Genome packaging in dsDNA viruses is regulated by a pressure-sensitive molecular switch

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Targeted mutagenesis of the P22 portal protein reveals the mechanism of signal transmission during DNA packaging

Text by Greg Bedwell

DNA packaging in dsDNA bacteriophage and herpesviruses relies on a powerful molecular motor to pump the viral genome into a pre-formed capsid.  In these viruses, the gateway into the capsid is formed and regulated by a ring-like structure referred to as the viral portal.  In its roles as both gateway and gatekeeper, the portal senses and responds to the density of DNA in the capsid interior and transmits that information to the motor on the capsid exterior.

The work presented here began in 2014 as an effort to better understand the aspect of signal transmission during DNA packaging. We knew of two single point mutations in the portal of bacteriophage P22 that increased the amount of DNA packaged into virions.  Mapping the location of these mutants onto the portal structure led us to realize that they were buried nearby the protein core.  Unable to interact with DNA directly, how were these mutations working to effectively “desensitize” the portal sensor?

We knew that the answer to this question lay in better understanding the sensing mechanism itself.  Using the already-identified overpackaging mutants as a springboard, we made additional mutants at these and other nearby residues to try to gain insight into how the sensing mechanism worked.  To our surprise, all of our amino acid substitutions resulted in fully viable phage and unique overpackaging phenotypes.  The trends that we observed led to the hypothesis that the overpackaging mutants were subtly altering the structure and/or dynamics of the protein in this region.  We confirmed this hypothesis with various biophysical measurements made on purified proteins.

We knew from structural data that the portal exists in two distinct pre- and post-packaging forms.  Comparison of these two structures in the context of our data suggested that the dynamic perturbations we observed in our mutants made the “natural” transition between forms more difficult to trigger due to changes in things like side chain packing.  This was the clue that we needed to better define the sensing mechanism itself.  DNA packaging is a continuous process that is accompanied by a continuous increase in pressure.  The P22 portal undergoes significant compression during the process of packaging.  Alterations in side chain packing are one of the key determinants in protein compressibility.  Our findings indicate that viral portal proteins are biological pressure transducers.  Pressure buildup inside the capsid during packaging induces gradual conformational changes in portal that eventually lead to packaging termination.

blogpicturetailmachine

Figure legend

During DNA packaging in P22, the dsDNA genome is pumped through the portal, a component of the tail machinery shown in the micrograph above.  The findings of our study indicate that the portal serves as a macromolecular pressure sensor that responds to the continuous increase in pressure that accompanies DNA incorporation into the viral capsid.

Introducing the authors

58779 YVIRO authors

Pictured: Greg Bedwell (left), Research Fellow, Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute. Boston MA. (current) and  Peter Prevelige, Professor, Department of Microbiology, University of Alabama at Birmingham, Birmingham AL.

About the research

Targeted mutagenesis of the P22 portal protein reveals the mechanism of signal transmission during DNA packaging
Gregory J. Bedwell, Peter E. Prevelige Jr.
Virology, Volume 505, May 2017, Pages 127–138