Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1

Abstract

Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro. Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates. IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

Positive‐strand RNA viruses—a Keystone Symposia report

“…Previous work demonstrated that EV‐D68, which can cause acute flaccid paralysis, can infect neuronal cells. Similar to EV‐71, the infection efficiency of EV‐D68 is related to a mutation at site VP1‐271, which is involved in heparan sulfate binding 63 . Wolthers showed unpublished data modeling EV‐D68 infection in brain organoids.…”

“…Similar to EV-71, the infection efficiency of EV-D68 is related to a mutation at site VP1-271, which is involved in heparan sulfate binding. 63 Wolthers showed unpublished data modeling EV-D68 infection in brain organoids. Finally, Wolthers's group is using organoids to model parechovirus infection.…”

Enterovirus D: A Small but Versatile Species

“…In addition to ICAM-5, a contemporary EV-D68 strain, EV-D68-947, was found to engage with sulfated glycosaminoglycans (sGAGs) and generate a productive infection in SA-deficient cell lines [142]. Interestingly, the interaction with sGAGs enables EV-D68 to bypass a common pan-EV uncoating host factor, PLA2G16, (Figure 4) indicating that the receptors engaged by the different strains of the virus can result in alternative infection pathways [142][143][144]. However, this engagement could be a result of cell line adaptation and sGAG might not represent a true receptor in vivo [144].…”

“…Interestingly, the interaction with sGAGs enables EV-D68 to bypass a common pan-EV uncoating host factor, PLA2G16, (Figure 4) indicating that the receptors engaged by the different strains of the virus can result in alternative infection pathways [142][143][144]. However, this engagement could be a result of cell line adaptation and sGAG might not represent a true receptor in vivo [144].…”

MYCN in Neuroblastoma: “Old Wine into New Wineskins”

“…A recent study showed that in neuroblastoma the human endogenous retroviruses was abundant, indicating a possible role in the ontogenesis of the disease [ 38 ]. Interestingly, another recent report has shown that enterovirii responsible for other conditions, are implicated in the nerve tissue and possibly in tumorigenesis, including neuroblastoma [ 39 ]. The role of viral genome in neuroblastoma is still under investigation and consists of an active research field.…”