Fred L. Homa, PhD
539 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219
Research in our lab is focused on understanding the mechanism of herpesvirus capsid assembly and DNA packaging. The herpesviruses comprise a large family of double stranded DNA viruses. Several of these viruses are important human pathogens and all remain with the host for life by residing in a latent or quiescent state where they avoid immune clearance. Primary or recurrent infections can be life threatening in immunocompromised patients, such as AIDS or transplant patients, where infection with human cytomegalovirus (HCMV) can result in retinitis, pneumonia, and gastrointestinal disease. Current treatments against herpes simplex viruses (HSV-1 and HSV-2) and the other human herpesviruses rely primarily on blocking viral DNA replication. Assembly of herpesvirus capsids involves highly specific interactions among at least five different proteins and seven additional proteins are involved in DNA packaging and cleavage. Most of the proteins involved in capsid assembly and DNA packaging are conserved suggesting that these mechanisms will also be similar for all herpesviruses.
Research in our lab is focused on understanding the mechanism of herpesvirus capsid assembly and DNA packaging. The structure of the HSV‑1 capsid was determined from three-dimensional image reconstructions computed from cryo-electron micrographs of capsids. The capsid shell is composed predominantly of four proteins, a major capsid protein, VP5, and three less abundant proteins, VP19C, VP23 and VP26. Herpesvirus DNA is incorporated into preassembled capsids through a ring-shaped portal present at a unique vertex. This process requires the action of six cleavage/packaging proteins that interact with the capsid either during capsid assembly or during DNA packaging. The terminase proteins (UL15, UL28, UL33) act as part of an ATP-dependent pump that drives DNA into the procapsid and cut the concatemeric DNA at specific sites yielding a capsid containing the intact genome. The final step in the process is “capsid completion” that results in the formation of a stable DNA-containing capsid. Of the seven HSV-1 proteins required for the cleavage/packaging reaction, only UL25 is required for maintaining the stable DNA-containing capsid; without UL25 the packaged DNA is lost resulting in “empty” A-capsids. The cleavage/packaging and capsid completion reactions can be viewed as separate steps in the overall process of generating a stable DNA-containing capsid. The main goals of this project are to determine the function(s) of the individual cleavage/packaging proteins in this process in order to achieve a detailed understanding of the HSV DNA cleavage and packaging mechanism.
Ongoing studies are focused at defining the role of the UL25 protein in DNA packaging with regards to its functions in retention of viral DNA by binding to capsid vertices through its interaction with the UL17 protein. Genetic and biochemical approaches are being be used to determine the role of UL28 in the assembly of a functional terminase complex and its interactions with UL15 and UL33. These studies utilize genetic, biochemical and structural (cryoEM) approaches to understand how the protein complexes assemble and carry out the cleavage/packaging reaction.
In collaboration with Dr. James Conway’s lab (Department of Structural Biology) molecular genetics and cryo-electron microscopy (cryoEM) are being used to obtain high resolution models of the HSV capsid and the essential minor proteins that interact with the capsid during and following DNA packaging. The locations of most of these essential minor proteins are not known nor are details of their interactions with each other and the capsid. Capsids incorporating specifically labeled subunits will be visualized by cryoEM to identify the locations of subunits. The knowledge obtained from these studies enables not only a significantly better understanding of herpesvirus capsid structure, but also provides the means to reveal aspects of how the viral DNA packaging machinery interacts with the capsid during and after DNA packaging. In addition, the essential minor proteins offer novel and highly specific structural targets for the development of antivirals. It is anticipated that the studies in this proposal will not only enhance our understanding of the mechanisms of genome maturation and encapsidation and lead to the development of novel strategies for antiviral therapy.