Insights into the Structure and Assembly of the Bacteriophage ϕ29 Double-Stranded DNA Packaging Motor

Abstract

The tailed double-stranded DNA (dsDNA) bacteriophage 29 packages its 19.3-kbp genome into a preassembled procapsid structure by using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near-crystalline densities within the confined space of the capsid requires that the packaging motor work against significant entropic, enthalpic, and DNA-bending energies. The motor consists of three phage-encoded components: the dodecameric connector protein gp10, an oligomeric RNA molecule known as the prohead RNA (pRNA), and the homomeric ring ATPase gp16. Although atomic resolution structures of the connector and different pRNA subdomains have been determined, the mechanism of self-assembly and the resulting stoichiometry of the various motor components on the phage capsid have been the subject of considerable controversy. Here a subnanometer asymmetric cryoelectron microscopy (cryo-EM) reconstruction of a connector-pRNA complex at a unique vertex of the procapsid conclusively demonstrates the pentameric symmetry of the pRNA and illuminates the relative arrangement of the connector and the pRNA. Additionally, a combination of biochemical and cryo-EM analyses of motor assembly intermediates suggests a sequence of molecular events that constitute the pathway by which the motor assembles on the head, thereby reconciling conflicting data regarding pRNA assembly and stoichiometry. Taken together, these data provide new insight into the assembly, structure, and mechanism of a complex molecular machine. IMPORTANCEViruses consist of a protein shell, or capsid, that protects and surrounds their genetic material. Thus, genome encapsidation is a fundamental and essential step in the life cycle of any virus. In dsDNA viruses, powerful molecular motors essentially pump the viral DNA into a preformed protein shell. This article describes how a viral dsDNA packaging motor self-assembles on the viral capsid and provides insight into its mechanism of action.

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

“…The ATPase must assemble to the head; in the other phages, the large subunit of the terminase interacts directly with the connector and perhaps the head shell, whereas in 29 it is the prohead binding domain of pRNA (Fig. 6C, blue shading) that binds directly to the head, interacting with the shell protein (14) and the connector (17)(18)(19). Prohead binding thereby positions the A-helices of pRNA (Fig.…”

“…1C) (16). It is a component of the prohead, making critical interactions with both the connector (17,18) and the head shell (14,19,20). Early showing the dodecameric connector (portal) in green, the pentameric pRNA ring in magenta, and the pentameric ring ATPase gp16 in blue.…”

“…pRNA assembles into a pentameric ring on the prohead via intermolecular base pairing between complementary loops of adjacent RNAs (14,15). The pRNA ring is positioned between the connector and ATPase components, with projecting spokes serving as the scaffold for assembly of the ring form of the packaging ATPase gp16 (Fig.…”

Breaking the symmetry of a viral capsid

“…Indeed, the viral genome itself is present as only a single copy and thus cannot assume the icosahedral symmetry of the surrounding capsid. Additionally, it is imperative that regions of the virus shell deviate from the global icosahedral symmetry of the capsid to successfully accomplish critical aspects of the viral life cycle such as genome packaging/release and host cell recognition/attachment (4,(6)(7)(8)(9). Until recently, structural biology has largely ignored these essential deviations from symmetry.…”

“…Coupled with the availability of powerful computing resources, these advances have made it possible to approach atomic resolution for certain well-ordered macromolecular assemblies and to reconstruct 3D volumes of viruses to subnanometer resolution without imposing any symmetry (6,14,18,19). Building on these advances, Gorzelnik et al have used cryo-EM to determine the structure of phage Qβ to 7-Å resolution without imposing any symmetry.…”