All Herpes viruses possess a proteinaceous layer termed tegument which lines the inner surface of their envelope. Tegument is acquired both at the inner nuclear membrane during primary envelopment, and in the cytoplasm during secondary envelopment. These tegument layers are morphologically distinct, however, little is known of how their molecular compositions differ. Furthermore, the mechanisms by which tegument proteins become bound to membranes and incorporated into the assembling HSV particle are poorly understood. We have identified a 42 amino acid membrane targeting motif in the amino terminus of vhs, a tegument component that is an important neurovirulence determinant. This membrane sorting motif is also sufficient to direct a GFP reporter into the HSV particle. Furthermore, we have shown that vhs binds to the cytoplasmic tail of the envelope glycoprotein gH. The aims of this proposal dissect the molecular details of membrane targeting by the amino terminus of vhs, tests the role of gH in this process and examines the nature of vhs in the perinuclear HSV particle. We also use newly developed methodologies to investigate the general composition and function of the perinuclear HSV particle, examine targeting of vhs to the perinuclear virion and compare the envelope and tegument of these poorly characterized particles with those of mature virions.