Molecular basis for the acid-initiated uncoating of human enterovirus D68

Abstract

Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 (“E1”) particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.

Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation

“…Hence, VP4 could contribute to the poorly ordered density on the inside of the capsid close to the vertices, attributed primarily to RNA, as well as to the density spanning the capsid. In corroboration of this finding, the presence of a tiny amount of VP4 was recently reported in A-particles of enterovirus D68 induced by acid treatment (49).…”

“…The first modeled residue of the VP1 N terminus (Asn 55) lies in close proximity to the unassigned density near the 2-fold axis inside the particle; therefore, the unassigned density could be the VP1 N termini now traversing the capsid and exposed on the surface of the treated capsid similarly to the interpretation for expanded particles of other enteroviruses (Fig. 6D, red arrows in the inset) (20,26,49). In addition, part of this poorly ordered density seen on the exterior could be attributed to a flexible VP3 loop (residues Thr 175 to Asp 183).…”

“…5D). Interestingly, this RNA-capsid interaction is in proximity to the N terminus of VP1 that appears to be extruded from the treated capsid, similarly to the VP1 in expanded poliovirus, coxsackievirus A16, and enterovirus D68 (20,26,49). VP4 could not be identified in the reconstruction, suggesting that it is no longer icosahedrally ordered or the occupancy is much reduced as suggested also by autoradiography.…”

The In Silico Prediction of Hotspot Residues that Contribute to the Structural Stability of Subunit Interfaces of a Picornavirus Capsid

“…It is generally accepted that the interfaces between pentamer subunits are major sites of structural rearrangement during capsid uncoating. For example, studies have widely documented the formation of pores at these interfaces [67][68][69][70][71][72][73] or the loss of one or more pentamer subunit(s) [53,58,74,75] upon exposure of the picornavirus capsid to denaturing conditions. To identify the hotspot residues that are critical to the binding specificity and stability of the pentamer interfaces, complexes of two opposing protomers at the two-fold axis and two adjacent protomers at the three-fold axis were analysed.…”

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

“…Unlike mature native virions (160S particles), whose capsids form a closed surface, the expanded 135S particles have holes in the capsid at the icosahedral 2-fold and quasi-3-fold axes. These openings permit the externalization of the membraneinteractive N-terminal extension of capsid protein VP1 [11,14] in poliovirus and in the 135S particles of other enteroviruses [19][20][21][22][23][24][25][26] and presumably VP4, which is myristoylated [27]. In vitro studies with model membranes have shown that these polypeptide chains are responsible for anchoring the virus to the host cell membrane [11,28].…”