Patricia Spear - US grants

Herpesviruses are etiologic agents or co-factors for certain cancers. In addition, reactivations of herpesviruses from the latent state are frequent in cancer patients and can result in severe and debilitating diseases. The long-range goals of this project are to understand the mechanisms by which herpes simplex virus (HSV) induces cell fusion, a process which facilitates the spread of virus in the human body as well as in cell culture. Experiments have been designed to define the viral and cell factors that influence whether cell fusion will occur after infection. The specific aims are (i) to determine which HSV proteins are necessary and sufficient to induce cell fusion; (ii) to identify viral and cell proteins that interact with cytoplasmic domains of two membrane glycoproteins (gB and gH) known to be required for cell fusion; (iii) to dissect the structure and function of HSV proteins, such as UL45, that may be required for cell fusion but not for infectivity; (iv) to identify the viral protein that interacts with glycosaminoglycans resulting in the activation of cell fusion; and (v) to investigate the role in cell fusion of a newly identified cell surface receptor that enhances HSV-1 entry into cells. The experimental approaches will include construction of plasmids expressing wild-type and mutant forms of viral proteins, for use in cell fusion assays; use of the two-hybrid method and co-immunoprecipitation to investigate protein-protein interactions; isolation and characterization of viral mutants and use of Chinese hamster ovary cells expressing a human cell surface receptor in cell fusion assays.

The ultimate objective of studies proposed here is to define the mechanism or mechanisms by which herpes simplex virus (HSV) invades a cell so as to initiate viral gene expression. Current efforts are directed toward identifying and characterizing cell surface components that influence HSV binding to cells and entry and also toward defining functional domains of the HSV glycoproteins that mediate HSV binding and entry. HSV makes its initial contact with cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans (PGs). The virion glycoprotein gC is principally responsible for this binding whereas other virion glycoproteins (gB, gD, gH and gL) are required for penetration. Most cultured cells are susceptible to HSV infection. One exception is the Chinese hamster ovary (CHO) cell line, which is partially resistant to entry of some HSV strains, such as HSV-1(KOS), but is fully susceptible to others. To better define the cell surface components that influence HSV binding and entry, we intend (i) to identify specific PGs that can serve as receptor (at least in part through their GAG chains) for HSV binding to cells; (ii) to explore further some preliminary evidence that GAGs may activate penetration of HSV, as well as serve as receptors for virus binding and (iii) to identify human genes that can render wild-type and mutant CHO cells susceptible to infection by strains of HSV to which the cells are resistant. To better define functional domains of HSV glycoproteins that mediate HSV infection, we intend to (iv) to isolate and characterize the genetic alterations of HSV mutants that exhibit altered specificity for cell binding; (v) to produce recombinant forms of the HSV glycoproteins that mediate virus binding, in order to explore the cell surface expression and distribution of ligands recognized by the viral glycoproteins with wild-type and altered specificity and (v) to determine which gene or genes of a virus that is competent to infect CHO cells must be transferred to HSV-1(KOS) in order to render HSV-1(KOS) competent for infection, thus identifying variable HSV genes that exhibit cell type- dependence in their ability to mediate viral entry. The results of these studies should better define the cell surface PGs to which HSV binds and the functional domains of viral glycoproteins that mediate the binding. In addition, other cell surface components required for HSV entry may be identified and viral genes that govern entry in a cell-specific fashion will be identified. The information and probes developed during the course of this study will permit a more thorough investigation, then is now possible, of some of the key determinants of HSV pathogenesis. Novel antiviral drugs and therapeutic or prophylactic strategies could result from better understanding the mechanism of cell invasion by HSV.

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) attaches to cells by binding to cell surface heparan sulfate chains and then engages any one of several other cell surface molecules to trigger fusion between the virion envelope and a cell membrane, which leads to viral entry. The other cell surface receptors (entry receptors) include HVEM, a member of the TNF receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and specific sites in heparan sulfate generated by certain 3-O-sulfotransferases. Virion gD is the ligand for all of these entry receptors. HSV-1 gD can bind to HVEM, nectin-1 and 3-O-sulfated heparan sulfate whereas HSV-2 gD can bind to HVEM, nectin-1 and nectin-2. Certain mutant forms of HSV-1 gD (Rid) have lost the ability to bind to HVEM and 3-O-sulfated heparan sulfate and acquired the ability to bind to nectin-2. The aims of the parent grant relate to the requirements for HSV entry into human cells and focus on structure/function studies of the entry receptors and characterization of their roles in HSV entry into specific human cell types. In this exploratory proposal, we intend to determine whether HSV-1 or HSV-1/Ridl gD can transduce or interrupt signals via any of the protein entry receptors and whether engagement of different entry receptors transduces different signals. Wild-type human fibroblasts expressing HVEM, nectin-1 and nectin-2 and mutant fibroblasts defective for nectin-1 expression will be used. These cells will be exposed to exogenous HSV-1 or HSV-1/Ridl gD or will be made to express each form of gD. RNAs will be isolated from the treated or mock-treated cells for comparisons of cell gene transcript levels by hybridization of labeled cRNAs to DNA oligonucleotide microarrays. Our hypothesis is that transduction or interruption of signals mediated by gD will result in changes in the levels of specific cell mRNAs and that the specific changes observed will depend on the particular receptor engaged by gD. If this proves to be the case, a long-term goal will be to determine whether signal transduction during viral entry influences post-entry events in viral infection in a receptor-dependent fashion. The studies described in this proposal are distinct from those supported by the parent grant but address questions that arise from findings made in the parent project.