Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection - PubMed (original) (raw)
Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection
Samuel Cordey et al. PLoS Pathog. 2012.
Abstract
Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP1₉₇ Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Replication efficiency of the lower respiratory tract (BAL) and stool isolates in Vero versus SH-SY5Y cells.
Vero cells (A–B) and SH-SY5Y cells (C–D) were infected with equivalent concentrations of BAL and stool isolates and replication was assessed by immunofluorescence with anti-EV71 monoclonal antibody.
Figure 2. Schematic representation of the infectious clones derived from the stool and BAL isolates and their derivatives and assessment of their replication efficiency in Vero versus SH-SY5Y cells.
(A) Representation of the infectious clones. (B–C) Vero cells (I–IV) and SH-SY5Y cells (V–VIII) were infected with infectious clones derived from the clinical isolates. (B) Replication was assessed by immunofluorescence with an anti-EV71 monoclonal antibody. The cell type and residue at positions VP197 and 2B38 are indicated on the left and bottom of the figure, respectively. The TCID50/ml performed after 5 days on the SH-SY5Y infected cell supernatant is indicated with a bar on the left side of each panel. (C) Increase in viral RNA load as quantified by real-time RT-PCR at different time points post infection with the four derivatives. Vertical bars indicate minimum/maximum values. Key for (A) +, positive charge, − negative charge, o neutral; * sequence corresponding to that of the BAL samples, ** sequence corresponding to that of the stool samples.
Figure 3. Competition between the stool and BAL infectious clone derivatives.
Vero cells, Caco-2 cells (A), and SH-SY5Y cells (B) were transfected by equimolar amounts of the stool and BAL infectious clone derivatives and virus present in the cell supernatant was analysed by sequencing post-transfection and repassage at different times. Substitutions are marked by red arrows and correspond to nucleotide (nt) and amino acid (aa) positions of the EV71 VP1 coding sequence (GenBank accession number: AAB39968.1).
Figure 4. EV71 VP1 substitution locations relative to known receptors and capsid symmetry.
(A) EV71 VP1 model highlighting the BC loop (green) and positions of VP197R (red circle) and VP1167E (orange circle) relative to known receptors (gray). Eight known picornavirus VP1-receptor complexes (PDB codes along their sides) were structurally aligned to our model using the VP1 coordinates in each structure file. The distance (∼12 Å) between EV71 VP1 residue 97 and receptor surfaces is marked by vertical black dotted lines (distance between VP1 residue 97 and the 3dpr receptor, also ∼12 Å, is not marked). (B) Five EV71 VP197R model monomers arranged in capsid symmetry based on poliovirus capsid VP1 orientations (PDB 3epf). BC loops (green) and positions of residue 97 (red circles) and residue 167 (orange circles) are highlighted. (C) Side view of VP197R capsid assembly in B, rotated 80° on the plane of this page. The curvature and thickness of the capsid surface (based on PDB 3epf capsid assembly, VIPERdb) is represented as a light gray arc. (D) Sequence alignment of VP1 clinical isolates, EV71 substrain BrCr (Genbank U22521), and polivirus (PV1 (Genbank V01149), PV2 (M12197), PV3 (K01392)) surrounding EV71 VP197 and VP1167 substitutions. Index numbers refer to EV71 VP1 residue positions.
Figure 5. EV71 binding assay.
SH-SY5Y cells (A) and Vero cells (B) were used to compare the cell-binding capacity of pCIVP197R2B38A and pCIVP197L2B38V. Two conditions were assessed: undiluted (1∶1) and 2-fold diluted (1∶2) standardized viral stocks. Quantification of bound virus was measured by the Entero/Ge/08 real-time RT-PCR assay and expressed relative to pCIVP197R2B38A (1∶1 condition). Vertical bars indicate minimum/maximum values.
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