A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates <i>ex vivo</i> tropism

<div><p>Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP1_97L or VP1_97R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient’s respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus’s dissemination.</p></div

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates <i>ex vivo</i> tropism - Fig 4

<p><b>Replication of EV71-VP1</b>_{<b>97R167G</b>}<b>, EV71-VP1</b>_{<b>97L167E</b>}<b>and EV71-VP1</b>_{<b>97R/L167G/E</b>}<b>in reconstituted airway epithelia developed from the upper (A) and lower (B) respiratory tract and HS/SCARB2 expression in the same tissues (C -D)</b>. <b>(A-B)</b> Viral loads were measured by RT-qPCR 5 dpi in the apically collected washes of the apically or basally inoculated tissues and are expressed as the mean ±SEM (N≥2). ***P<0.001, **P<0.01, *P<0.05. Frequency plots are shown on the right (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007190#ppat.1007190.g003" target="_blank">Fig 3</a> for details) <b>(C-D)</b> Tissue sections were colored with hematoxylin and eosin (upper panel) or labeled by immunofluorescence. SCARB2 is stained in green, HS in red and cell nuclei in blue. Scale bar = 20 μm.</p

Differential cell tropism of EV71-VP1_97R167G and EV71-VP1_97L167E is linked to HS binding and correlates with its expression level at the cell surface.

<p><b>(A).</b> Binding of EV71-VP1_97R167G (left panel) and EV71-VP1_97L167E (right panel) in the presence or absence of HS. Binding <b>(B)</b> and replication <b>(C)</b> of a viral stock composed of equivalent amounts of EV71-VP1_97R167G and EV71-VP1_97L167E in different cell lines and under conditions preventing attachment to HS. Viral loads were measured by RT-qPCR (real-time quantitative reverse transcription PCR) in whole cell extracts for binding assay and both cell extracts and supernatants for replication assay, and expressed as the mean (±SEM, N≥2) relative to the EV71-VP1_97R167G load in non-treated RD cells (100%). <b>(D).</b> Quantification of HS-expressing (positive) cells was achieved through an analysis using MetaMorph software (left panel) based on the immunofluorescence labeling (right panel) of HS attachment receptors at the surface of Vero, RD, SH-SY5Y cells and Caco-2 cells. More than 500 cells were included in the analysis. Scale bar = 20 μm. <b>(E)</b> Binding of EV71-VP1_97R/L167G/E to heparin sepharose beads. Viral population present in the input, flow-through and elute population was characterized by sequencing. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. For B, C and E, the frequency plot [<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>] of codons encoding VP1 aa 97, 98 and 167 are shown and are representative of biological replicates (N≥2). Arginine (R) at position 97 is encoded by CGC, Leucine (L) by CTC; Glutamate (E) at position 98 is encoded by GAA, Lysine (K) by AAA; Glycine (G) at position 167 is encoded by GGG, Glutamate (E) by CTC.</p

SCARB2 is necessary for effective infection by EV71-VP1_97R167G and EV71-VP1_97L167E.

<p><b>A)</b> Mean (N = 2) viral titers (±SEM) expressed as log TCID₅₀/ml (median tissue culture infective dose) obtained 5 dpi in cells expressing (control) or not (ΔSCARB2) SCARB2. Wells with less than 50 infected cells were considered as negative. <b>B)</b> Western blot showing SCARB2 expression in ΔSCARB2 cells and control cells. ****P<0.0001, **P<0.01, *P<0.05.</p

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates <i>ex vivo</i> tropism - Fig 5

<p><b>Replication of EV71-VP1</b>_{<b>97R167G</b>}<b>, EV71-VP1</b>_{<b>97L167E</b>}<b>and EV71-VP1</b>_{<b>97R/L167G/E</b>}<b>in reconstituted small intestine tissues (A) and HS/SCARB2 expression in the same tissues (B and C)</b>. <b>(A)</b> Viral loads were measured by RT-qPCR 5 dpi in the apically collected washes of the apically or basally inoculated tissues and are expressed as the mean (±SEM) N = 2. **P<0.01, *P<0.05. The frequency plots are shown on the right (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007190#ppat.1007190.g003" target="_blank">Fig 3</a> for details) <b>(B)</b> Tissue sections were colored with hematoxylin and eosin (upper panel) or labeled by immunofluorescence. SCARB2 is stained in green, HS in red and cell nuclei in blue. Scale bar = 20 μm. <b>(C)</b> Quantification of HS expression at the apical and basal surface of intestinal tissues was achieved through an analysis using MetaMorph software based on the immunofluorescence labeling of HS attachment receptor in sectioned tissues.</p

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature

<div><p>Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. <i>In vitro</i> differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential <i>in vivo</i> replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world’s most prevalent pathogens and could aid target selection for vaccine or antiviral development.</p></div

Differential cell tropism EV71-VP1_97R167G and EV71-VP1_97L167E.

<p>The mean (N = 2) viral titers (±SEM) expressed as log TCID₅₀/ml (median tissue culture infective dose) obtained 4 dpi are indicated. *P<0.05.</p

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates <i>ex vivo</i> tropism - Fig 7

<p><b>Viral infectivity of EV71-VP1</b>_{<b>97R167G</b>}<b>and EV71-VP1</b>_{<b>97L167E</b>}<b>in Vero cells (A) and of EV71-VP1</b>_{<b>97R167G</b>}<b>, EV71-VP1</b>_{<b>97L167E</b>}<b>and EV71-VP1</b>_{<b>97L/R167E/G</b>}<b>in neural cells (B) in the presence of increasing doses of ı-carrageenan.</b> The percent of infection was measured by an immunocytochemistry assay in Vero cells (A) and by RT-qPCR on cell lysate 1 dpi in neural cells (B) and expressed as the mean (±SEM) compared to non-treated viruses. (N = 2). **P<0.005. The frequency plots are shown on the right (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007190#ppat.1007190.g003" target="_blank">Fig 3</a> for details).</p

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates <i>ex vivo</i> tropism - Fig 6

<p><b>Replication of EV71-VP1</b>_{<b>97R167G</b>}<b>, EV71-VP1</b>_{<b>97L167E</b>}<b>and EV71-VP197</b>_{<b>R/L167G/E</b>}<b>in 2D (A) and 3D (B) neural tissues and HS/SCARB2 expression in the same 2D tissues (C). (A-B)</b> Viral load was measured by RT-qPCR and expressed as the mean (±SEM). (N = 2). ****P<0.0001, ⁺⁺P<0.01 or ^#P<0.05. (⁺relative to EV71-VP1_97L167E and ^# relative to EV71-VP1_97R/L167G/E at the same time point). The frequency plots are shown on the right (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007190#ppat.1007190.g003" target="_blank">Fig 3</a> for details) <b>(C)</b> 2D neural cultures were labeled by immunofluorescence. SCARB2 is stained in green, HS in red and cell nuclei in blue. Scale bar = 20 μm.</p

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature - Fig 1

<p><b>A.</b> Schematic representation of the artificially engineered chimeric EV-D68/EV-D94 viruses. Construct names are indicated on the left. EV-D68 regions are represented by white boxes; EV-D94 regions are in grey. Results obtained upon transfection and passage in HeLa cells are indicated on the right, as is the percentage of nucleotide and amino acid sequence identity between the exchanged regions. V, viable construct; X, non-viable construct; V/X, viable unfit construct. <b>B.</b> Non-synonymous adaptation mutations observed in viral stocks after 6 passages in HeLa cells. Sequencing was performed from nt 45 to 7344. Production of viral stocks by transfection at both 33°C and 37°C was attempted for all viruses, but only EV-D94 could be recovered at 37°C. For EV-D94, the stock subsequently used for phenotypic assessment is the one prepared at 33°C. HeLa 33 and HeLa 37: viral stocks transfected and amplified at 33°C and 37°C respectively.</p