Viral DNA Retention and Ejection Controlled by Capsid Stability

2018-12-04T18:01:35Z (GMT) by Krista Freeman
My thesis work explores the importance of metastability in the lifecycle of DNA viruses. Metastability<br>refers to the fact that DNA viruses spend the majority of their lifetime in an energetically unfavorable state –<br>that is, with a significant amount of stored internal energy in the form of tightly packaged DNA. This presents<br>both a challenge and an advantage to the virus: the viral capsid must be both stable enough to retain its<br>pressurized load during transit through harsh environments to the host, but also be unstable enough to<br>quickly eject its genome into a host to begin the infection cycle. Here, I present data discussing both the<br>destabilization occurring during the DNA ejection process and the mechanical stability needed to retain<br>pressurized DNA.<br>To study the controlled destabilization a capsid undergoes during the infection process, I have used<br>a combination of light scattering, x-ray scattering and cryo-electron microscopy to track the dynamics of viral<br>DNA ejection. I showed first that receptor-bound phages eject their DNA stochastically with temperaturedependent<br>rates correlated to an activation energy barrier [results published in the Journal of Physical<br>Chemistry B (DOI: 10.1021/acs.jpcb.6b04172)]. In addition to this temperature dependence, the rate of the<br>stochastic DNA ejection events is also greatly influenced by internal DNA pressure. A greater DNA pressure<br>corresponds to more internal energy exerted on the portal, and thus a smaller excess energy barrier to<br>overcome in destabilizing and opening the portal. This result suggests that DNA ejection occurs only after a<br>2-step unlocking process: the bacteriophage must not only bind to its receptor, but also acquire sufficient<br>energy to critically destabilize the portal through DNA pressure and heat.<br>To study the capsid stability necessary to retain pressurized DNA, I used atomic force microscopy<br>to measure the critical mechanical strength of herpes simplex virus type 1 (HSV-1) capsids with varying<br>degrees of mechanical reinforcement. The data reveals that the capsid gains critical mechanical strength<br>from external stabilization by the minor capsid protein UL25. To achieve full stability, the capsid must be fully<br>occupied by a sufficient number of full-length UL25 copies. That is, without this full occupation of the capsid<br>by UL25 proteins, infectious, DNA-containing virions cannot be formed. This suggests that the capsid<br>structure and genetic coding are finely tuned to create a viral particle which is just strong enough and stable<br>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<br>requires an extremely strong capsid. There must be a balance between storing enough energy (as DNA<br>pressure) to power DNA ejection and storing more energy than the capsid can hold within its walls. This<br>balance of pressure has been optimized through evolution, and results in the finely tuned and highly<br>reproducible replication cycle of viruses. Understanding the purpose of and structural requirements for this<br>stored energy will help the overall understanding of the mechanisms of viral infection. <br>