Replacement of the P1 amino acid of human immunodeficiency virus type 1 Gag processing sites can inhibit or enhance the rate of cleavage by the viral protease

Abstract

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1' amino acid. These results point to a trans effect between the P1 and P1' amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

FIG. 1.

Processing of the HIV-1 Gag precursor in vitro. (A) Schematic representation of the Pr55 Gag precursor with the five major processing sites shown as vertical lines. The sequence of the individual sites from the P4 through P3′ is shown above. The rates of cleavage of the five sites relative to the initially cleaved p2/NC site is shown below, as determined by cleavage in vitro with recombinant protease (36). (B) Representative SDS-PAGE gel showing ordered processing in vitro of the wild-type Gag precursor as a function of time in the presence of recombinant HIV-1 protease. The positions of the Gag precursor, processing intermediates, and mature products are shown on the left. The positions of molecular mass markers are shown on the right. (C) Plot of the time course of cleavage of the five processing sites as derived from densitometric analysis of gels of the processing reaction. The plot is derived from the summation of the percent initially uncleaved substrate containing each individual processing site over time.

FIG. 2.

Effect of substitutions of the P1 amino acid on the cleavage of the p2/NC site in vitro. The plot shows the percentage of initially uncleaved p2/NC site (as precursor and intermediates) over the time course of the assay. Only substitutions that allowed some degree of cleavage are shown in the plot.

FIG. 3.

Effect of substitutions of the P1 amino acid on the cleavage of the MA/CA site in vitro. The plot shows the amount of initially uncleaved MA/CA processing site over the time course of the assay. Only substitutions that allowed some degree of cleavage are shown in the plot.

FIG. 4.

Cleavage of the NC/p1 site in vitro and in vivo. (A) Time courses of cleavage in vitro of the wild type (WT) and P1 mutants with mutations of the NC/p1 site. Gag processing intermediates (including NC-p1) are shown on the left, as well as the mature products (including NC). The P1 substitution mutant is noted above the relevant gel. (B) Extent of processing of NC-containing Gag products in released virus with P1 substitutions at the NC/p1 site. Virion-associated precursors, intermediates, and products containing NC were detected with a specific antibody reagent in a Western analysis as described in Material and Methods. The compositions of NC-containing precursors and products are shown on the left, and the positions of molecular mass markers are shown on the right. The P1 substitution mutation of the NC/p1 cleavage site is shown at the top of the relevant lane.

FIG. 5.

Summary of the activity of Gag P1 processing site mutations. The values presented in Table 1 were corrected for the rate of cleavage of the individual sites relative to each other, as described in the legend to Fig. 1 and reference . After correcting for the relative rates, all rates were multiplied by 100 so that the rate of the first site cleaved (p2/NC) has a value of 100. For each site, the rate of cleavage of the wild-type sequence is shown by the black bar. The individual sites and the substitutions that supported detectable cleavage are shown at the bottom of the graph. The asterisks indicate that the actual rate was greater than the rate shown but by an unknown amount.