An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins.

The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-Å resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers T _end = 13 laevo for the icosahedral caps and T _mid = 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein ( hoc ) and small outer capsid protein ( soc ), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc ^– and hoc ^– soc ^– T4 structures.

Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead's portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL's N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage ϕ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics.

Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage φ29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for φ29, averaging ≈700 bp/s and ranging up to ≈2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in 300 s ⁻¹ . The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.