In these studies we have characterized the role of the P1 amino acid in determining the rate of cleavage by the HIV-1 protease at the five predominant processing sites in the HIV-1 Gag precursor. Approximately 15 different amino acids were tested at each of these sites for their ability to support cleavage by the viral protease. These analyses were carried out using full-length Gag precursor as substrate with more physiological reaction conditions of neutral pH and low salt concentration rather than the conditions of low pH and high salt concentration that are typically used in the analysis of cleavage of peptide substrates. Our results confirmed previous observations that the P1 position prefers a hydrophobic and non-beta-branched amino acid. In addition, we were able to add sufficient quantitation to these studies to be able to draw several unexpected conclusions.
One surprising outcome of these studies is the observation that for three of the five sites the rate of cleavage could be improved with the introduction of specific alternative amino acids at the P1 position (summarized in Fig. ). Only the p2/NC site and the p1/p6 site could not be improved. This observation provides clear evidence that the rate of cleavage at these sites, and perhaps their order of cleavage, is regulated by the use of suboptimal amino acids in the cleavage sites. Using peptide substrates, Ridky et al. (45
) observed a 42% increase in the rate of cleavage at the CA/p2 site with a P1 Phe in place of the wild-type Leu, although they observed a decrease in the rate of cleavage with a P1 Tyr at this site. Tritch et al. (55
) observed an increase in the rate of cleavage of the MA/CA site with a P1 Phe in place of Tyr, similar to our observation (Fig. ), and Partin et al. (34
) observed that Phe readily substituted for Tyr at this site. Both increased and decreased rates of cleavage for a P1 Phe have been reported for equivalent peptide substrates of the MA/CA site (3
FIG. 5. Summary of the activity of Gag P1 processing site mutations. The values presented in Table were corrected for the rate of cleavage of the individual sites relative to each other, as described in the legend to Fig. and reference (more ...)
There are constraints that may dictate the use of some suboptimal amino acids. The P1′ Pro in the MA/CA site is required to participate in a specific interaction that is unique to the processed form of CA and an integral part of virion maturation (10
). Pro is the P1′ amino acid in several processing sites, although this distinctive requirement for Pro after cleavage is known only for CA, and this is a conserved feature of the upstream cleavage site of CA among retroviruses (37
). It may be that the requirement for Pro in this postcleavage interaction provides the selective pressure to be able to accommodate Pro in the P1′ site of the protease target sequence, defining a maximum rate of cleavage even with an optimal P1 amino acid. The P1 amino acid Asn of the NC/p1 site contributes its third codon position U to the slippery sequence that directs the −1 frameshifting event that allows expression of the Gag-Pro-Pol precursor (18
). The potential importance of this P1 amino acid (or its underlying coding sequence) can be seen in the fact that a compensatory mutation to increase the rate of cleavage at this site in the presence of a protease inhibitor-resistant protease occurs through a change in the P2 position (Ala to Val) (8
). Introduction into a virus (with a wild-type protease) of the P2 position Val at the NC/p1 site has a small negative effect on infectivity (8
). However, the NC/p1 site does not appear to be overly sensitive to up regulation since a Phe or Tyr substitution at the P1 site retains significant infectivity (data not shown).
The other surprising conclusion derived from these experiments is that the processing sites appear to fall into two groups, based on the pattern of activity associated with a specific subset of P1 amino acids: Phe, Met, Tyr, and Leu. In group 1 sites (p2/NC and NC/p1), the aliphatic amino acids Met and Leu confer activity that is more similar to Phe, while Tyr is the least active of this group of amino acids. In contrast, the group 2 sites (MA/CA and CA/p2) display activity with Tyr similar to that with Phe, while Met and Leu show lesser activity. For the p1/p6 site, Met is significantly less active than Phe, with Leu being inactive; Tyr was not tested at this site, but overall the site appears to follow the group 2 pattern (Fig. ).
An examination of the amino acids flanking the P1 position in the cleavage sites suggests an explanation for the existence of these two groups of sites. In group 1 sites (P1 Phe/Met/Leu), the P1′ amino acid is large, either Phe or Met. In the group 2 sites (P1 Phe/Tyr), the P1′ amino acid is smaller (Pro, Ala, and Leu). Given that the substrate cleavage site assumes a beta-sheet-like conformation in the active site of the protease (60
), this linkage suggests a significant trans
interaction between the P1 and P1′ amino acids flanking the scissile bond. The trans
interaction between P1 and P1′ appears to provide a better explanation of the data than the trans
interaction between P1 and P2 or the cis
interactions between P1 and P3 or P1 and P2′. In some HIV-1 isolates, the P1′ Leu is replaced with Pro in the p1/p6 site, maintaining an amino acid in the small group (group 2). In contrast, increases in the rate of processing of the p1/p6 site in the presence of a protease inhibitor-resistant protease are effected by a P1′ change of Leu to Phe (8
), introducing a P1′ amino acid from the large group (group 1).
The issue of cis
interactions has been addressed with the HIV-1 protease by Ridky et al. (44
), who examined the effects of single and double substitutions in peptide substrates representing the Rous sarcoma virus NC/PR cleavage site (PAVS/LAM). They noted that a P1 Trp or a P1 Leu improved the activity of a substrate with a P1′ Ala beyond what was expected and suggested that the large P1 amino acid positioned the small Ala side chain deeper in the S1′ subsite. We considered a similar structural model to explain the observed trans
effect inferred from our studies. However, in comparing the crystal structures of the protease with six peptide substrates (representing the cleavage sites at MA/CA, CA/p2, p2/NC, p1/p6, RT/RH, and RH/IN) (40
), we found no evidence for the physical displacement of either the P1 or the P1′ amino acid side chain due to a trans
effect. As an alternative model, we have considered the possibility that the sizes of the P1 and P1′ amino acids are somehow additive and can be too large for optimal cleavage. Thus, a P1 Tyr would be most active with small P1′ amino acids and less active with Tyr or Phe in the P1′ position. We previously observed that aliphatic amino acids at the P1′ position of the MA/CA cleavage site (with a P1 Tyr) were more active than Phe or Tyr (22
), although other studies using similar peptide substrates have not observed this rank order (3
). Ridky et al. (45
) reported that aliphatic amino acids and Phe, when used instead of the P1′ Ala in the CA/p2 site, enhanced the rate of cleavage. In the substrate-PR crystal structures, Tyr makes a significant number of contacts in either the P1 or P1′ position, and it is also able, through its side chain hydroxyl, to make additional contacts with solvent, perhaps enhancing its binding energy. The additive effects of the P1 and P1′ amino acids in defining the rate of cleavage and in passing through an optimum are consistent with the data obtained in this study (Table ).
Hydrophobic beta-branched amino acids are never found in naturally occurring protease cleavage sites (37
), and a structural reason for their exclusion has been proposed (54
). In contrast, a site with a P1 Ile substitution of the RSV NC/PR is cleaved by the Rous sarcoma virus protease (47
). We have now observed a low level of cleavage by the HIV-1 protease at the NC/p1 site with either Val or Ile at the P1 position (Table ; Fig. ). We did not see cleavage at the CA/p2 site with either Val or Ile, even though the rates of cleavage at the NC/p1 and CA/p2 wild-type sequences are similar. This difference may be due to the large P1′ amino acid in the NC/p1 site contributing to an enhanced rate of cleavage with suboptimal P1 amino acids. Thus, we confirm that the protease can cleave a peptide bond with a beta-branched P1 amino acid, although in our case we did not analyze the position of the cleaved site by protein sequencing.
In summary, our results point to significant trans
interactions between the P1 and P1′ side chains of HIV-1 protease substrates in determining the rate of cleavage. Phe appears best suited to productively interacting with the S1 subsite and perhaps the S1′ subsite. Met and Leu are more active when an amino acid with a larger P1′ side chain occupies the S1′ subsite, and Tyr is more active with amino acids with smaller P1′ side chains occupying the S1′ subsite. These results suggest a basis for two groupings of the diverse cleavage site sequences as an alternative to older classification schemes that had focused largely on the presence or absence of a P1′ Pro (13