The life cycles of many viruses include a self-assembly stage in which a powerful molecular motor must pack the DNA genome into the virus's preformed shell (the capsid). How it manages this intricate feat has been subject to debate, but we know that the DNA passes into the capsid shell through a channel formed by a structure called the connector. Scientists have speculated that rotation of the connector complex might feed the DNA into the capsid as it turns.
In new research published online this week in the open access journal PLoS Biology, researchers Thorsten Hugel, Jens Michaelis, Craig Hetherington, and Carlos Bustamante present their detailed investigation into how the Bacillus subtilis bacteriophage f29 crams its DNA into the capsid following replication.
Using their innovative system, Hugel and colleagues were able to observe capsids as they pack their DNA to directly test the connector rotation hypothesis. They found that it is more than likely not the mechanism by which the DNA is packed. The researchers combined single-molecule fluorescence polarization with "magnetic tweezers" to glue the end of the capsid farthest from the hole to a slide using antibodies, and then they draw out the DNA being packaged in the opposite direction by attaching a magnetic bead to its loose end and applying a magnetic field. Importantly, they also labeled the connector complex with fluorescent dye molecules so they could observe its motion using single-molecule fluorescence polarization spectroscopy.
The researchers then looked at connector movement during DNA packaging in six f29 mutants. After attaching the fluorescent molecules to the connector complex, they set the mutants to work packaging DNA in a flow chamber. As the connectors functioned, the researchers shone homogeneously polarized light on them and recorded the pattern of fluorescence produced in two channels at right angles to one other.