A Proline-Rich Motif (PPPY) in the Gag Polyprotein of Mason-Pfizer Monkey Virus Plays a Maturation-Independent Role in Virion Release

In all retroviruses, the gag gene products are translated from unspliced, genome-length mRNA as polyprotein precursors. While the size and sequence content of the precursors vary among the different retrovirus families, all retroviral Gag precursors contain at least three domains: the matrix domain (MA), the major capsid domain (CA), and the nucleocapsid domain (NC) (19). Several studies in a number of systems have shown that expression of the gag gene alone results in the efficient assembly and release of membrane-enveloped virions (10, 13, 15, 20, 26, 32, 39). Thus, the product of this gene has the necessary structural information to mediate intracellular transport, to direct assembly of the capsid shell, and to catalyze the process of membrane extrusion known as budding. In some retroviruses, the regions and modifications of Gag polyproteins required for capsid assembly, intracellular transport, and membrane association have been identified. However, little is known about the viral and cellular requirements for retrovirus budding and release. Mason-Pfizer monkey virus (M-PMV) represents the prototypical type D retrovirus, characterized by the assembly of immature capsids or procapsids within the cytoplasm of the infected cell (37). Although a full complement of structural and enzymatic proteins together with the viral genomic RNA are required for infectivity, most are dispensable for viral assembly. The Gag polyprotein (Pr78) can form procapsids in the absence of other viral products in both mammalian and insect cells (30, 32). M-PMV procapsid assembly has also been observed in prokaryotic cells and following in vitro translation of Gag polyproteins (18, 28). Mutagenesis studies have shown that portions of the MA and CA domains are indispensable for virion assembly (24, 33). Moreover, in M-PMV, a novel Gag polyprotein domain, p12, is also important for efficient assembly of capsids (32). Following assembly, the immature capsids located within the cytoplasm are transported to the cell membrane. Both myristylation of MA and specific amino acid sequences within this domain of Gag play crucial roles in mediating the intracytoplasmic transport of preassembled procapsids to their normal site of budding and release at the plasma membrane (24, 27). The separately processed and exported Env protein complex is incorporated into the virion envelope via an interaction with a domain of the Gag polyprotein. It seems likely that a specific association between MA and some portion of the transmembrane protein directs incorporation of the Env complex into virions (5, 6, 25). Newly budded-off virions undergo a maturation process to acquire infectivity. During the process of virus maturation, the Gag precursors are cleaved by the viral proteinase to yield the individual virion proteins. As in other replication-competent retroviruses, these include the matrix protein (p10 [MA]), the major viral capsid protein (p27 [CA]), and the nucleocapsid protein (p14 [NC]). In addition, the type D retrovirus Gag polyprotein encodes p4, a short C-terminal protein of unknown function; p12, the Gag domain involved in procapsid assembly; and pp24, a phosphoprotein which in M-PMV is further cleaved to yield a second phosphoprotein (pp16). These mature gag gene products are arranged in the order NH2-p10- pp24/16-p12-p27-p14-p4-COOH on the Gag precursor Pr78 (4). Similarly, the Gag-Pro and Gag-Pro-Pol precursors are cleaved to yield the enzymatic components of the virion, thus preparing the system for reverse transcription when it encounters the proper environment. The function of the pp24 and pp16 proteins (pp24/16) in the process of viral replication has not been defined. The pp16 protein is a C-terminal-cleavage product of pp24 and contains a proline-rich motif, PPPY, that is conserved among a wide variety of retroviral Gag proteins (22, 38). In this study, we examined the role of pp24/16 in capsid assembly and postassembly steps as well as the importance of the PPPY motif in the function of this Gag precursor domain. The results presented here show that the PPPY motif is dispensable for most aspects of M-PMV assembly but plays a critical role in the final release step of virus budding.