Multifunctionality of Structural Proteins in the Enterovirus Life Cycle

Abstract

Members of the genus Enterovirus have a significant effect on human health, especially in infants and children. Since the viral genome has limited coding capacity, Enteroviruses subvert a range of cellular processes for viral infection via the interaction of viral proteins and numerous cellular factors. Intriguingly, the capsid-receptor interaction plays a crucial role in viral entry and has significant implications in viral pathogenesis. Moreover, interactions between structural proteins and host factors occur directly or indirectly in multiple steps of viral replication. In this review, we focus on the current understanding of the multifunctionality of structural proteins in the viral life cycle, which may constitute valuable targets for antiviral and therapeutic interventions.

Full text

Perspective
For reprint orders, please contact: reprints@futuremedicine.com
Multifunctionality of structural proteins in
the enterovirus life cycle
Xingjian Wen‡,1,2 ,DiSun
‡,1, Jinlong Guo‡,1, Fabian Elgner2, Mingshu Wang1,Eberhard
Hildt2& Anchun Cheng*,1
1Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, Sichuan, PR China
2Paul-Ehrlich-Institut, Department of Virology, Langen, Germany
*Author for correspondence: chenganchun@vip.163.com
‡Authors contributed equally
Members of the genus Enterovirus have a signicant effect on human health, especially in infants and
children. Since the viral genome has limited coding capacity, Enteroviruses subvert a range of cellular pro-
cesses for viral infection via the interaction of viral proteins and numerous cellular factors. Intriguingly,
the capsid–receptor interaction plays a crucial role in viral entry and has signicant implications in viral
pathogenesis. Moreover, interactions between structural proteins and host factors occur directly or indi-
rectly in multiple steps of viral replication. In this review, we focus on the current understanding of the
multifunctionality of structural proteins in the viral life cycle, which may constitute valuable targets for
antiviral and therapeutic interventions.
First draft submitted: 4 May 2019; Accepted for publication: 19 July 2019; Published online:
1 August 2019
Keywords: antiviral strategies •assembly •autophagy •capsid •cytolytic •enterovirus •neuropathogenesis •
receptor •structural protein •viral tropism
The genus Enterovirus (EV) includes a large group of enveloped RNA viruses within the family Picornaviridae,
including species EV A–L and rhinovirus A–C [1–3]. A spectrum of pathogens in this genus are emerging as causative
agents of many human and veterinary diseases outbreaks worldwide. Among these pathogens, EV71, coxsackievirus
A6 (CVA6) and CVA16 are the most common causative pathogens of hand, foot and mouth disease, which affects
millions of people each year – especially infants and young children [4–7]. Indeed, EVs are commonly associated with
mild infections. However, they are also associated with various severe diseases, which frequently result from acute
infections [8,9]. Additionally, persistent viral infections appear to be involved in myocarditis, dilated cardiomyopathy
and Type 1 diabetes [10]. Despite these burdens, there are no approved antiviral medications or vaccines available
to combat nonpolio EVs infection. Furthermore, some EVs could be used as oncolytic viruses due to their tumor
cytotoxic properties [11,12]. It will be valuable to focus on the viral life cycle for the development of potent antiviral
strategies and oncolytic agents in the coming decades.
Since viral genomes have limited coding capacity, EVs invade host cells by interacting with various host cellular
proteins and signaling pathways [13,14]. Capsid proteins interact with host factors to subvert a range of cellular
processes for viral infection, according to the characteristics of the virus. Specifically, there is substantial evidence
suggesting that a strong relationship between the capsid and receptor determines the cellular and tissue tropism [15–
20]. Moreover, recent reports indicate that the molecular determinants of viral pathogenesis are tightly related to
tissue tropism and cytolytic capacity [12,18,21–24]. Thus, comprehensively understanding the roles of the structural
proteins in viral replication and propagation may provide valuable information for viral pathogenesis. Previously, we
discussed how the structural proteins manipulate host cell cycle progression [25]. In this review, we focus on recently
identified host cellular factors that interact with the viral structural proteins in the viral life cycle, regulate viral
propagation, and appear to be involved in viral pathogenesis. An improved understanding of the multifunctionality
of structural proteins in viral pathogenesis may facilitate the development of potential novel antiviral therapeutics
and therapeutic interventions.
Future Microbiol. (Epub ahead of print) ISSN 1746-091310.2217/fmb-2019-0127 C
2019 Future Medicine Ltd

Perspective Wen, Sun, Guo et al.
Promoter (5S)
(VP0, VP1, VP3)
Pentamer (14S)
5x (VP0, VP1, VP3)
Procapsid (80S)
12x (VP0, VP1, VP3)
Provirion (150S)
12x (VP0, VP1, VP3) + RNA
The viral genome
Mature virus (160S)
12x (VP2, VP4, VP1, VP3) + RNA
Empty particle (80S)
(VP2, VP1, VP3)
The expanded 1 particle (160S)
12x (VP2, VP4, VP1, VP3) + RNA
‘A particle’ (135S)
12x (VP2, VP1, VP3) + RNA
VPg P1 P2 P3
VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D
2A3C3CD
?
5'UTR 3'UTR
AAAn
ORF
Figure 1. A schematic representation of the enterovirus genome and virion structure. (A) The model of enterovirus
genome and polyprotein organization. (B) A schematic representation of enterovirus virion assembly and its
intermediates with different features throughout the life cycle.
Enterovirus virion structure
EVs are a group of positive-sense ssRNA viruses, which contain a viral genome enclosed in the nonenveloped
capsid. The structure of the viral genome and the architecture of virions are shown in Figure 1. The capsids are
asymmetric icosahedrons with pseudo-T = 3, containing 60 copies of each of the four structural proteins (VP1–
VP4). The viral genome is expected to be highly condensed in mature virus particles, which are composed of
VP1–VP3 located on the outer surface, and VP4 located in the interior of the capsid. The symmetry axes of the
capsid are represented by fivefold, threefold and twofold axes, as shown in Figure 1B. The adjacent protomers of
VP1 and VP3 form the fivefold axis, which is surrounded by a (‘canyon’). The twofold axis constitutes adjacent
VP2 and VP3 protomers. As the capsid, the structural proteins of EVs preserve the viral genome from challenging
environmental conditions and deliver them to the host cells. There are consequently distinct configurations for EV
virions and their intermediates with different features throughout the life cycle, and distinguishable sedimentation
coefficients [26–36]. With the development of cryo-electron microscopy, recent studies have demonstrated that there
are various forms of EV capsid [26–36], including the mature virion (160S), the expanded one particle (intermediate
in the transition from full native virion to ‘A-particles’), ‘A-particles’ (uncoating intermediates, 135S) and empty
particles (80S). Compared with that of mature virions, the capsid radius of empty- and A-particles is markedly
expanded [37–40].
Receptor binding: enterovirus entry
EVs exploit host cellular metabolism throughout their entire life cycle (Figure 2), primarily relying on interactions
between viral proteins and host factors. The majority of EV infections primarily occur in the gastrointestinal tract
10.2217/fmb-2019-0127 Future Microbiol. (Epub ahead of print) future science group

Multifunctionality of EV structural proteins Perspective
Nucleus
Golgi
Mitochondria
Assembly
ER
Uncoating
Entry
Replication Translation
Lysosome
Autolysosome
Non-lytic release
Lytic release
Virus receptor
Attachment
Autophagosomes
Figure 2. A schematic representation of the enterovirus life cycle. Enterovirus exploits host cellular metabolism
throughout their entire life cycle, including attachment, endocytosis, uncoating, genome translation and proteolytic
processing, genome replication, virion assembly, virion maturation and release.
via the fecal-oral route, and EVs adopt different strategies to invade CNS from the primary infection site [41].
However, infection by certain EV types, especially rhinoviruses, can occur in the upper respiratory tract via the
airway epithelium [42]. The intestinal epithelial cells serve as the primary entry portal for viruses. After the initial
replication, the newly formed virions spread to other tissues and species if the appropriate receptors are present,
leading to the second phase of viral replication. Correct receptor binding is necessary for the viral life cycle to
start a productive infection; thus, correct receptor presence is a determinant of viral infectivity. Moreover, evidence
suggests that the capsid-receptor interactions play decisive roles in viral attachment, internalization, and entry,
determining cell types, tissues and species tropisms [12,15–24]. Over the past two decades, many receptor candidates
and entry factors have been found for human EVs, as shown in Table 1. In addition, there are also host factors that
play critical roles in various steps of the viral life cycle; for example, prohibitin is involved in both EV71 viral entry
and replication in neuronal cells, as well as in neuropathogenesis [23]. As a soluble factor, galectin-1 could facilitate
viral replication and the stability of EV71 virions [43].
The receptors of EV71 infection have been well elucidated in vitro [19]. Before entry into cells, EV71 initially
recognizes specific cellular factors, such as PSGL-1 [47],HS[54],Anx2[55], sialylated glycan [56], nucleolin [57],
fibronectin [58] and vimentin [59] on the cell surface, which may serve as attachment receptors to support virion
binding. Subsequently, SCARB2 serves as the major receptor that play roles in the internalization and initiation of
virion uncoating [60]. The attachment receptors capture virions on the cell surface and deliver them to functional
receptor SCARB2, which is mainly localized on endosomal/lysosomal membranes [61]. Importantly, amino acid
variation at VP1-145 of EV71 enables considerable flexibility in receptor usage and binding ability and thereby
modulates ex vivo tropism [15,62–65]. Notably, a G or a Q at VP1 residue 145 is present in PSGL-1-binding strains,
whereas strains with an E at this position do not bind PSGL-1. In addition, the majority of EV71 strains utilize
SCARB2 for entry, while EV71 strains with E145G, V146I and S241L mutations in VP1 and the T494C mutation
in the 5-UTR exhibit increased binding to PSGL-1 and lack the ability to infect and cause disease [66]. In addition,
a recent study identified that hWARS is also an EV71 susceptibility determinant in the process of viral entry [48,49].
future science group 10.2217/fmb-2019-0127

Perspective Wen, Sun, Guo et al.
Table 1. Specic receptors for enteroviruses.
Virus Species Receptor Functions Ref.
Coxsackievirus A10 Enterovirus A (major group) KREMEN1 Entry [24]
Coxsackievirus A14 Enterovirus A (minor group) SCARB2 Entry [44]
Coxsackievirus A16 Enterovirus A (minor group) SCARB2 Entry [44]
HS Attachment [45]
Coxsackievirus A7 Enterovirus A (minor group) SCARB2 Entry [44]
Enterovirus 71 Enterovirus A -3 (minor group) SCARB2 Entry [46]
PSGL-1 Attachment [47]
hWARS Entry [48,49]
HS Attachment Reviewed in [25]
Anx2 Attachment Reviewed in [25]
Prohibitin Attachment [23]
Coxsackievirus A9 Enterovirus B Integrin ␣V-␤3 Entr Reviewed in [25]
Integrin ␣V-␤6 Entry Reviewed in [25]
Coxsackievirus B3 Enterovirus B DAF Attachment Reviewed in [25]
Echovirus 1 Enterovirus B Integrin ␣2-␤1 Attachment Reviewed in [25]
Echovirus 6 Enterovirus B Neonatal Fc receptor Entry [21,22]
PVR (CD155) Attachment Reviewed in [25]
Echovirus 7 Enterovirus B DAF Entry Reviewed in [25]
Echovirus 9 Enterovirus B Integrin ␣-␤3 Entry Reviewed in [25]
Poliovirus Enterovirus C PVR (CD155) Entry Reviewed in [25]
Coxsackievirus A21 Enterovirus C ICAM-1 Entry [50]
DAF Attachment
Coxsackievirus A24 Enterovirus C ICAM-1 Entry [18]
Sialic acid Attachment [20]
Enterovirus D68 Enterovirus D ICAM-5 Entry [51]
␣2,6- and ␣2,3-linked sialic acid Attachment [52]
Enterovirus 70 Enterovirus D Sialic acid 5- N-acetyl-neuraminic
acid
Attachment Reviewed in [25]
Majority of rhinovirus A and all
rhinovirus A B types
Rhinovirus A (major group) and
rhinovirus B
ICAM-1 Entry Reviewed in [25]
Rhinovirus A (minor group) Rhinovirus A (minor group) Low density lipoprotein receptor Entry Reviewed in [25]
Rhinovirus C Rhinovirus C CDHR3 Entry [53]
Enterovirus uncoating & genome release
Upon direct binding to one or multiple designated host factors, EVs enter the cytoplasm through receptor-
mediated endocytosis. Subsequently, the virions undergo an irreversible structural rearrangement in the intracellular
environment for uncoating. Eventually, the viral genome is released from the capsid into the cytosol to initiate
infection. Significantly, it has been shown that the uncoating process of some EVs may not be solely mediated
by virion-receptor binding but may also rely on low pH to initiate genome release. Recent discoveries indicate
that receptor binding might destabilize the virus particle, facilitating the low-pH uncoating of the virus in the
endosome/lysosome and subsequent genome release into the cytosol [30]. Capsid proteins are the only determinants
of acid sensitivity in EVs [17]. It has been reported that acidification of the endosome is essential for EV71 and
human rhinovirus, while it is unnecessary for poliovirus and group B coxsackieviruses.
Using cryo-electron microscopy, the process of genome release from has been visualized for EVs, including
PV [67,68],EV71[29,38],CVA7[69], EV-D68 [28] and echovirus 18 (E-18) [27]. It has been proposed that the
virion-receptor interaction, induces virion conformational rearrangements that trigger RNA genome release [28,70].
Correspondingly, there are diverse uncoating intermediates with somewhat distinct structural features. By external-
izing the N-terminus of VP1, the native virion forms the expanded 1 particle (intermediate in the transition from
full native virion ‘A particle’). Subsequently, by expelling the full VP4 protein, the virion ‘A-particles’ (uncoating
intermediates, 135S). Following the release of the viral RNA genome, an empty particle (80S) is left behind. It has
10.2217/fmb-2019-0127 Future Microbiol. (Epub ahead of print) future science group

Multifunctionality of EV structural proteins Perspective
been reported that VP4 contains membrane pore-forming activity [71], and it is assumed that the egression of VP4
induces liposome disruption to aid viral genome delivery into the cytoplasm. In contrast to release via the channels
on the ‘A-particle’, the genome of E-18 and E-30 exits from the capsid via a loss of one, two or three adjacent
capsid-protein pentamers [27].
Enterovirus translation & genome replication
In recent years, the steps of viral replication have been described in detail. After entering into the cytoplasm via
receptor-mediated endocytosis, EVs hijack various cellular proteins and signaling pathways to facilitate viral RNA
genome delivery and initiate viral translation and replication [72,73]. For example, the lipid-modifying enzyme
PLA2G16 prevents the clearance of the viral genome [74]. Utilizing the host translational machinery, the viral RNA
genome is immediately translated. The formation of replication complexes then occurs following the replication of
the viral genome [75]. The negative strand of viral genome then serves as a template to produce multiple copies of the
positive strand, which can be used either as additional mRNA or as genetic material for nascent viral particles that
are released from the cell and can go on to infect other cells [76]. The viral genome encodes a precursor polyprotein
that is subsequently cleaved into eleven proteins by both viral and host cell proteases. Recent studies have revealed
that EV infection induces the formation of a membranous web associated with the cellular autophagy pathway
to facilitate viral replication and assembly [77]. Autophagy is an essential process involved in the degradation of
protein aggregates and damaged organelles. However, it is also known that the autophagy process plays critical
roles in viral infection, including the promotion of viral replication and immune evasion [78–81]. The formation of
replication complexes occurs following the replication of the viral genome [75]. More recently, connections between
structural proteins and autophagy have been identified. For example, EV71 VP1 activates endoplasmic reticulum
stress, which activates autophagy in neuronal cells [65,82].
Enterovirus virion assembly
Virion assembly/morphogenesis of EVs is a stepwise process that involves consecutive oligomerization of structural
proteins [83]. Through the cleavage of the precursor P1 region by the viral proteases 2A and 3C, three structural
proteins, VP0 (precursor of VP4 and VP2), VP1 and VP3 [84,85], assemble into protomers (5S), pentamers (14S) and
genome-free empty procapsids (75S–80S). Alternatively, the formation of a full provirion (150S) occurs following
the assembly of 12 pentamers around the newly synthesized viral RNA genome. The virion maturation process
causes VP0 precursor cleavage; however, the mechanism underlying this process is still unclear [86].Ithaslong
been considered that pentamers self-assemble into empty capsids. However, recent publications demonstrate that
the RNA genome plays a functional role in virion assembly, related to packaging signals of viral genome [87,88].In
addition, a short motif (YCPRP in foot-and-mouth disease virus, WCPRP in EVs) within the capsid precursor is
highly conserved among picornaviruses and helps to maintain the structure of precursors and capsid assembly [89].
Moreover, recent studies have shown that positively charged residues in VP1 play a key role in the maturation of
infectious virus particles [90,91].
Recently, it has been shown that multiple other viral and cellular components appear to affect the formation
of virions [92]. For example, the 2A protein regulates the kinetics of viral polyprotein processing and permits
virion assembly [93]. Additionally, a study identified that an interaction between 2C and VP3 is involved in viral
morphogenesis [94]. Moreover, it has also been reported that the members of heat shock protein family, including
HSP70 and HSP90, play a crucial role in P1 processing [95].
Enterovirus virion release
Most EVs are cytolytic and induce cell death in the later stages of infection [84]. A recent study showed that
EV71 VP1 plays a role in the viral-induced brainstem neuronal cell damage via VP1-induced cell surface-exposed
calreticulin upregulation [65]. However, recent studies have shown that several EVs exit cells within extracellular
vesicles by a nonlytic release mechanism, which has implications for pathogenesis [96]. Notably, the newly produced
virions can be released from intact cells via the exosomal pathway, resulting in nonlytic dissemination in the
CNS [97–100]. Quasi-enveloped virions can be released from cells packaged within a single vesicle from host-derived
membranes, thereby enabling their escape from capsid-specific antibodies and promoting viral spread from cell to
cell. These naked and quasi-enveloped progeny viruses use similar endocytic pathways for cell entry, but uncoat in
different compartments and release their genomes into the cytosol [101]. Thus far, the functional roles of structural
proteins in this process remain elusive and should be further explored.
future science group 10.2217/fmb-2019-0127

Perspective Wen, Sun, Guo et al.
Advances in capsid-targeting antiviral strategies
It has been proposed that all of the viral and host factors involved in viral infection are targets of antiviral
research [102]. For several decades, structure-based designs of antiviral strategies, including drugs (inhibitors),
protective neutralizing antibodies and vaccines, have been developed. However, currently, there are no approved
antivirals and vaccines available to combat nonpolio EV infections. The interaction of the viral capsid with
receptors is an attractive target for antiviral therapeutics (reviewed in [103]). Several viral capsid inhibitors, including
pleconaril [104], pirodavir [104], vapendavir (BTA798) [105], pocapavir (V-073 or SCH48973) [106],NLD[107],
NF449 [108], the tannins chebulagic acid [109], punicalagin [109], imidazolidinone derivative [110], and aminopyridyl
1,2,5-thiadiazolidine 1,1-dioxides [111], auraptene [112], formononetin [112] and yangonin [112], have been shown to
be useful for the restriction of viral infections. In addition, stabilizing RNA-capsid interactions and virion assembly
could be valuable approaches for antiviral drugs.
With receptor-competing activity, antibodies are the major protective immune response to EVs. Mutations in
the structural proteins have been found to be related to antibody sensitivity in various EVs [63,113]. Thus, it has
been proposed that differences in the resistance of the variants to neutralizing antibodies may be one of the reasons
for the difference in virulence. It has been hypothesized that antibody-mediated neutralization plays a critical role
in viral clearance. Thus, efficient interruption of viral attachment could serve as a novel approach for antiviral
development. As the major capsid protein, VP1 contains key neutralizing determinants, located in the N-terminal
region [113,114]. In addition, neutralizing antibodies targeting VP3 [115,116] and VP4 [67] could prevent virus infection
in vitro. Furthermore, pentamer-based nanoparticles [117] and virus-like particles [118–121] could serve as vaccine
candidates.
Future perspective
The EVs are emerging as a persistent global health threat, particularly in humans, and especially in children. The
EVs are commonly associated with mild infections. However, they are also associated with various neurological
disorders, such as acute flaccid myelitis, encephalitis, paralytic poliomyelitis and nonpolio flaccid paralysis. The
majority of EV infections primarily occur in the gastrointestinal tract via the fecal-oral route, and EVs adopt
different strategies to invade the CNS from the primary infection sites. The life cycle of EVs is related to different
viral and cellular proteins. In this review, we focused on the current understanding of the multifunctionality of EV
capsid proteins in various stages of the viral life cycle. These insights into the relationship between the structural
proteins and the pathogenesis of viruses may ultimately inform the development of novel antivirals. Ideally, the
development of a cocktail of different types of antibodies and drugs is recommended for thorough virus clearance
based on their various targets. In addition, multivalent and broad-spectrum antibodies targeting different forms of
capsids may provide an unexpected synergistic activity against infecting viruses.
Author contributions
X Wen, D Sun, and J Guo conceived the idea. X Wen and D Sun drew the gures. X Wen, D Sun and J Guo wrote the paper. F
Elgner, M Wang, E Hildt and A Cheng reviewed the manuscript. All authors have read and approved the manuscript.
Financial and competing interests disclosure
This work was supported by grants from National Key Research and Development Program of China (grant num-
ber 2017YFD0500800), China Agricultural Research System (CARS-42-17), Sichuan Veterinary Medicine and Drug Innovation
Group of China Agricultural Research System (CARS-SVDIP). Medical writing support was provided by American Journal Ex-
perts, and was funded by X Wen. The authors have no other relevant afliations or nancial involvement with any organization or
entity with a nancial interest in or nancial conict with the subject matter or materials discussed in the manuscript apart from
those disclosed.
10.2217/fmb-2019-0127 Future Microbiol. (Epub ahead of print) future science group

Multifunctionality of EV structural proteins Perspective
Executive summary
Virion structure of EV
•As the capsid, enterovirus (EV) structural proteins preserve the viral genome from challenging environmental
conditions and deliver them to the host cells. Therefore, there are distinct congurations of EV virions and their
intermediates with distinct features throughout the life cycle.
Receptor binding: EV entry
•Binding to the appropriate receptor is necessary for the initiation of a productive infection, and plays decisive
roles in viral attachment, internalization, entry, and determines cell type, tissue and species tropism.
EV uncoating & genome release
•The uncoating process of some EVs may not be solely mediated by virion-receptor binding but may also rely on
low pH to initiate genome release.
EV translation & genome replication
•EVs hijack various cellular proteins and signaling pathways to facilitate viral RNA genome delivery and initiate
viral translation and replication.
EV virion assembly
•Virion assembly/morphogenesis of EVs is a stepwise process that involves the consecutive oligomerization of
structural proteins.
EV virion release
•Most EVs are cytolytic and induce cell death in the later stages of infection. However, recent studies have shown
that several EVs can exit cells within extracellular vesicles by a nonlytic release mechanism, which has implications
for pathogenesis.
Advances in capsid-targeting antiviral strategies
•Structure-based designs for antivirals, including drugs (inhibitors), protective neutralizing antibodies and
vaccines, have been developed.
References
Papers of special note have been highlighted as: •of interest; •• of considerable interest
1. Lukashev AN, Vakulenko YA, Turbabina NA, Deviatkin AA, Drexler JF. Molecular epidemiology and phylogenetics of human
enteroviruses: is there a forest behind the trees? Rev. Med. Virol. 28(6), e2002 (2018).
2. Lukashev AN, Vakulenko YA. Molecular evolution of types in nonpolio enteroviruses. J. Gen. Virol. 98(12), 2968–2981 (2017).
3. Nikolay B, Cauchemez S. Enterovirus outbreak dynamics. Science 361(6404), 755–756 (2018).
•Enteroviruses are highly predictable pathogens, with outbreaks largely driven by serotype-specific long-term immunity and birth
rates.
4. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of
enterovirus 71. Lancet Infect. Dis. 10(11), 778–790 (2010).
5. Chong P, Liu CC, Chow YH, Chou AH, Klein M. Review of enterovirus 71 vaccines. Clin. Infect. Dis. 60(5), 797–803 (2015).
6. Mao Q, Wang Y, Yao Xet al. Coxsackievirus A16: epidemiology, diagnosis, and vaccine. Hum. Vaccin. Immunother. 10(2), 360–367
(2014).
7. Kimmis BD, Downing C, Tyring S. Hand-foot-and-mouth disease caused by coxsackievirus A6 on the rise. Cutis 102(5), 353–356
(2018).
8. Bearden D, Collett M, Quan PL, Costa-Carvalho BT, Sullivan KE. Enteroviruses in X-linked agammaglobulinemia: update on
epidemiology and therapy. J. Allergy Clin. Immunol. Pract. 4(6), 1059–1065 (2016).
9. Rudolph H, Schroten H, Tenenbaum T. Enterovirus infections of the central nervous system in children: an update. Pediatr. Infect. Dis.
J. 35(5), 567–569 (2016).
10. Rodriguez-Calvo T. Enterovirus infection and Type 1 diabetes: unraveling the crime scene. Clin. Exp. Immunol. 195(1), 15–24 (2019).
11. Gromeier M, Nair SK. Recombinant poliovirus for cancer immunotherapy. Ann. Rev. Med. 69(1), 289–299 (2018).
12. Lipatova AV, Le TH, Sosnovtseva AO, Babaeva FE, Kochetkov DV, Chumakov PM. Relationship between cell receptors and tumor cell
sensitivity to oncolytic enteroviruses. Bull. Exp. Biol. Med. 166(1), 58–62 (2018).
13. Baggen J, Thibaut HJ, Strating J, van Kuppeveld FJM. The life cycle of nonpolio enteroviruses and how to target it. Nat. Rev.
Microbiol. 16(6), 368–381 (2018).
14. Huang PN, Jheng JR, Arnold JJ, Wang JR, Cameron CE, Shih SR. UGGT1 enhances enterovirus 71 pathogenicity by promoting viral
RNA synthesis and viral replication. PLoS Pathog. 13(5), e1006375 (2017).
15. TseligkaED,SoboK,StoppiniLet al. A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate
binding ability and modulates ex vivo tropism. PLoS Pathog. 14(8), e1007190 (2018).
future science group 10.2217/fmb-2019-0127

Perspective Wen, Sun, Guo et al.
16. Royston L, Essaidi-Laziosi M, P´
erez-Rodr´
ıguez FJ et al. Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism,
acid sensitivity and optimal growth temperature. PLoS Pathog. 14(4), e1006375 (2018).
17. Lin JY, Shih SR. Cell and tissue tropism of enterovirus 71 and other enteroviruses infections. J. Biomed. Sci. 21, 18 (2014).
18. Baggen J, Hurdiss DL, Zocher GBet al. Role of enhanced receptor engagement in the evolution of a pandemic acute hemorrhagic
conjunctivitis virus. Proc. Natl Acad. Sci. USA 115(2), 397–402 (2018).
19. Yamayoshi S, Fujii K, Koike S. Receptors for enterovirus 71. Emerg. Microbes Infect. 3(7), e53 (2014).
20. Nilsson EC, Jamshidi F, Johansson SM, Oberste MS, Arnberg N. Sialic acid is a cellular receptor for coxsackievirus A24 variant, an
emerging virus with pandemic potential. J. Virol. 82(6), 3061–3068 (2008).
21. Morosky S, Wells AI, Lemon K et al. The neonatal Fc receptor is a pan-echovirus receptor. Proc. Natl Acad. Sci. USA 116(9), 3758–3763
(2019).
22. Zhao X, Zhang G, Liu S et al. Human neonatal Fc receptor is the cellular uncoating receptor for Enterovirus B. Cell 177(6),
1553–1565.e1516 (2019).
23. Too IHK, Bonne I, Tan EL, Chu JJH, Alonso S. Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis. PLoS Pathog.14(1),
e100677100678 (2018).
24. Staring J, van den Hengel LG, Raaben M, Blomen VA, Carette JE, Brummelkamp TR. KREMEN1 is a host entry receptor for a major
group of enteroviruses. Cell Host Microbe 23(5), 636–643.e5 (2018).
25. Wen X , C he n g A, et al. Recent advances from studies on the role of structural proteins in enterovirus infection. Future Mi crobiol. 10(9),
1529–1542 (2015).
26. Myllynen M, Kazmertsuk A, Marjomaki V. A novel open and infectious form of echovirus 1. J. Virol. 90(15), 6759–6770 (2016).
27. Buchta D, Fuzik T, Hrebik D et al. Enterovirus particles expel capsid pentamers to enable genome release. Nat. Commun. 10(1), 1138
(2019).
•• The exit of the RNA from the particle of echovirus 18 results in a loss of one, two or three adjacent capsid-protein pentamers,
this finding uncovers a mechanism of enterovirus genome release that could become target for antiviral drugs.
28. Liu Y, Sheng J, van Vliet ALW, Buda G, van Kuppeveld FJM, Rossmann MG. Molecular basis for the acid-initiated uncoating of human
enterovirus D68. Proc. Natl Acad. Sci. USA 115(52), E12209–E12217 (2018).
29. Ross CJ, Atilgan AR, Tastan Bishop O, Atilgan C. Unraveling the motions behind enterovirus 71 uncoating. Biophys. J. 114(4), 822–838
(2018).
30. Zhou D, Zhao Y, Kotecha Aet al. Unexpected mode of engagement between enterovirus 71 and its receptor SCARB2. Nat.
Microbiol. 4(3), 414–419 (2019).
31. Ruokola P, Dadu E, Kazmertsuk A, Hakkanen H, Marjomaki V, Ihalainen JA. Raman spectroscopic signatures of echovirus 1
uncoating. J. Virol. 88(15), 8504–8513 (2014).
32. Organtini LJ, Makhov AM, Conway JF, Hafenstein S, Carson SD. Kinetic and structural analysis of coxsackievirus B3 receptor
interactions and formation of the A-particle. J. Virol. 88(10), 5755–5765 (2014).
33. LyuK,DingJ,HanJFet al. Human enterovirus 71 uncoating captured at atomic resolution. J. Virol. 88(6), 3114–3126 (2014).
34. Butan C, Filman DJ, Hogle JM. Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane
interaction and RNA release. J. Virol. 88(3), 1758–1770 (2014).
35. Shakeel S, Seitsonen J, Kajander T et al. Structural and functional analysis of coxsackievirus a9 integrin αvβ6 binding and uncoating. J.
Virol. 87(7), 3943–3951 (2013).
36. RenJ,WangX,HuZet al. Picornavirus uncoating intermediate captured in atomic detail. Nat. Commun. 4, 1929 (2013).
37. Zhu L, Sun Y, Fan J et al. Structures of Coxsackievirus A10 unveil the molecular mechanisms of receptor binding and viral uncoating.
Nat. Commun. 9(1), 4985 (2018).
•• The mature virus structure for the KREMEN1-dependent subgroup of HEV-A viruses, providing implications for receptor
binding; and have captured the dynamics in CVA10 uncoating, suggesting an ordered signal transmission mechanism for
enterovirus uncoating.
38. Shingler KL, Yoder JL, Carnegie MS et al. The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of
picornavirus uncoating. PLoS Pathog. 9(3), e1003240 (2013).
39. Garriga D, Pickl-Herk A, Luque D et al. Insights into minor group rhinovirus uncoating: the x-ray structure of the HRV2 empty
capsid. PLoS Pathog. 8(1), (2012).
40. Myllynen M, Kazmertsuk A, Marjomaki V. A novel open and infectious form of Echovirus 1. J. Virol. 90(15), 6759–6770 (2016).
41. Xue YC, Feuer R, Cashman N, Luo H. Enteroviral Infection: the forgotten link to amyotrophic lateral sclerosis? Front. Mol.
Neurosci. 11(63), 63 (2018).
42. Basnet S, Palmenberg AC, Gern JE. Rhinoviruses and their receptors. Chest 155(5), 1018–1025 (2019).
10.2217/fmb-2019-0127 Future Microbiol. (Epub ahead of print) future science group

Multifunctionality of EV structural proteins Perspective
43. Lee PH, Liu CM, Ho TSet al. Enterovirus 71 virion-associated galectin-1 facilitates viral replication and stability. PLoS
ONE 10(2), e0116278 (2015).
44. YamayoshiS,IizukaS,YamashitaT et al. Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus
71. J. Virol. 86(10), 5686–5696 (2012).
45. Zhang X, Shi J, Ye X et al. Coxsackievirus A16 utilizes cell surface heparan sulfate glycosaminoglycans as its attachment receptor. Emerg.
Microbes Infect. 6(7), e65 (2017).
46. Yamayoshi S, Yamashita Y, Li J et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat. Med. 15(7), 798–801 (2009).
47. Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H. Human P-selectin glycoprotein ligand-1 is a functional
receptor for enterovirus 71. Nat. Med. 15(7), 794–797 (2009).
48. Yeung ML, Jia L, Yip CCY et al. Human tryptophanyl-tRNA synthetase is an IFN-gamma-inducible entry factor for enterovirus. J.
Clin. Invest. 128(11), 5163–5177 (2018).
•• The results extend the knowledge of the pathogenicity of EV-A71 in relation to entry factor expression upon IFN-γstimulation
and the therapeutic options for treating severe EV-A71–associated complications.
49. Perlman S, Gallagher T. Not your usual tRNA synthetase: hWARS serves as an enterovirus entry factor. J. Clin. Invest. 128(11),
4767–4769 (2018).
50. Xiao C, Bator-Kelly CM, Rieder E et al. The crystal structure of coxsackievirus A21 and its interaction with ICAM-1. Structure 13(7),
1019–1033 (2005).
51. Wei W, Guo H, Chang J et al. ICAM-5/telencephalin is a functional entry receptor for enterovirus D68. Cell Host Microbe 20(5),
631–641 (2016).
52. Liu Y, Sheng J, Baggen J et al. Sialic acid-dependent cell entry of human enterovirus D68. Nat. Commun. 6, 8865 (2015).
53. Bochkov YA, Watters K, Ashraf S et al. Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates
rhinovirus C binding and replication. Proc. Natl Acad. Sci. USA 112(17), 5485–5490 (2015).
54. Tan CW, Poh CL, Sam I-C, Chan YF. Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor. J.
Virol. 87(1), 611–620 (2013).
55. Yang SL, Chou YT, Wu CN, Ho MS. Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity. J.
Virol. 85(22), 11809–11820 (2011).
56. Yang B, Chuang H, Yang KD. Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells. Virol.
J. 6(1), 141 (2009).
57. Su PY, Wang YF, Huang SW et al. Cell surface nucleolin facilitates enterovirus 71 binding and infection. J. Virol. 89(8), 4527–4538
(2015).
58. He QQ, Ren S, Xia ZC, Cheng ZK, Peng NF, Zhu Y. Fibronectin facilitates enterovirus 71 infection by mediating viral entry. J.
Virol. 92(9), (2018).
59. Du N, Cong H, Tian H et al. Cell surface vimentin is an attachment receptor for enterovirus 71. J. Virol. 88(10), 5816–5833 (2014).
60. Lin YW, Lin HY, Tsou YL et al. Human SCARB2-mediated entry and endocytosis of EV71. PLoS ONE 7(1), e30507 (2012).
61. He Y, Ong KC, Gao Z et al. Tonsillar crypt epithelium is an important extra-central nervous system site for viral replication in EV71
encephalomyelitis. Am. J. Pathol. 184(3), 714–720 (2014).
62. Kobayashi K, Sudaka Y, Takashino A, Imura A, Fujii K, Koike S. Amino acid variation at VP1-145 of enterovirus 71 determines
attachment receptor usage and neurovirulence in human scavenger receptor B2 transgenic mice. J. Virol. 92(15), (2018).
63. Fujii K, Sudaka Y, Takashino A et al. VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and
resistance to neutralizing antibody during cynomolgus monkey infection. J. Virol. 92(15), e00682–00618 (2018).
•VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.
64. van der Sanden SMG, Sachs N, Koekkoek SM et al. Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral
infectivity determinant. Emerg. Microbes Infect. 7(1), 84 (2018).
65. Hu DD, Mai JN, He LY et al. Glucocorticoids prevent enterovirus 71 capsid protein VP1 Induced calreticulin surface exposure by
alleviating neuronal ER Stress. Neurotox. Res. 31(2), 204–217 (2017).
66. Chang CK, Wu SR, Chen YC et al. Mutations in VP1 and 5’-UTR affect enterovirus 71 virulence. Sci. Rep. 8(1), 6688 (2018).
67. Levy HC, Bostina M, Filman DJ, Hogle JM. Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate
characterized by cryo-electron microscopy. J. Virol. 84(9), 4426–4441 (2010).
68. Bostina M, Levy H, Filman DJ, Hogle JM. Poliovirus RNA is released from the capsid near a twofold symmetry axis. J. Virol. 85(2),
776–783 (2011).
69. Seitsonen JJ, Shakeel S, Susi P et al. Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating. J.
Virol. 86(13), 7207–7215 (2012).
future science group 10.2217/fmb-2019-0127

Perspective Wen, Sun, Guo et al.
70. Ross C, Knox C, Tastan Bishop O. Interacting motif networks located in hotspots associated with RNA release are conserved in
enterovirus capsids. FEBS Lett. 591(12), 1687–1701 (2017).
71. Panjwani A, Asfor AS, Tuthill TJ. The conserved N-terminus of human rhinovirus capsid protein VP4 contains membrane pore-forming
activity and is a target for neutralizing antibodies. J. Gen. Virol. 97(12), 3238–3242 (2016).
72. Groppelli E, Levy HC, Sun E et al. Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer
across cellular and model membranes. PLoS Pathog. 13(2), e1006197 (2017).
73. Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology 479–480 (2015).418–433
74. Staring J, von Castelmur E, Blomen VA et al. PLA2G16 represents a switch between entry and clearance of picornaviridae. Nature 541,
412 (2017).
75. Melia CE, van der Schaar HM, de Jong AWM et al. The origin, dynamic morphology, and PI4P-independent formation of
encephalomyocarditis virus replication organelles. MBio 9(2), e00420 (2018).
76. Warden MS, Cai K, Cornilescu G et al. Conformational flexibility in the enterovirus RNA replication platform. RNA 25(3), 376–387
(2019).
77. Strating JR, van Kuppeveld FJ. Viral rewiring of cellular lipid metabolism to create membranous replication compartments. Curr. Opin.
Cell. Biol. 47, 24–33 (2017).
78. Corona Velazquez A, Corona AK, Klein KA, Jackson WT. Poliovirus induces autophagic signaling independent of the ULK1 complex.
Autophagy 14(7), 1201–1213 (2018).
79. Lai JK, Sam IC, Chan YF. The autophagic machinery in enterovirus infection. Viruses 8(2), 32 (2016).
80. Mohamud Y, Luo H. The intertwined life cycles of enterovirus and autophagy. Virulence 10(1), 470–480 (2019).
81. Mohamud Y, Shi J, Qu J et al. Enteroviral infection inhibits autophagic flux via disruption of the SNARE complex to enhance viral
replication. Cell Rep. 22(12), 3292–3303 (2018).
82. Li P, Yang S, Hu D et al. Enterovirus 71 VP1 promotes mouse Schwann cell autophagy via ER stressmediated PMP22 upregulation. Int J
Mol Med. 44(2), 759–767 (2019).
83. Jiang P, Liu Y, Ma HC, Paul AV, Wimmer E. Picornavirus morphogenesis. Microbiol. Mol. Biol. Rev. 78(3), 418–437 (2014).
84. Sun D, Chen S, Cheng A, Wang M. Roles of the picornaviral 3C proteinase in the viral life cycle and host cells. Viruses 8(3), 82 (2016).
85. YangX,ChengA,WangMet al. Structures and corresponding functions of five types of picornaviral 2A proteins. Front.
Microbiol. 8(1373), 1373 (2017).
86. Cao J, Liu H, Qu M et al. Determination of the cleavage site of enterovirus 71 VP0 and the effect of this cleavage on viral infectivity and
assembly. Microbial. Pathogenesis 134, 103568 (2019).
87. Song Y, Gorbatsevych O, Liu Y et al. Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus
genomes. Proc. Natl Acad. Sci. USA 114(41), E8731–E8740 (2017).
88. Dykeman EC. A model for viral assembly around an explicit RNA sequence generates an implicit fitness landscape. Biophys. J. 113(3),
506–516 (2017).
89. Kristensen T, Belsham GJ. Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at
distal sites. PLoS Pathog. 15(1), e1007509 (2019).
•• A short, highly conserved, motif is required for picornavirus capsid precursor processing at distal sites; it may represent a site
that interacts with cellular chaperones.
90. Yuan S, Li G, Wang Y et al. Identification of positively charged residues in enterovirus 71 capsid protein VP1 essential for production of
infectious particles. J. Virol. 90(2), 741–752 (2016).
91. Zhang YX, Huang YM, Li QJ et al. A highly conserved amino acid in VP1 regulates maturation of enterovirus 71. PLoS
Pathog. 13(9), e1006625 (2017).
92. Tijsma A, Thibaut HJ, Franco D, Dallmeier K, Neyts J. Hydantoin: the mechanism of its in vitro anti-enterovirus activity revisited.
Antiviral Res. 133, 106–109 (2016).
93. Adeyemi OO, Sherry L, Ward JC et al. Involvement of a nonstructural protein in poliovirus capsid assembly. J.
Virol. 93(5), e01447–01418 (2019).
94. Liu Y, Wang C, Mueller S, Paul AV, Wimmer E, Jiang P. Direct interaction between two viral proteins, the nonstructural protein 2C and
the capsid protein VP3, is required for enterovirus morphogenesis. PLoS Pathog. 6(8), e1001066 (2010).
95. Tsou YL, Lin YW, Chang HW et al. Heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for
therapy. PLoS ONE 8(10), e77133 (2013).
96. Bird SW, Kirkegaard K. Escape of non-enveloped virus from intact cells. Virology 479, 444–449 (2015).
97. van der Grein SG, Defourny KAY, Slot EFJ, Nolte-’t Hoen ENM. Intricate relationships between naked viruses and extracellular vesicles
in the crosstalk between pathogen and host. Semin. Immunopathol. 40(5), 491–504 (2018).
10.2217/fmb-2019-0127 Future Microbiol. (Epub ahead of print) future science group

Multifunctionality of EV structural proteins Perspective
98. van der Grein SG, Defourny KAY, Rabouw HH et al. Picornavirus infection induces temporal release of multiple extracellular vesicle
subsets that differ in molecular composition and infectious potential. PLoS Pathog. 15(2), e1007594 (2019).
99. Raab-Traub N, Dittmer DP. Viral effects on the content and function of extracellular vesicles. Nat. Rev. Microbiol. 15(9), 559–572
(2017).
100. ChenYH,DuW,HagemeijerMCet al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 160(4),
619–630 (2015).
101. Rivera-Serrano EE, Gonzalez-Lopez O, Das A, Lemon SM. Cellular entry and uncoating of naked and quasi-enveloped human
hepatoviruses. Elife 8 (2019).
102. Thibaut HJ, De Palma AM, Neyts J. Combating enterovirus replication: state-of-the-art on antiviral research.
Biochem. Pharmacol. 83(2), 185–192 (2012).
103. Benschop KS, van der Avoort HG, Duizer E, Koopmans MP. Antivirals against enteroviruses: a critical review from a public-health
perspective. Antivir. Ther. 20(2), 121–130 (2015).
104. Bernard A, Lacroix C, Cabiddu MG, Neyts J, Leyssen P, Pompei R. Exploration of the anti-enterovirus activity of a series of
pleconaril/pirodavir-like compounds. Antivir. Chem. Chemother. 24(2), 56–61 (2015).
105. Tijsma A, Franco D, Tucker S et al. The capsid binder vapendavir and the novel protease inhibitor SG85 inhibit enterovirus 71
replication. Antimicrob. Agents Chemother. 58(11), 6990–6992 (2014).
106. Torres-Torres S, Myers AL, Klatte JM et al. First use of investigational antiviral drug pocapavir (v-073) for treating neonatal enteroviral
sepsis. Pediatr. Infect. Dis. J. 34(1), 52–54 (2015).
107. Kelly JT, De Colibus L, Elliott L et al. Potent antiviral agents fail to elicit genetically-stable resistance mutations in either enterovirus 71
or Coxsackievirus A16. Antiviral Res. 124, 77–82 (2015).
108. Nishimura Y, McLaughlin NP, Pan J et al. The suramin derivative NF449 interacts with the 5-fold vertex of the enterovirus A71 capsid
to prevent virus attachment to PSGL-1 and heparan sulfate. PLoS Pathog. 11(10), e1005184 (2015).
109. Lin CJ, Liu CH, Wang JY et al. Small molecules targeting coxsackievirus A16 capsid inactivate viral particles and prevent viral binding.
Emerg. Microbes Infect. 7(1), 162 (2018).
110. Ho JY, Chern JH, Hsieh CF et al. In vitro and in vivo studies of a potent capsid-binding inhibitor of enterovirus 71. J. Antimicrob.
Chemother. 71(7), 1922–1932 (2016).
111. Li P, Yu J, Hao F et al. Discovery of potent EV71 capsid inhibitors for treatment of HFMD. ACS Med. Chem. Lett. 8(8), 841–846
(2017).
112. LiG,GaoQ,YuanSet al. Characterization of three small molecule inhibitors of enterovirus 71 identified from screening of a library of
natural products. Antiviral Res. 143, 85–96 (2017).
113. Ku Z, Ye X, Shi J, Wang X, Liu Q, Huang Z. Single neutralizing monoclonal antibodies targeting the VP1 GH loop of enterovirus 71
inhibit both virus attachment and Internalization during viral entry. J. Virol. 89(23), 12084–12095 (2015).
114. Zheng Q, Zhu R, Xu L et al. Atomic structures of enterovirus D68 in complex with two monoclonal antibodies define distinct
mechanisms of viral neutralization. Nat. Microb iol. 4(1), 124–133 (2019).
115. Shingler KL, Cifuente JO, Ashley RE, Makhov AM, Conway JF, Hafenstein S. The enterovirus 71 procapsid binds neutralizing
antibodies and rescues virus infection in vitro.J. Virol. 89(3), 1900–1908 (2015).
116. JiaQ,NgQ,ChinWet al. Effective in vivo therapeutic IgG antibody against VP3 of enterovirus 71 with receptor-competing
activity. Sci. Rep. 7, 46402 (2017).
117. Xue L, Liu J, Wang Q et al. Purification and assembling a fused capsid protein as an enterovirus 71 vaccine candidate from inclusion
bodies to pentamer-based nanoparticles. Biochem. Eng. J. 117, 139–146 (2017).
118. Adeyemi OO, Nicol C, Stonehouse NJ, Rowlands DJ. Increasing type 1 poliovirus capsid stability by thermal selection. J. Virol. 91(4),
(2017).
119. Zhao H, Li H-Y, Han J-F et al. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus
71 and coxsackievirus A16 in mice. Sci. Rep. 5, 7878 (2015).
120. Dai W, Zhang C, Zhang X et al. A virus-like particle vaccine confers protection against enterovirus D68 lethal challenge in mice.
Vaccine 36(5), 653–659 (2018).
121. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin. Exp. Vaccine Res. 6(1), 4–14 (2017).
future science group 10.2217/fmb-2019-0127