Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.bacteriophage T7 maturation | DNA packaging intermediates | noncovalent topological linking | procapsid | single-particle cryo-EM M any dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1-6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7-9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the shorttail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From lowresolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-...

Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used singleparticle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.T he tailed dsDNA bacteriophages and human dsDNA viruses, such as herpesviruses and adenoviruses, share many features of structural organization and life cycle. Viruses of these diverse families evolved from a common ancestor that existed before the divergence of prokaryotes, archaea, and eukaryotes, the three domains of life (1-3). Assisted by a scaffolding protein, these viruses assemble a procapsid with a symmetrical, usually icosahedral, outer shell. One of the shell's 12 fivefold vertices is replaced by a symmetry-mismatched 12-fold portal. The dsDNA genome is subsequently pumped into the capsid chamber through the portal channel, accompanied by exit of scaffolding proteins. The energydependent DNA packaging process is performed by a complex machinery involving multiple, stacked layers at the portal vertex. Functions of the layers include DNA binding/loading (small terminase), ATPase hydrolysis (large terminase), DNA channel (portal), and DNA condensation (internal proteins, sometimes in several layers). The detailed structural basis and functional mechanism of the DNA packaging process are under extensive investigation (4-6).Structural analysis of phage portal vertex and associated proteins has impact beyond understanding viral assembly. Symmetry mismatches also occur in the capping and branching of actin filaments to control...

The tightly packaged dsDNA genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼10 -4 of packaged DNA in all particles) and are initially detected by non-denaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy (cryo-EM) of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNAcapsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) forms a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings like those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection.

Evidence is presented here that in vivo bacteriophage T3 DNA packaging includes capsid hyperexpansion that is triggered by lengthening of incompletely packaged DNA (ipDNA). This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNAcontaining capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids is based on non-denaturing two-dimensional agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDSPAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The hypotheses are presented that (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.