Herpes simplex viruses (HSVs) cause a variety of human diseases, including cold stores, eye and genital infections, neonatal infections and encephalitis. The nervous system plays a central role in the pathogenesis of HSV. Both serotypes of the virus, HSV-1 (the oral form) and HSV-2(the genital form) establish lifelong latent infections within sensory peripheral ganglia. Reactivation of latent virus causes recurrent epithelial eruptions and virus shedding. In addition, the nervous system is the major target of morbidity and mortality resulting from herpetic encephalitis and neonatal herpes. This proposal concerns the mechanism by which HSV enters cells of neuronal origin to initiate infection. It is divided into an analysis of the roles of the viral glycoproteins in this process (Aims 1 and 2) and the identification of the corresponding cellular receptor(s) (Aim 3). HSV virions contain a number of glycoproteins in their liquid envelope which are important determinants of virus pathogenicity.
In Aim 1, we will examine the role of viral glycoproteins in HSV entry into neuronal cell lines that are unstimulated or stimulated to differentiate. We will use a two-chamber system for studying HSV entry into neuritic processes in both primary neurons and established neuronal cell lines.
In Aim 2, we will determine whether glycoproteins which are essential or non-essential for virus infection of non-neuronal cells are important for HSV entry into mouse neurons in vivo.
In Aim 3, we will address three questions concerning neuronal cell receptors for HSV, 1) Which glycoproteins interact with cell surface molecules? 2) What are the characteristics of cell surface molecules that bind to HSV glycoproteins; and 30 What is the cell receptor(s) for HSV? We will focus on three major contenders for interaction of HSV with cell surface molecules: g?, Gc and gD. The major approach is to use the glycoproteins as affinity reagents to isolate and characterize cell surface molecules which bind. Preliminary experiments have identified a 62K protein on SY5Y cell surfaces which binds specifically to Gd. Experiments are planned to follow up on this observation. A second approach will be to screen monoclonal antibodies to surface molecules on neuronal cells for ability to lock virus infectivity. A third approach is to screen a CDNA expression library from SY5Y cells for neuronal proteins which bind to specific glycoproteins, particularly Gd, as well as Gb and Gc. Once one or more molecules are identified, we will scale up the purification and isolate the putative receptor protein. Thus, the specific aims are: (1) To study HSCV entry into cells of neuronal origin and to define the role of individual HSV glycoproteins in this process; (2) To study the role of HSV glycoproteins in viral pathogenesis using the mouse model of HSV latency; and (3) to identify receptors on cells of neuronal origin which are necessary for HSV entry.
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