The herpes simplex virus (HSV) major tegument structural protein VP22 resides in multiple subcellular regions during productive infection. During an analysis of the molecular determinants of these localizations, we observed that a transfected fusion of the C-terminal portion of VP22, containing its pat4 nuclear localization signal, with GFP lacked nucleolar sparing compared to GFP alone. Thus, the initial goal was to determine whether VP22 associates with nucleoli. Using an optimized indirect immunofluorescence system to visualize nucleolin and viral proteins, we observed that VP22 present in VP22-expressing Vero (V49) cells "surrounded" nucleolin. These two initial findings implied that VP22 might associate directly with nucleoli. We next analyzed HSV-infected cells and observed that at late times, anti-nucleolin immune reactivity was dispersed throughout the nuclei while it retained uniform, circular staining in mock-infected cells. Time course infection experiments indicated that nucleolin initiated its transition from uniform to dispersed structures between 2 and 4 hpi. Comparison of Hoechst stained nuclei showed bright anti-nucleolin staining localized to regions of marginalized chromatin. These effects required de novo infected cell protein synthesis. A portion of VP22 detected in nuclei at 4 and 6 hpi localized to these areas of altered nucleolin and marginalized chromatin. VP22 was excluded from viral replication compartments containing the viral regulatory protein ICP22. Finally, altered nucleolin and marginalized chromatin were detected with a VP22-null virus, indicating that VP22 was not responsible for these nuclear architecture alterations. Thus, we conclude that nuclear VP22 targets unique subnuclear structures early (<6hpi) during herpes simplex virus 1 (HSV-1) infection.