Human Enterovirus 71 Uncoating Captured at Atomic Resolution

Abstract

Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2-and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses. IMPORTANCE Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD Human enterovirus 71 (EV71) is the leading causative agent for severe hand-foot-and-mouth diseases (HFMD) in infants and young children (1), and EV71 infection has caused significant morbidity and mortality in the Asia-Pacific regions. During 2008 to 2012, numerous outbreaks of HFMD occurred in mainland China, with over 2,000 fatal cases reported (www.chinacdc.cn). However, vaccines and effective drugs remain unavailable. EV71 belongs to Enterovirus species A within the Enterovirus genus of the Picornaviridae (2). The EV71 virion contains a singlestranded positive-sense RNA genome of 7.5 kb. The viral capsid, which has a diameter of approximately 300 Å, is composed of 60 copies each of VP1 to VP4 (3, 4) that are organized onto a quasi-Tϭ3 icosahedral lattice. The capsid proteins VP1, VP2, and VP3 each possess a ␤-sandwich jelly roll fold and form the outer surface of the capsid shell, whereas VP4 is situated inside the shell. The capsid surface is characterized by a depression encircling each 5-fold symmetry axis, which is referred to as the "canyon" and often contains the receptor-binding site in picornaviruses (5, 6). The hydrophobic pocket of VP1, which is located at the bottom of the canyon, contains a lipid moiety termed the "pocket factor," which likely stabilizes the mature virion. As observed in poliovirus and certain picornaviruses, receptor bindin...

Crystal Structures of Enterovirus 71 (EV71) Recombinant Virus Particles Provide Insights into Vaccine Design

“…Virus Purification-EV71 and the recombinant viruses (EV71-NE1 and EV71-N6) were all grown and purified as described previously (20). The virions and empty particles were separated by sedimentation through a discontinuous 10 -50% sucrose gradient by no-brake ultracentrifugation at 26,000 rpm for 3 h. Fractions were collected and resolved by 12% SDS-PAGE.…”

“…2, A-C). Biochemical analyses showed that the empty particle contains VP0, VP1 (or mutant VP1), and VP3, whereas the virion contains VP1-4 (20). EM analyses showed that the empty particle exhibits only an empty shell, whereas the virion displays a dark center within the shell due to the RNA genome inside (Fig.…”

“…The empty particle structure was also compared with that of the virion (Protein Data Bank code 4N53) (20). The calculated r.m.s.d.…”

“…One notable difference lies in the N-terminal portion of VP2 (amino acids 47-52) near the bottom of the junction, which was modeled as a loop region in the empty particle but is disordered in the uncoating intermediate, suggesting more drastic conformational changes during uncoating (20). Crystal Structure of the EV71-NE1 Empty Particle-The crystal structure of the EV71-NE1 empty particle was determined to assess the effects of the insertion on the capsid structure.…”

“…Recently, we and others have reported the structures of the virion, the naturally occurring empty particle, an uncoating intermediate or the A-particle, and the empty particle after the uncoating of EV71 as determined by x-ray crystallography or cryo-EM techniques (17)(18)(19)(20)(21). Despite its elusive role during the viral life cycle, the empty particle shares similar structural features with the virion and the uncoating intermediate.…”