The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule-the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.The basic phosphoprotein (BPP) is an abundant constituent of the cytomegalovirus (CMV) virion. On the basis of its presence in preparations of virions, noninfectious enveloped particles, and cytoplasmic nucleocapsids, but not immature nuclear capsids or dense bodies, BPP was classified as a tegument protein (13,16,21). More recent evidence from cryoelectron microscopy suggests that it contacts the capsid through the distal end of the capsomers or through the triplex subunits that interlink them (8,43). BPP is phosphorylated in vivo (12,24,35), like many other herpesvirus tegument proteins (e.g., references 17, 29, and 31), and is a predominant phosphate acceptor in vitro for the virion-associated protein kinase(s) (35).BPP homologs are recognized in other betaherpesviruses (e.g, see Fig. 2) but not in alpha-or gammaherpesviruses. The human CMV (HCMV) BPP homolog is encoded by open reading frame (ORF) UL32 (7, 23) and is predicted to have a mass of 113 kDa. However, its relative electrophoretic mobility is closer to that of the 150-kDa major capsid protein (MCP; pUL86) and is influenced by the specific conditions of electrophoresis (21, 24), potentially complicating its identification on the basis of size or relative electrophoretic mobility alone. BPP is expressed late (6, 36) and has been detected in the nuclei of infected cells (20,33), although at later times it accumulates in the perinuclear region of the cytoplasm (20,36,38). Unlike its simian CMV (SCMV) counterpart, HCMV BPP is modified by the attachment of O-linked N-acetylglucosamine to two serines near its carboxyl end (2, 19). It elicits a strong humoral immune response (16,24,27), and there is evidence from the effects of antisense sense RNAs (30) and a spontaneous mutation in the gene...