Herpes simplex virus 1 (HSV-1) causes diseases that range from painful skin lesions to keratitis and encephalitis. HSV-1 encodes an essential protein called ICP27 that is involved in a diversity of functions during viral infection. ICP27 has the intriguing ability to interact with viral mRNA and a multiplicity of host cell proteins, hijacking hem to benefit virus production. Thus, ICP27 could conceivably serve as a target for antiviral intervention. The future development of antivirals requires an understanding of how the cooperative assembly of these essential multicomponent complexes occurs and how the assembly is regulated. We will study the role of cooperativity in protein-protein and protein-RNA interactions mediated by ICP27 and characterize the binding interfaces of its complexes to determine how ICP27 is assembled in different complexes during viral infection, with the goal of revealing the molecular mechanism of its function. The structural information will be used to explore the role of interaction interface residues in ICP27 function during infection. ICP27 consists of a number of structured as well as intrinsically-unstructured domains, which participate in a large number of diverse protein-protein and protein-RNA interactions. We hypothesize that these interactions may be multi-site and therefore cooperative, mediated by regions that may be distant in the primary sequence. We postulate that transient interactions also are possible, especially when unfolded regions are involved. We hypothesize that at least some of ICP27's interactions are regulated by phosphorylation and/or arginine methylation. We will test these hypotheses in two specific aims: 1) To study the direct binding interfaces of ICP27 with cellular proteins and viral mRNA by generating shorter protein constructs in which structural stability is not perturbed, and expressing them in amounts sufficient for structural biology studie including traditional and novel Isotopically-Discriminated (IDIS) NMR spectroscopy as well as biophysical approaches. Cooperativity will be analyzed in interactions between short functional fragments of intrinsically unstructured N-terminal ICP27 and partners that interact within this region, including viral RNA and host cell proteins. In vitro post-translational modifications of ICP27 will be performed to explore how ICP27-multi-protein complex assembly is affected by phosphorylation and arginine methylation. We will also endeavor to perform structural studies of folded domains from the C-terminal part of ICP27 and to characterize interactions with its cellular protein partners. Because ICP27 undergoes a head-to-tail intramolecular interaction in vivo, a chimera containing appropriate fragments of the N- and C-termini could be created to study how these regions interact. 2) To explore the role of interaction interface residues in ICP27 function during viral infection, recombinant viruses will be constructed bearing point mutations at interaction interfaces and will be characterized for in vivo interactions and ICP27 functional activities and effects on viral infection.

Public Health Relevance

Herpes simplex virus 1 (HSV-1) causes diseases that range from recurrent painful skin lesions to blindness resulting from keratitis and morbidity and mortality due to encephalitis. Recent studies suggest that HSV-1 is a contributing factor in Alzheimer's disease. Further, HSV infection increases the risk for HIV acquisition. Current antivirals do not prevent or fully suppress HSV replication, which raises a major health concern. All human herpesviruses encode an essential multifunctional protein, which is called ICP27 in HSV. ICP27 has the intriguing ability to be involved in a diversity of functions during infection, involving hijacking host macromolecular complexes to benefit virus production. ICP27 interacts with viral mRNA and a multiplicity of host cell proteins and thus could be a target for antiviral intervention. How the cooperative assembly of ICP27 in these complexes occurs and how it is regulated, is unclear, but is required for future development of targeted antivirals directed at interaction interfaces. Further, what we learn about ICP27 may be helpful in designing drug targets for other herpesviruses, such as KSHV, EBV and HCMV, which encode ICP27 homologues.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Challberg, Mark D
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University of California Irvine
Schools of Medicine
United States
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