Two lineages of viral RNA-dependent RNA polymerases (RDRPs) differing in the organization (canonical vs. noncanonical) of the palm subdomain have been identified. Phylogenetic analyses indicate that both lineages diverged at a very early stage of the evolution of the enzyme [Gorbalenya AE, Pringle FM, Zeddam JL, Luke BT, Cameron CE, Kalmakoff J, Hanzlik TN, Gordon KH, Ward VK (2002) J Mol Biol 324:47–62]. Here, we report the x-ray structure of a noncanonical birnaviral RDRP, named VP1, in its free form, bound to Mg ²⁺ ions, and bound to a peptide representing the polymerase-binding motif of the regulatory viral protein VP3. The structure of VP1 reveals that the noncanonical connectivity of the palm subdomain maintains the geometry of the catalytic residues found in canonical polymerases but results in a partial blocking of the active site cavity. The VP1–VP3 peptide complex shows a mode of polymerase activation in which VP3 binding promotes a conformational change that removes the steric blockade of the VP1 active site, facilitating the accommodation of the template and incoming nucleotides for catalysis. The striking structural similarities between birnavirus (dsRNA) and the positive-stranded RNA picornavirus and calicivirus RDRPs provide evidence supporting the existence of functional and evolutionary relationships between these two virus groups.

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. Human Rhinoviruses (HRVs), members of the Picornaviridae family, are small non-enveloped viruses possessing an icosahedral capsid that protects the single-stranded RNA genome. Although much is known about their binding to cell receptors and their uptake into the host cell, the mechanism by which their genomic RNA leaves the capsid and arrives to the cytosol to initiate replication is poorly understood. In HRV2, a member of the minor group HRVs, upon binding to lipoprotein receptors (LDL-R) on the cell surface virions are taken up into vesicles and directed to early endosomes. The low pH conditions found in the endosome, and not the binding to LDL-R, catalyze the delivery of the viral genome. The crystal structure of the HRV2 empty particle, representing the last stage of the uncoating process, unveils the structural rearrangements produced in the viral capsid during the externalization of the VP1 N-terminus and the delivery of the genomic RNA. We propose that RNA exit occurs through large capsid disruptions that are produced at the particle two-fold symmetry axes. Our data also suggests that the VP1 N-terminus would be externalized through a new pore, opening at the canyon floor.

Significance: Most antibiotics do not interfere with viral infections. Rifampicin is a notable exception, as it inhibits several poxviruses, including the causative agent of smallpox. However, the inhibition of viral assembly is unrelated to the antibacterial activity of rifampicin against microbial RNA polymerases. Here, we reveal how the antibiotic prevents the recruitment of an essential scaffolding protein to nascent viral membranes. Based on these results, we provide a structural model of membrane assembly that is distinct from budding through cellular membranes and is most likely conserved in many large DNA viruses. Together, the mechanism of membrane assembly and structural models provide avenues to develop broad spectrum inhibitors against human and animal poxviruses.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 Å resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.

Significance: Human rhinoviruses (HRVs) cause the common cold and exacerbate chronic pulmonary diseases. Their single-stranded RNA genome is protected by an icosahedral capsid and must be released into the host cell cytosol for translation and replication. Using X-ray and cryo-EM analyses, we identified structural alterations that take place in the virus architecture during infection. In acidic endosomes in vivo and in our experimental conditions, the native virion is converted into the expanded, porous uncoating intermediate A-particle. This is accompanied by altered RNA–protein contacts at the inner capsid wall, leading to major changes in RNA conformation that result in a well-organized RNA layer. These rearrangements suggest that the RNA–protein interactions prepare RNA and facilitate its subsequent egress via a well-ordered mechanism.