Abstract
Author SummaryPhage T4 is a prototype for tailed bacteriophages, the most abundant organisms on the planet, as well as for large eukaryotic viruses such as herpes viruses. These viruses encode powerful machines to package their genomes tightly inside an icosahedral-shaped capsid “head.” Packaging into the capsid occurs via a dodecameric portal, localized in one of the vertexes of the capsid. Packaging requires precise orchestration of a series of steps: assembly of an empty prohead, concatemer cutting and attachment of the motor-DNA complex to the portal vertex, ATP-fueled DNA translocation until the head is full, DNA cutting to terminate packaging, detachment of the motor, and sealing of the packaged head by “neck” assembly. Sequential conformational changes, particularly in the portal, are thought to drive these transitions such that assembly proceeds directionally and irreversibly. Here, we found that the phage T4 packaging machine on various capsids is highly promiscuous, translocating DNA into proheads but also, unexpectedly, into previously filled virus heads. Other studies have shown that in filled viral capsids the structure of the portal is fundamentally altered, and it was thought that the packaging mechanism on full heads would be irreversible. We show that full heads, or heads that are emptied of most of their packaged DNA, can reassemble the packaging machine and use it to re-fill the capsid with any DNA molecules. These results challenge the classic sequential virus assembly models, suggest an explanation for the evolution of viral genomes that fit capsid volume, and point the way to a novel nanocapsid delivery system in which the viral packaging machine (portal and motor) could be used to translocate DNA and other therapeutic molecules into synthetic capsids.
Highlights
Tailed bacteriophages are ubiquitously distributed in nature and are the most abundant organisms on the planet [1]
The proteins gp13, gp14, and gp15 assemble into a neck that seals off packaged heads, with the gp13 protein directly interacting with the portal protein gp20 following DNA packaging and gp14 and gp15 assembling on the gp13 platform
According to the well-accepted sequential assembly models, the heads following completion of DNA packaging are expected to have the least affinity for the packaging motor but high affinity for the neck proteins
Summary
Tailed bacteriophages are ubiquitously distributed in nature and are the most abundant organisms on the planet [1]. These, in particular, bacteriophage T4, are excellent models to elucidate the mechanisms of DNA condensation and decondensation in living organisms. A capsid of precise dimensions is first assembled, often with a single type of protein subunit polymerizing around a protein scaffold (Figure 1). A cone-shaped dodecameric portal initiates assembly and remains at the special five-fold vertex of the isometric capsid (prehead), facilitating all subsequent transactions: DNA entry, tail attachment, and DNA ejection [7,8]. The scaffold is removed, creating an empty space inside the capsid (prohead or procapsid) for encapsidating the viral genome (Figure 1A). A packaging ATPase motor, known as the ‘‘terminase,’’ recognizes and cuts the concatemeric viral DNA and docks at the narrow protruding end of the prohead portal, inserting the DNA end into the
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