Date Published: May 9, 2019
Publisher: Public Library of Science
Author(s): Liang Sun, Hyunwook Lee, Hendrik Jan Thibaut, Kristina Lanko, Eva Rivero-Buceta, Carol Bator, Belen Martinez-Gualda, Kai Dallmeier, Leen Delang, Pieter Leyssen, Federico Gago, Ana San-Félix, Susan Hafenstein, Carmen Mirabelli, Johan Neyts, Z. Hong Zhou.
Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.
Since the first large outbreak in 1997, enterovirus A71 (EV-A71) (genus Enterovirus, family Picornaviridae) has been reported to cause 2–3 year cyclic epidemics in the Asia-Pacific region [1,2]. In the last two decades, the increasing number of EV-A71 cases and the spread of the virus across Asia have raised major concerns about its pandemic potential. The virus is primarily transmitted by the oral-fecal route [3,4]. Most EV-A71 infections are characterized by mild symptoms, with the typical signs of the hand, foot and mouth disease (HFMD): slight fever, red rashes on the palms of hand and soles of feet, and ulcers in the mouth. However, EV-A71 infections are also associated to severe neurological complications (such as encephalitis, aseptic meningitis and poliomyelitis-like syndrome) and acute pulmonary edema, which may be highly limiting and fatal particularly in children under the age of 5 years [5,6]. In 2010, a large outbreak of HFMD in China resulted in an estimated 1.7 million cases and 905 deaths  and an outbreak in Cambodia in 2012 resulted in the death of 54 children [8,9]. A sub-genogroup C4 EV-A71-inactivated vaccine has recently been approved in China, but worldwide coverage and long-term protection still need to be addressed [10–12]. There are no antiviral agents approved against EV-A71 nor against any other enteroviruses.
We report here on the mechanism of action of MADAL385, the lead compound of a novel class of tryptophan dendrimers with exquisitely potent in vitro antiviral activity against EV-A71. Cryo-EM studies revealed that the highly conserved lysine residue at position 244 of VP1 (VP1_244K), near the icosahedral 5-fold vertex, is closely connected to the density of MADAL385. This residue also plays a key role in the interaction of EV-A71 with PSGL1 and HS. Both receptors are sulfated molecules (i.e. endowed with a negative charge at physiological pH) whose interaction with the positively charged VP1_244K capsid residue is thought to involve a strong electrostatic interaction. As a result of this high-affinity interaction, we showed that MADAL385 inhibits EV-A71 binding with PSGL1 and HS. Together with biochemical evidence, we also demonstrate that MADAL385 inhibits the binding of EV-A71 to human SCARB2- or PSGL1-expressing L929 cells. We observed that the activity of MADAL385 in L929-SCARB2 cells was exclusively dependent on the inhibition of HS binding since the activity of MADAL385 was lost in cells treated with sodium chlorate (NaClO3), a molecule that prevents cell-surface sulfation. In addition, this experiment also demonstrates the importance of HS binding for efficient entry and replication of EV-A71 in both L929-SCARB2 and L929-PSGL1 overexpressing cells.