Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNAprotein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp 38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.genome uncoating | X-ray analysis | 3D cryo-EM | picornavirus H uman rhinoviruses (HRVs) cause the common cold. Although seldom severe, this disease is widespread and frequent in man; HRVs thus have considerable economic impact due to expenditure on medication and lost working days. More than 150 serotypes belong to the genus Enteroviruses (EVs) of the Picornaviridae family, which includes serious human and animal pathogens. In addition to phylogenetic classification into species A, -B, and -C, HRVs are divided into a minor receptor group (12 HRV-A) that bind low-density lipoprotein receptors (LDLRs), and a major receptor group (more than 89 HRV-A and -B serotypes) that use intercellular adhesion molecule 1 (ICAM-1) for cell entry (1). HRV-C binds an unknown receptor (2).The EV icosahedral shell is built from four viral proteins (VP1-4) that encase a single-stranded (+)-sense RNA genome. Sixty copies each of these four polypeptides assemble on a T = 1 (pseudo T = 3) lattice, ∼30 nm in diameter. VP1, VP2, and VP3 are surface-exposed; the small myristoylated VP4 is internal. In the mature virion, the N-terminal extensions of VP1, VP2, and VP3, together with the entire VP4, interact in an intricate network beneath the shell (Fig. S1) (3, 4).In the cytosol, the viral RNA is translated into a ∼250 kDa precursor polyprotein that is processed by viral proteinases. Assembly of the viral shell involves immature pentamers built from VP0, VP1, and VP3. VP2 and VP4 arise late in infection through VP0 cl...

birnavirus replication ͉ infectious bursal disease virus ͉ noncanonical palm ͉ virus evolution ͉ double-stranded RNA viruses