The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions

Abstract

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly

“…Our amino-terminal capsid interface mutants also exhibited Gag assembly defects, which may have contributed to their reduced infectivity (as has been observed previously, e.g. Kaplan et al, 1993;Pettit et al, 1994;Fouchier et al, 1997). Thus, our data are consistent with the hypothesis that the amino-terminal end of capsid also participates in important Gag-Gag interactions in the immature virion.…”

“…It is increasingly clear that retroviral maturation proceeds via a highly ordered pathway (Mervis et al, 1988;Erickson-Viitanen et al, 1989;Göttlinger et al, 1989;Gowda et al, 1989;Tritch et al, 1991;Pettit et al, 1994;Kräusslich et al, 1995;Wiegers et al, 1997). This is perhaps not surprising, given that the viral core assembles de novo at very high Gag protein concentrations (Ͼ5 mM in the virion), where non-specific aggregation may pose a significant problem.…”

“…This is perhaps not surprising, given that the viral core assembles de novo at very high Gag protein concentrations (Ͼ5 mM in the virion), where non-specific aggregation may pose a significant problem. The rates of cleavage at the different HIV-1 Gag processing sites differ considerably (Mervis et al, 1988;Erickson-Viitanen et al, 1989;Gowda et al, 1989;Kräusslich et al, 1989;Pettit et al, 1994), and these differences may serve to release various Gag domains as they are needed for assembly of the mature virion. Consistent with this model, viral maturation arrests at morphologically distinct stages when the different Gag processing sites are blocked (Wiegers et al, 1997).…”

“…Our work shows that this cleavage event also refolds the capsid amino-terminus and allows formation of a new CA-CA interface that is essential for assembling the mature capsid core. Finally, processing at the CA-p2 junction frees the capsid carboxy-terminus and allows core assembly to proceed to completion (Pettit et al, 1994;Kräusslich et al, 1995).…”

Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

“…Pr160 gag-pol cleavage by PR yields reverse transcriptase (RT) and integrase (IN) in addition to Gag products. The PRmediated proteolytic cleavage of Pr55 gag and Pr160 gag-pol , known as virus maturation, is essential for the acquisition of viral infectivity [9,10,11,12,13].…”