Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1.

Abstract

The effects on human immunodeficiency virus type 1 virion morphogenesis and on virus replication of mutations that affect posttranslational processing of the capsid precursor protein are described. A change in the glycine residue at position two from the N terminus abolishes the myristoylation of the precursor proteins and also prevents virus particle release. Mutations in the viral protease gene abolish proteolytic cleavage of the capsid precursor but do not prevent the formation and budding of virion particles of immature appearance. Mutations that alter the sequence of the sites normally used for cleavage of the major capsid protein p24 from the capsid precursor alter virion morphogenesis and prevent virus replication.Electron microscopy of infected cells reveals that particle formation of the human immunodeficiency virus type 1 (HIV-1) begins when an electron-dense structure assembles in patches under the cell membrane (1-3). As the structure assumes a more spherical shape, a portion of the cell membrane pinches off to surround the budding structure. During the course of budding, no clear central core of the virus particle is apparent. After release from the cell, a coneshaped inner core particle is formed (2).What features of the capsid proteins and their precursor polypeptides determine this maturation process? The precursor of the virion capsid proteins is a 55-kDa polyprotein (p55) encoded by the gag region of the viral genome (4). A 160-kDa polyprotein, which is made as a consequence of a -1 frameshift between the gag and pol frames (5), contains precursor forms of the enzymes necessary for virus replication in addition to the capsid proteins. The N terminus ofboth the 55-kDa and 160-kDa proteins is myristoylated (6). Addition of myristic acid to proteins typically occurs cotranslationally (7) and is specified by the sequence ofthe N terminus of the protein (8).Cleavage of both the capsid precursor to yield the mature p17, p24, p9, and p6 capsid proteins as well as the precursor of the replication activities is at least in part specified by the viral protease (9, 10). The viral protease is encoded by the N terminus of the pol reading frame. The protease is reported to be active only as a dimer (11) and has been demonstrated to exhibit a preference for certain natural cleavage sites over others (12).The effects of mutations that prevent the capsid protein myristoylation and the protease activity of HIV-1 on virus assembly and virion morphology have not been reported. The experiments reported here reveal the effects on capsid protein processing, virion morphogenesis, and virus replication of a series of mutations that prevent myristoylation, inactivate the viral protease, or alter the sequence of the cleavage sites. MATERIALS AND METHODSConstruction of Mutants. A 1.3-kilobase (kb) Sac I-Apa I gag fragment from pHXB-SV (13) was subcloned into pBluescriptSK(+) (Stratagene) to generate pSK'gag as a target plasmid for the mutagenesis of the myristoylation site and the N-terminal p24 cleavage site. A 4...

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