Attenuation hotspots in neurotropic human astroviruses

Figures

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  Fig 1. Classification and attenuation of MLB astroviruses. (A) Simplified phylogenetic tree for the Astrovirus genus. The tree is based on full nucleotide sequences available for indicated species. The pictogram of the intestine or neuron indicates the tropism associated with astrovirus strains. Some astrovirus genotypes labeled with the neuron icon are associated with neuropathology. Neurotropic MLB strains are shown in red. (B) The evolution experiment was performed for MLB1 and MLB2 astroviruses. (C) The coevolution experiment was performed for MLB1 and MLB2 astroviruses. (D) Nucleotide and amino acid sequences of MLB1 and MLB2 viruses containing deletions identified in evolved MLB virus stocks.

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  Fig 2. Purification of CPNTD and immunodetection of MLB1- and MLB2-infected Huh7.5.1 cells and iPSC-derived neurons. (A) Schematic representation of MLB1 genome and location of CPNTD. Lower panel represents the sequence of recombinant CPNTD. ORF, open reading frame; CP, capsid polyprotein; NTD, N-terminal domain. (B) Coomassie-stained SDS-PAGE profile of the purified CPNTD from E. Coli. (C) Huh7.5.1 cells were infected with MLB1 and MLB2 viruses at MOI 0.1 and incubated for 48 h. CP was detected using the antibody generated against CPNTD. Purified CPNTD was used for detection limit assessment (1–9 ng). (D) Experimental setup to determine the CPE and titration for MLB1 and MLB2 infection. (E) Huh7.5.1 cells were infected at an MOI 1 and incubated for indicated periods, then stained and imaged. Hoechst-stained nuclei were counted from 12 images (approximately 200 cells per image) and normalized to mock-infected samples. Data are mean ± SD. ***p < 0.001, ****p < 0.0001 using two-way ANOVA test against mock. (F) Huh7.5.1 cells were seeded on 96-well plate and infected with 10-fold serial dilutions of MLB1 and MLB2 astroviruses, fixed at 20 hpi, permeabilized, stained with anti-CP antibody,…

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  Fig 3. Generation and validation of RG system for MLB1 and MLB2 astroviruses. (A, B) Schematic representation of MLB1 (A) and MLB2 (B) genomes used to generate infectious clones. MLB genome elements: ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; CP, capsid polyprotein; FS, frameshift site; SG, subgenomic promoter. (C) Strategy for a plasmid-derived RG system for MLB1 and MLB2. MLB cDNAs contain the entire genome flanked by the T7 promoter and XhoI linearization site. Huh7.5.1 cells were electroporated with full-genome T7 transcripts, the collected virus was used for serial passages in the same cell line. (D, E) Multistep growth curves of MLB1 (D) and MLB2 (E) on Huh7.5.1 cells (the second passage). Cells were infected at an MOI 0.1, and virus titer was measured from the intracellular and extracellular fractions in triplicates. Data are mean ± SD. (F) Cells were infected at an MOI 0.1, harvested at 48 hpi, and analyzed by western blotting with anti-CP and anti-tubulin antibodies. (G) Huh7.5.1 cells were infected with MLB1 and MLB2 at an MOI 0.1 and incubated for 72 h. The cell- and media-derived samples were harvested and analyzed by western blotting. (H, I) Huh7.5.1 cells…

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  Fig 4. Analysis of C-terminal part of MLB1 and MLB2 astrovirus CP. (A) Analysis of publicly available sequences of C-terminal part of MLB2 CP. (B) Analysis of publicly available sequences of C-terminal part of MLB1 CP. The deletion region is indicated on top of MLB1 and MLB2 alignments (A, B). (C) Analysis of the C-terminal part of MLB1 and MLB2 CP for putative caspase cleavage sites using Procleave software [33]. The sequences in bold indicate caspase 1, 3, and 6 putative cleavage sites with a probability score of >0.7. The regions of MLB1 (top) and MLB2 (bottom) deletions are shown. (D) Huh7.5.1 cells were infected with indicated recombinant viruses of MLB1 (left) and MLB2 (right) at an MOI 0.5 in triplicates, the virus was collected at indicated times post infection, and titer was measured for both extracellular and intracellular fractions. Data are mean ± SD. (E) Analysis of CP expression in Huh7.5.1 cells infected with second passage of MLB1 and MLB2 wt and mutant viruses (MOI 0.1). Cell lysates were harvested at 48 hpi and analyzed by western blotting with anti-CP and anti-tubulin antibodies. GNN is RdRp knock-out recombinant virus (GDD to GNN). (F) The effect of pan-caspase…

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  Fig 5. RNA replication properties in truncated MLB replicons and viruses. (A) The prediction of the RNA secondary structure and location of identified deletions in MLB1 3′ UTR. (B) Schematic of the MLB1 and MLB2 astrovirus replicons. The 2A-RLuc cassette is fused in the ORF2 followed by the stop codon and extended 3′ UTR. (C) Huh7.5.1 cells were transfected with MLB1 and MLB2 replicons expressing mCherry, incubated for 24 h, fixed and imaged, nuclei were counterstained with Hoechst (blue). Scale bars are 100 μm. (D) Relative MLB1 and MLB2 replicon luciferase activities were measured after RNA transfection of Huh7.5.1 or HEK293T cells. Values are normalized so that the mean wt replicon value at each time point is 100%. The replication fold difference between wt and GNN mutant replicon is provided for the final time point. (E) Relative MLB1 and MLB2 replicon luciferase activities were measured after RNA transfection of Huh7.5.1 or HEK293T cells. The resulting replicon activities were normalized to wt replicon values at each time point. (F, G) Extracellular and intracellular RNA levels were measured for MLB1 (F) and MLB2 (G) infections. Huh7.5.1 cells were infected with indicated…

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  Fig 6. Infection of iPSC-derived i3Neurons with MLB astroviruses. (A) The schematic representation of the experiment where partially differentiated i3Neurons were seeded on IBIDI (imaging) or 12-well (other analyses) plates, differentiated into mature i3Neurons, infected with indicated MLB1 and MLB2 recombinant viruses at MOI 0.5. (B, C) iPSC-derived neurons were infected with MLB2 (B) and MLB1 (C). Representative confocal images of fixed and permeabilized cells visualized for MLB CP (green) and MAP2 (magenta). Nuclei were stained with Hoechst (blue). Scale bars are 50 μm. (D) At least 15 images (150–250 cells per image) were analyzed for CP-positive cells. ***p < 0.001, ****p < 0.0001, ns nonsignificant, using two-tailed Mann–Whitney test against wt virus infection. (E) iPSC-derived neurons were infected with MLB viruses in quadruplicate at MOI 0.5 and passaged in neurons using 1/100th volume of the previous passage. Each virus was titrated on Huh7.5.1 cells. *p < 0.05, ****p < 0.0001, using two-way ANOVA test against wt infection. (F) Representative confocal images of fixed and permeabilized infected i3Neurons were taken, scale bars are 25 μm. (G) Intracellular RNA levels were…