Attenuation hotspots in neurotropic human astroviruses

Abstract

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

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.

Fig 2. Purification of CP_NTD and immunodetection of MLB1- and MLB2-infected Huh7.5.1 cells and iPSC-derived neurons.

(A) Schematic representation of MLB1 genome and location of CP_NTD. Lower panel represents the sequence of recombinant CP_NTD. ORF, open reading frame; CP, capsid polyprotein; NTD, N-terminal domain. (B) Coomassie-stained SDS-PAGE profile of the purified CP_NTD 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 CP_NTD. Purified CP_NTD 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, and imaged by LI-COR. (G) Huh7.5.1 cells were infected with MLB1 and MLB2 viruses and incubated for 24 h. Representative confocal images of fixed and permeabilized cells visualized for CP (green) and stained for nuclei (Hoechst, blue) are shown. Scale bars are 10 μm. (H) Huh7.5.1 cells were infected with MLB1 and MLB2 virus stocks at MOI 0.1 and incubated for 72–120 h. Virus titers were determined from 6 independent experiments. Data are mean ± SD. (I) Partially differentiated i³Neurons were seeded on IBIDI plates, differentiated into mature glutamatergic neurons, infected with MLB1 and MLB2 viruses, and incubated for 48 (MLB2) or 96 (MLB1) h. Representative confocal images of fixed and permeabilized cells visualized for MLB CP (green), neuronal marker MAP2 (magenta) and stained for nuclei (Hoechst, blue) are shown. Scale bars are 50 μm. All uncropped images can be found in the Supporting information file as S1 Raw Images. All individual quantitative observations that underlie the data can be found in S1 Data file. CPE, cytopathic effect; hpi, hours post infection; MOI, multiplicity of infection.

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 were infected in triplicates with MLB1 (H) and MLB2 (I) at an MOI 0.1 and incubated for 72–120 h until full CPE. Total virus titers of 10 serial passages were determined in triplicates (n = 2 independent experiments). Data are mean ± SD. (J, K) Analysis of CP expression in Huh7.5.1 cells infected with 2–10 passage of MLB1 (J) and MLB2 (K). Cells were infected at an MOI 0.1, harvested at 48 hpi, and analyzed by western blotting. All uncropped images can be found in the Supporting information file as S1 Raw Images. All individual quantitative observations that underlie the data can be found in S1 Data file. CPE, cytopathic effect; hpi, hours post infection; MOI, multiplicity of infection; RG, reverse genetics.

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 inhibitor z-VAD-fmk on CP processing during infection with classical human astrovirus 4 (HAstV4), MLB1 and MLB2 astroviruses. Caco2 cells were infected with HAstV4 and Huh7.5.1 cells were infected with MLB1 and MLB2 astroviruses at MOI 1 in the presence or absence of z-VAD-fmk. Cell lysates were harvested at indicated hpi and analyzed by western blotting with 8E7 antibody against HAstV CP (for HAstV4), anti-CP (for MLB), and anti-tubulin antibodies. The average inhibition of CP cleavage was quantified from 3 independent experiments. (G) The schematic representation of the virus competition experiment where Huh7.5.1 cells were coinfected with wt and mutant viruses and passaged. Virus RNA was isolated and used for RT-PCR to detect virus-specific fragments. (H) The RT-PCR fragments from the competition experiment were analyzed by agarose electrophoresis. The fragments corresponding to the expected size of each PCR product are shown on the right. All uncropped images can be found in the Supporting information file as S1 Raw Images. All individual quantitative observations that underlie the data can be found in S1 Data file. CP, capsid polyprotein; hpi, hours post infection; MOI, multiplicity of infection; wt, wild-type.

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 recombinant viruses of MLB1 and MLB2 at an MOI 0.5 in triplicate, the virus was collected at indicated times post infection, and RNA levels were measured for both extracellular and intracellular fractions. These samples were collected in parallel with those shown in Fig 4D to match the observations. Data are mean ± SEM (n = 3, ≥3 independent experiments, graphs D–G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using two-way ANOVA test against wt replicon/infection (E–G). All individual quantitative observations that underlie the data can be found in S1 Data file. MOI, multiplicity of infection; ORF, open reading frame; UTR, untranslated region; wt, wild-type.

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 quantified using qPCR. The virus-specific signal was normalized to GAPDH RNA and calculated as fold increase to the input RNA levels. *p < 0.05, ***p < 0.001, using one-way ANOVA test against wt infection (MLB1) and **p < 0.01, using Student’s t test (MLB2). Data are mean ± SD (D, E, G). (H) Schematic representation of MLB genome organization. MLB genome elements: ORF, open reading frame; NTD, N-terminal domain; TM, transmembrane domain; Pro, protease; VPg, virus protein genome-linked; HVR, hypervariable region; RdRp, RNA-dependent RNA polymerase; XP, X protein; FS, frameshift site; SG, subgenomic promoter. All individual quantitative observations that underlie the data can be found in S1 Data file. CP, capsid polyprotein; iPSC, induced pluripotent stem cell; MOI, multiplicity of infection; wt, wild-type.