Mutational analysis of the C-terminal Gag cleavage sites in human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6(Gag) proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9(NC/SP2) protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8(SP2/p6) or p15(NC/SP2/p6) protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6(Gag) site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6(Gag) and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9(NC/SP2) species in some aspect of integration.