incubation with eight sera pools ranging from 0 to 35-72 dpi. (B) ASFV-pI215LWT and eight multi-point ASFV-pI215L mutants incubated with sera pool from 0 or 21-30 dpi, for detection of total IgG with antigen affinity to ASFV-pI215L.

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  Figure 5.. ELISA quantification of total IgG and IgM in sera from ASFV-infected pigs, grouped according to dpi, with affinity to the biotin-tagged ASFV-pI215L peptide library (25-residue length), for selection of ASFV-pI215L regions of interest highly recognized by ASFV-related immunoglobulins (marked with black arrows). (A) Detection of total IgG in each sera pool binding to each peptide, normalized to absorbance measurement with 0 dpi pool (n = 3). (B) Detection of total IgM in sera binding to each peptide, normalized to 0 dpi (n = 3). A graphical representation of the measured absorbance units for IgG and IgM detection, without normalization to 0 dpi, can be consulted in Supplementary Figure S2.

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  Figure 6.. ELISA quantification of immunoglobulin affinity of ASFV-infected sera to the PS-tagged ASFV-pI215L peptide library (9-residue length) suggests that the ASFV-pI215L 61–69 region, marked with a black arrow, contains a B-cell epitope recognized by ASFV-related IgG and IgM. (A) Absorbance measurement of total IgG after incubation of selected PS-tagged peptides with pools of sera from ASFV-infected pigs, grouped according to dpi (n = 3). (B) Absorbance measurement of total IgM after incubation of selected PS-tagged peptides with sera pools (n = 3). (C) Quantification of total IgG with affinity to PS-tagged peptide of the ASFV-pI215L 61–69 region, after incubation with individual sera from 0 and 13–30 dpi (n = 3) (D) Quantification of total IgM with affinity to the 61–69 region peptide, using individual sera from 0 and 7–16 dpi (n = 3).

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  Figure 7.. ELISA screening of immunoglobulin affinity of ASFV-infected sera to the PS-tagged peptide library containing ASFV-pI215L 61–69 wildtype (WT) and mutant sequences (A to X), with mutant peptides E and Q presenting a significant decrease in recognition by both IgG and IgM. (A) Quantification of total IgG after incubation of wildtype and mutant sequence peptides with pools of sera from ASFV-infected pigs, grouped according to dpi (n = 3). (B) Quantification of total IgM after incubation of wildtype and mutant sequence peptides with sera pools (n = 3). (C) Incubation of peptide of the ASFV-pI215L 61–69 region wildtype sequence (WT), or selected mutants E and Q, with individual sera from 0 and 10–30 dpi for quantification of total IgG with antigen affinity to peptide (n = 3) (D) ASFV-pI215L 61–69 wildtype (WT) or selected mutants E and Q incubated with individual sera from 0 to 20 dpi for quantification of total IgM (n = 3). A more comprehensive graphical representation of quantified IgG and IgM concentration after peptide incubation with individual sera, sorted by sera sample and grouped according to each infected animal, can be consulted in Supplementary Figure S3.

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  Figure 8.. Structure and function analysis of mutant ASFV-pI215L containing residue substitutions for suppressing epitope recognition and further impairing ubiquitin-conjugating activity, with placement of epitope mutation E showing severe impairment of ubiquitin-conjugating capacity and mutation Q resulting in loss of function. (A) Front and back view of a 3D representation of the protein surface of the ASFV-pI215L 1–189 residue region (RCSB PDB 7WLH), showing the 85-cysteine active centre in yellow, the 11–15 residue region targeted for functional mutations (11A, 11B, 11C) in green, the 130–134 residue region targeted for functional mutation (130A) in blue, residues 62 and 63 altered in epitope mutation E and mutation Q shown in pink, and the 66–69 residue region additionally altered in epitope mutation Q shown in red. (B) Graphical representation of the western blot band intensities of pI215L-ubq conjugates after 2-minutes or 1-hour ubiquitination of ASFV-pI215L epitope mutants (full-length protein containing the epitope mutation E or Q alone or combined with functional mutations 11A,11B, 11C or 130A), normalized to the band intensity from ASFV-pI215LWT. Band detection of ubiquitinated…