These results indicate that 2-helix is responsible for ORF52 dimerization, which, in turn, is critical to MHV-68 ORF52 function. == Fig 6. of three -helices followed by one -strand. To understand the structural requirements for ORF52 function, we constructed mutants of ORF52 and examined their ability to match an ORF52-null MHV-68 computer virus. Mutations in conserved residues in the N-terminal 1-helix and C terminus, or deletion of the 2-helix, resulted in a loss-of-function phenotype. Furthermore, the 1-helix was crucial for the predominantly punctate cytoplasmic localization of 5(6)-FITC ORF52, while the 2-helix was a key domain name for ORF52 dimerization. Immunoprecipitation experiments exhibited that ORF52 interacts with another MHV-68 tegument protein, ORF42; however, a single point mutation in R95 in the C terminus of ORF52 led to the loss of this conversation. Moreover, the homologues of MHV-68 ORF52 in Kaposi’s sarcoma-associated herpesvirus and 5(6)-FITC Epstein-Barr computer virus match the defect in ORF52-null MHV-68 and interact with MHV-68 ORF52. Taken together, these 5(6)-FITC data uncover the relationship between the -helical structure and the molecular basis for ORF52 function. This is the first structure-based functional domain mapping study for an essential gammaherpesvirus tegument protein. == INTRODUCTION == Herpesviruses constitute an ancient virus family consisting of three subfamilies,Alpha-,Beta-, andGammaherpesvirinae. The herpesvirus virion consists of four morphologically distinct components: the double-stranded DNA genome in the core, an icosahedral capsid shell, the outer lipid-glycoprotein envelope, and an electron-dense tegument between the capsid and the envelope (18). Virion morphogenesis for herpesviruses is a multistep process that is generally classified into four distinct stages: nucleocapsid assembly in the nucleus, primary envelopment at the nuclear membrane followed by de-envelopment and egress into the cytoplasm, secondary envelopment, and egress from the cell (15). Most studies on herpesvirus morphogenesis have focused on alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabiesvirus (PrV). In contrast, relatively little is known about the morphogenesis of beta- and gammaherpesviruses. Productive replication (i.e., the complete lytic phase) of two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), is limited in cultured cells. Murine gammaherpesvirus-68 (MHV-68) is closely related to KSHV and EBV (8,20,23); approximately 90% of MHV-68 genes have homologous counterparts in KSHV and EBV, particularly lytic genes (26). MHV-68 can establish productive infections in a variety of fibroblast and epithelial cell lines, so it provides an excellent model for investigating the basic biology of gammaherpesviruses (20). By using MHV-68 genome cloned as a bacterial artificial chromosome (BAC), the functions of MHV-68 proteins have been studied in the context of viral infection via a genetics approach (1,4,22,28). Compared with capsids and glycoproteins, little is known about the structure and composition of the virion tegument proteins. Tegument proteins are also less conserved, as a herpesvirus in each subfamily encodes a number of tegument proteins that are absent in other subfamilies. In recent years, emerging evidence has suggested that the tegument is an organized structure built through specific protein-protein interactions (7,13,19,27). Furthermore, tegumentation is a key step for virion maturation, during which nascent nucleocapsids are wrapped and bud into the lumen of cytoplasmic compartments, forming a nearly complete virion 5(6)-FITC within the lumen. Mature virions are then released into the extracellular space in a manner resembling exocytosis (10). Although specific tegument proteins apparently play an important role in the herpesvirus tegumentation and envelopment process, the molecular mechanisms responsible for the assembly of tegument proteins into nascent herpesvirus particles are poorly understood. MHV-68ORF52encodes a tegument protein that is abundantly present in virions (5). ORF52 has no homologue in the alpha- or betaherpesviruses and thus is unique to gammaherpesviruses. By constructing anORF52-null MHV-68/BAC (52S BAC) genome, we have previously shown thatORF52encodes a highly expressed late protein, with an essential function after viral genome replication, viral DNA cleavage/packaging, and nucleocapsid assembly in the nucleus but prior to complete virion tegumentation and envelopment PLA2G5 in the cytoplasm and egress of infectious virions from the cell (4). Without the ORF52 protein, cytoplasmic viral particles cannot form mature virions and be released from the cell. Partially tegumented capsids produced by theORF52-null mutant contain all of.