The alphaherpesvirus subfamily includes human herpes simplex virus type-1 (HSV-l) and -2 (HSV-2) and varicella zoster virus (VZV), the causative agent of chickenpox. Subsequent to lytic infection of the host's epithelial tissue, alphaherpesviruses establish a reactivatable latent infection in the peripheral nervous system. The natural neurotropism of these viruses has led to the current use of HSV-1 in the treatment of inoperable human gliomas. However, the inherent toxicity of HSV-1 hinders many other potential therapeutic applications. Our understanding of alphaherpesvirus assembly and spread in the nervous system is still in its early stages. Pseudorabies virus (PRV) is an alphaherpesvirus closely related to HSV and VZV. PRV does not infect humans and higher primates making it a safe and effective model for the study of alphaherpesvirus assembly in the mammalian nervous system. Previous studies have established that formation of the viral envelope around the tegument and capsid requires the presence of either glycoprotein M (gM) or the cytoplasmic tail of gE (gEct). The experiments outlined in this proposal present a combined biochemical and genetic approach to investigate the steps of capsid envelopment in neurons at the molecular level. Mutations that rescue the growth of PRV lacking both gM and gEct will identify specific viral proteins involved in capsid envelopment in epithelial cells. Proteins involved in capsid envelopment likely serve additional functions in neuronal infection. PRV mutants produced and characterized in genetic and biochemical analyses will be assayed for efficient axonal transport and capsid envelopment in primary cultured neurons. These studies will pave the way for future investigation of the consequences of capsid assembly during alphaherpesvirus infection of the mammalian nervous system.