Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense, mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner, double-layered particle (DLP) into the cyotosol. DLP structures determined by x-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a “fivefold hub” of more structured density projecting inward from the VP2 shell of the DLP along each of the 12 fivefold axes. Analysis of the x-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the x-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the fivefold axis. The template strand enters from within the particle, and the dsRNA product of the initial, replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

The MoxR family of AAA+ ATPases is widespread throughout bacteria and archaea but remains poorly characterized. We recently found that the Escherichia coli MoxR protein, RavA (Regulatory ATPase variant A), tightly interacts with the inducible lysine decarboxylase, LdcI/CadA, to form a unique cage-like structure. Here, we present the X-ray structure of RavA and show that the αβα and all-α subdomains in the RavA AAA+ module are arranged as in magnesium chelatases rather than as in classical AAA+ proteins. RavA structure also contains a discontinuous triple-helical domain as well as a β-barrel-like domain forming a unique fold, which we termed the LARA domain. The LARA domain was found to mediate the interaction between RavA and LdcI. The RavA structure provides insights into how five RavA hexamers interact with two LdcI decamers to form the RavA-LdcI cage-like structure.acid stress | alarmone P roteins of the AAA+ superfamily (ATPases Associated with diverse cellular Activities) are highly ubiquitous and found in all kingdoms of life. These proteins are characterized by the structural conservation of a central ATPase domain of about 250 amino acids called the AAA+ module (1, 2). AAA+ ATPases employ the energy derived from ATP hydrolysis to remodel proteins, DNA, or RNA. Typically, the AAA+ domain can be divided into two structural subdomains, an N-terminal P-loop NTPase αβα subdomain that is connected to a smaller C-terminal all-α subdomain. The αβα subdomain adopts a Rossman fold and contains several motifs involved in ATP binding and hydrolysis, including Walker A, Walker B, and Sensor 1 signature sequences (3-6). The all-α subdomain, which contains the Sensor 2 motif (7), is much less conserved across AAA+ proteins.AAA+ proteins form oligomers, usually hexameric rings, in the presence of nucleotides (8). The ATP-binding pocket is located at the interface between two neighboring subunits. A highly conserved arginine from one subunit, called an "arginine finger," contacts the γ-phosphate of bound ATP of the neighboring subunit (9). AAA+ proteins typically go through a cycle of ATP binding, hydrolysis, and release of products. This reaction cycle results in a series of conformational changes and mechanical movements that allow these proteins to exert their activity either directly or through domains attached to the AAA+ domain (3, 10).The RavA protein (Regulatory ATPase Variant A) belongs to the MoxR AAA+ family (11). Limited experimental data suggest a function of MoxR AAA+ proteins as chaperones in the assembly of multimeric complexes and a possible role in small molecule cofactor insertion/removal (11). However, how these proteins act is not clear. In Escherichia coli, the ravA gene is in an operon with another gene of unknown function, which we termed viaA, and the operon is under the control of σ S promoter, suggesting a function of RavA and ViaA under stress conditions (12). This is further substantiated by our discovery that RavA physically interacts with the inducible lysine decarboxylase enzyme, LdcI/CadA...