The Herpes Virology Group

Carnegie Mellon University, Department of Physics
Principal Investigator: Prof. Alex Evilevitch

http://www.cmu.edu/physics/index.html
 

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Herpes Virology Research

Herpesviruses are a leading cause of human viral disease, second only to influenza and cold viruses. Herpesviruses consist of a double-stranded (ds) DNA genome contained within a protein shell, termed the capsid, that is surrounded by an unstructured protein layer (the tegument) and a lipid-envelope. During viral replication, an ATP-dependent motor packages the genome into a preformed capsid through a unique opening created by the portal complex. Herpes Simplex virus type 1 (HSV-1) is a prototypical model system to study the general infection mechanisms of herpesviruses and other viruses that release their genome into the cell nucleus without capsid disassembly. We have recently shown that HSV-1 genome packaging creates an internal pressure of tens of atmospheres within the viral capsid. This pressure results from bending stress and repulsive forces acting on the tightly packaged DNA molecule. We also found that despite its liquid crystalline state inside the capsid, the DNA is fluid-like which facilitates its ejection into the cell nucleus during infection. The fluidity or, equivalently, mobility of the closely packaged DNA strands caused by interstrand repulsive interactions is regulated by the ionic environment of the cellular cytoplasm.

Between rounds of replication, the virion must be sufficiently stable to ensure that the packaged genome is retained within the capsid. Conversely, during infection the virion must be unstable enough to allow genome release into the cell nucleus. A precise balance between these physical aspects of the viral capsid and its encapsidated genome is crucial to the viral replication cycle. Using HSV-1 as our primary model system, we investigate the roles of intracapsid DNA mobility, internal DNA pressure and capsid stability for viral replication with respect to retention of the packaged genome inside the capsid and its subsequent ejection during infection. These studies provide new insights into the key mechanisms facilitating as well as inhibiting viral infectivity.

Physical Virology of Herpesviruses and Phage

Viruses are simple lifeless entities that cannot reproduce on their own and therefore depend on host cells to provide them with the necessary life support mechanisms. Simplified, all viruses consist of a protein shell (capsid) that protects the viral genome (DNA or RNA). To infect, the viral genome must enter the cell, where it hijacks the host cell’s machinery and synthesizes multiple copies of virions. This can lead to cell lysis, which is a lethal event.

Physical virology is a rather new field that seeks to define the physical mechanisms controlling virus development. This knowledge can provide information essential to the rational design of new anti viral strategies with less specificity for a limited number of viruses. Furthermore, biological and physical simplicity relative to other biological systems have made viruses an attractive physical model system to study fundamental prosperities of DNA compaction and translocation as well as protein self-assembly using viral capsids.

Our Biophysical Tools

Evilevitch's Herpes virology group investigates fundamental physical principles that control viral encapsidation and genome release. Our group has discovered a way to determine genome pressure in viral capsid and found that to be as high as 20 atm in human Herpes Simplex Type 1 virus, a pressure equivalent to that at a depth of 600 ft in the ocean. We are specifically interested in determining the physical nature of genome packaging and release, the kinds of pressures involved, the strengths and elastic properties of the capsids, and limits on the amount of material that can be encapsidated. The main tools in the lab are: Atomic Force Microscopy (AFM), microcalorimetry, high resolution cryo electron microscopy and single molecule fluorescence.

*Cryo-EM: 3D Cryo-electron microscopy reconstructions of DNA-filled and empty phage capsid.
* AFM: Atomic Force Microscopy high-resolution imaging of page capsids and breaking the phage with an AFM cantilever.
* ITC: Isothermal Titration Calorimetry setup showing the titration of phage solution (from the syringe) into the LamB receptor solution in the sample cell.