Encephalomyocarditis virus may use different pathways to initiate infection of primary human cardiomyocytes

Abstract

Encephalomyocarditis virus (EMCV) can infect a wide range of vertebrate species including swine and non-human primates, but few data are available for humans. We therefore wanted to gain further insight into the mechanisms involved in EMCV infection of human cells. For this purpose, we analyzed the permissiveness of primary human cardiomyocytes towards two strains of EMCV; a pig myocardial strain (B279/95) and a rat strain (1086C). In this study, we show that both strains productively infect primary human cardiomyocytes and induce complete cytolysis. Binding and infection inhibition experiments indicated that attachment and infection are independent of sialic acid and heparan sulfate for B279/95 and dependent for 1086C. Sequence comparison between the two strains and three-dimensional analysis of the capsid revealed that six of the seven variable residues are surfaceexposed, suggesting a role for these amino acids in binding. Moreover, analysis of variants isolated from the 1086C strain revealed the importance of lysine 231 of VP1 in the attachment of EMCV to cell-surface sialic acid residues. Together, these results show a potential for EMCV strains to use at least two different binding possibilities to initiate infection and provide new insights into the mechanisms involved in primary human cell recognition by EMCV.

A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection

“…Our data support that ADAM9 is a crucial protein for EMCV infection; specifically, ADAM9 knockout cells from two mammalian species are not infected by two different EMCV strains, reconstitution of ADAM9 permits EMCV infection, and virus replication is unaffected by ADAM9 deficiency when viral RNA is transfected directly into the cytosol. EMCV may directly bind to ADAM9, but we and others (57) have observed polysaccharide-related binding that may facilitate productive infection. Also, as a disintegrin, ADAM9 is known to affect expression of other cell surface proteins (27, 38); thus, expression of ADAM9 could lead to uncovering of another protein that is the actual binding “receptor” protein.…”

“…Previous EMCV studies suggest that different strains of EMCV may bind to different receptors and that some EMCV strains may use multiple pathways (56, 57). In addition, EMCV strains with different tissue tropism have been identified (1).…”

“…However, cells lacking VCAM-1 have also been shown to be susceptible to EMCV infection via a yet-uncharacterized 70-kDa glycoprotein hypothesized to be responsible for mediating virus binding and attachment in these cells (56). Infection of human cardiomyocytes with two different strains of EMCV demonstrated a requirement for sialic acid and heparan sulfate in one EMCV strain but not the other, raising the possibility that different EMCV strains may bind to different polysaccharides in order to initiate infection (57). While different molecules have been implicated as receptors or attachment factors for specific strains of EMCV, the provenance of these strains has been difficult to authenticate, and we have not been able to obtain many of the strains previously reported to have specific receptor or attachment requirements.…”

Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses

“…The viruses, which were cultured in baby hamster kidney-21 (BHK-21) cells, produced two groups of variants. Group I with Lys231 in VP1 that becomes Sia-dependent can bind to and infect primary human cardiomyocytes more efficiently than can group II with Arg231 in VP1, which is not a Sia-dependent group [92]. In addition, the rat strain 1086C appeared to acquire the ability to replicate effectively in buffalo rat liver (BRL) cells as a result of 3 amino acid mutations, Lys49Glu, Leu142Phe, and Ile180Ala, in VP1 that provided the ability to bind to the sialylated BRL cell 512 Nongluk Sriwilaijaroen and Yasuo Suzuki surface after 29 passages [87].…”

“…The crystal structures of the viral capsid proteins, VP1, VP2, VP3, and VP4, in complex with a sialyl ligand have shown that a Sia-binding pocket is in VP1 of ERAV [83], in a pit between VP1 and VP3 of EV-D68 [89], in VP1 of CVA24v [90], and mainly in the VP2 puff B of low-neurovirulent TMEV [88,91]. Analysis of EMCV mutants/ variants has indicated that amino acids in VP1 are responsible for interactions with Sia on the host cell surface [87,92]. Typically, binding of the viral capsid proteins of picornaviruses to receptors triggers endocytosis.…”