Abstract
Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.
Highlights
Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors
The large terminase protein docks onto the portal protein, which is embedded in the capsid wall, and translocates DNA using free energy liberated from ATP hydrolysis
P23-45 and P74-26 belong to the genus P23virus, and are closely related to bacteriophage G20c, all of which infect Thermus thermophilus [20]
Summary
Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Akey step in the assembly pathway of double-stranded DNA viruses, including bacteriophage and evolutionarily related herpesviruses, is the packaging of viral genomic DNA into the procapsid [1]. During this process, the large terminase protein docks onto the portal protein, which is embedded in the capsid wall, and translocates DNA using free energy liberated from ATP hydrolysis. Structural information on the portal protein, through which the genome is packaged, demonstrates that the capsid influences the portal’s conformation This has implications for understanding how processes inside and outside the capsid can be coordinated
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