Herpes viruses are a family of human pathogens that establish lifelong latent infections from which viruses periodically reactivate, causing a number of ailments. Reactivations are responsible not only for a significant disease burden but also for a high rate of new infections. During reactivation, multiple infectious progeny virions are assembled and released from the cell in a process called egress. The first step in this process is nuclear egress whereby assembled nucleocapsids exit the nucleus into the cytoplasm. Although key viral nuclear egress participants have been identified, critical mechanistic details are lacking. The long-term goal of this research is to elucidate the process of nuclear egress at the atomic level for two alpha herpes viruses, herpes simplex (HSV) and pseudorabies (PRV). The objective of this proposal is to initiate a biophysical, biochemical, and structural characterizatin of the HSV-1 nuclear egress complex UL31/UL34 (NEC). This research is driven by a hypothesis that the NEC enables primary envelopment by a mechanism similar to that of matrix proteins of negative-stranded RNA viruses and cellular ESCRT-III proteins, which promote budding by deforming the membrane in an oligomerization-dependent manner.
Aim 1 will focus on the determination of the affinity and stoichiometry of UL31/UL34 interactions, mapping of the minimal fragment of each protein that can form the NEC, and initiation of crystallization trials with the goal of determining the structures of UL31, UL34 and the UL31/UL34 complex.
Aim 2 will test the hypothesis that purified HSV-1 NEC can deform membranes in vitro in the absence of other viral or host proteins, in oligomerization-dependent manner. The proposed studies will provide important insights into the structure of the NEC and the mechanism by which it enables membrane deformation during nuclear egress. Obtaining the crystal structures of UL31, UL34, and the NEC would be a significant achievement that would have a major impact in the field of herpes virology. Known functional data can be mapped onto the structures to obtain insight into the structural basis of functional phenotypes. In the future, this knowledge will allow for the structure-guided design of drugs targeting these interfaces. Showing that the purified NEC alone can deform membranes in vitro not only will demonstrate that it is necessary and sufficient for this process but will also allow the development of an in vitro assay in which the membrane deformation activity of UL31 and UL34 mutants can be correlated with their in vivo budding phenotypes. In the future, the results obtained in the course of the proposed work will form the foundation for a subsequent R01 application to elucidate fully the mechanism of herpes virus egress.
Herpes viruses cause lifelong infections whereby viruses remain in a dormant state from which they periodically reactivate causing a number of ailments ranging from cold sores and genital herpes to blindness, encephalitis, cancers, and life-threatening conditions in the immunocompromised individuals and in newborns. The subject of this proposal is herpes virus nuclear egress - a critical stage in the process by which infected cells produce and release new viral particles. In this proposal we will study the nuclear egress complex, the key player in nuclear egress, and its components. New knowledge obtained as the result of the proposed work may lead to successful therapeutic interventions based on blocking the release of new viral particles.
|Bigalke, Janna M; Heldwein, Ekaterina E (2016) Nuclear Exodus: Herpesviruses Lead the Way. Annu Rev Virol 3:387-409|
|Bigalke, Janna M; Heldwein, Ekaterina E (2015) The Great (Nuclear) Escape: New Insights into the Role of the Nuclear Egress Complex of Herpesviruses. J Virol 89:9150-3|
|Bigalke, Janna M; Heldwein, Ekaterina E (2015) Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J 34:2921-36|
|Bigalke, Janna M; Heuser, Thomas; Nicastro, Daniela et al. (2014) Membrane deformation and scission by the HSV-1 nuclear egress complex. Nat Commun 5:4131|