Viral DNA Retention and Ejection Controlled by Capsid Stability

2017-08-14T00:00:00Z (GMT) by Krista Freeman
My thesis work explores the importance of metastability in the lifecycle of DNA viruses. Metastability
refers to the fact that DNA viruses spend the majority of their lifetime in an energetically unfavorable state –
that is, with a significant amount of stored internal energy in the form of tightly packaged DNA. This presents
both a challenge and an advantage to the virus: the viral capsid must be both stable enough to retain its
pressurized load during transit through harsh environments to the host, but also be unstable enough to
quickly eject its genome into a host to begin the infection cycle. Here, I present data discussing both the
destabilization occurring during the DNA ejection process and the mechanical stability needed to retain
pressurized DNA.
To study the controlled destabilization a capsid undergoes during the infection process, I have used
a combination of light scattering, x-ray scattering and cryo-electron microscopy to track the dynamics of viral
DNA ejection. I showed first that receptor-bound phages eject their DNA stochastically with temperaturedependent
rates correlated to an activation energy barrier [results published in the Journal of Physical
Chemistry B (DOI: 10.1021/acs.jpcb.6b04172)]. In addition to this temperature dependence, the rate of the
stochastic DNA ejection events is also greatly influenced by internal DNA pressure. A greater DNA pressure
corresponds to more internal energy exerted on the portal, and thus a smaller excess energy barrier to
overcome in destabilizing and opening the portal. This result suggests that DNA ejection occurs only after a
2-step unlocking process: the bacteriophage must not only bind to its receptor, but also acquire sufficient
energy to critically destabilize the portal through DNA pressure and heat.
To study the capsid stability necessary to retain pressurized DNA, I used atomic force microscopy
to measure the critical mechanical strength of herpes simplex virus type 1 (HSV-1) capsids with varying
degrees of mechanical reinforcement. The data reveals that the capsid gains critical mechanical strength
from external stabilization by the minor capsid protein UL25. To achieve full stability, the capsid must be fully
occupied by a sufficient number of full-length UL25 copies. That is, without this full occupation of the capsid
by UL25 proteins, infectious, DNA-containing virions cannot be formed. This suggests that the capsid
structure and genetic coding are finely tuned to create a viral particle which is just strong enough and stable
enough to ensure successful infection and replication, with no excess material carried or created. Thus, we see that pressure is essential for efficient infection by viruses but also that pressure
requires an extremely strong capsid. There must be a balance between storing enough energy (as DNA
pressure) to power DNA ejection and storing more energy than the capsid can hold within its walls. This
balance of pressure has been optimized through evolution, and results in the finely tuned and highly
reproducible replication cycle of viruses. Understanding the purpose of and structural requirements for this
stored energy will help the overall understanding of the mechanisms of viral infection.