Enteroviruses are a group of viruses responsible for numerous pandemics, posing significant public health threats. Among them, Enterovirus 71 (EV71), Coxsackievirus A16, and Coxsackievirus A6 are the primary causes of hand, foot, and mouth disease (HFMD), with EV71 being the major pathogen responsible for outbreaks. First identified in 1969, EV71 is a single-stranded RNA virus approximately 7.5 kb in length, belonging to the genus Enterovirus within the family Picornaviridae.

EV71 primarily infects infants and young children under the age of five. While HFMD caused by EV71 is typically self-limiting, some cases progress to severe complications, such as aseptic meningitis, brainstem encephalitis, and other neurological disorders. Since its discovery, EV71 has caused numerous outbreaks and epidemics worldwide, particularly in the Asia-Pacific region, including countries like China, Korea, Singapore, Japan, and Vietnam. Despite the availability of an EV71 vaccine, no effective clinical drugs have been developed to treat HFMD caused by EV71.

The EV71 genome contains only one open reading frame, which encodes 2193 amino acids. During the EV71 replication process, the polyprotein is subdivided into P1, P2, and P3 regions. P1 encodes four structural proteins (VP1–VP4), whereas P2 and P3 encode seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). These 11 proteins are closely related to EV71 infection, inflammatory responses, and host immune responses.

Fig. 1 Structure of the enterovirus 71 (EV71) genome {Wang, 2019 #1195}

VP1

The VP1 protein of EV71 contains 297 amino acids and exhibits complete genetic diversity corresponding to the viral serotype, which can be used as the basis for EV71 serotype classification. Human scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1) are two cell receptors for EV71, while heparan sulfate and vimentin serve as attachment receptors.

The N-terminal region of the VP1 capsid protein contains an important antigenic site, which is highly immunogenic. The peptides corresponding to amino acids 66-77 and 208-222 in the C-terminal region of VP1 may stimulate the production of neutralizing antibodies. VP1 is the main virus-neutralizing determinant, directly determining the virus's antigenicity. Therefore, it is a suitable candidate for EV71 vaccines.

VP2-VP4

EV71 virus capsid proteins VP2 and VP3, which are important parts of the shell protein, are associated with the antigenicity of the virus. The VP4 package is embedded in the inside of the virus shell, is closely connected with the virus core, and exhibits an extended spatial conformation feature, which is a bridge connecting the inside and outside.

Protease 2A

The EV71 2A protease is a cysteine protease consisting of 150 amino acids. It cleaves at its own N-terminus, specifically at the junction between VP1 and 2A of the polyprotein. The 2A protease is primarily involved in disrupting host cell functions, including shutting down host protein synthesis, inhibiting nucleocytoplasmic transport, and evading innate immunity.

Fig. 2 Cleavage site of Protease 2A {Cai, 2013 #1217}

Protein 2B

The EV71 2B protein, a small hydrophobic ion channel protein with 99 amino acid residues, may mediate a chloride-dependent rather than calcium-dependent current in oocytes. A hydrophilic region of 14 amino acids in the N-terminal region of 2B is important for Bax interaction and subsequent activation.

Protein 2C

The 2C protein of EV71 is one of the most highly conserved nonstructural proteins, containing 329 amino acid residues. 2C harbors an adenosine triphosphatase (ATPase) domain, a zinc finger structure, and an alpha helix at the end of the C-terminal region. The ATPase domain, which belongs to SF3 helicases of the AAA+ ATPase superfamily, contains Walker motifs and motif C.

Fig. 3 Overall structure of EV71 2C {Guan, 2017 #1187}

Protein 3A

The EV71 3A protein, composed of 86 amino acids, is a membrane-binding protein that regulates intracellular transport in host cells. It likely localizes to the surface of intracellular membrane vesicles, which are derived from the endoplasmic reticulum and induced following viral infection, serving as sites for viral RNA replication.

Protein 3B

The 3B protein, also known as a VPg protein, is a small protein that contains 22 amino acid residues. VPg protein can interact with the polymerase 3D, and VPg is catalyzed by 3D polymerase to uridine acidification, which is a primer for viral RNA synthesis.

Protease 3C

The EV71 3C protein, consisting of 183 amino acids, exhibits both serine protease and cysteine protease activities. It is responsible for nearly all of the proteolytic processes during viral replication. Additionally, 3C protease plays a key role in evading and suppressing host defense mechanisms. Its critical functions in the EV71 life cycle make protease 3C a valuable therapeutic target.