Ordered Processing of the Human Immunodeficiency V
http://www.100md.com
病菌学杂志 2005年第16期
Departments of Medicine Microbiology and Immunology
Lineberger Comprehensive Cancer Center
Center for Infectious Diseases, University of North Carolina School of Medicine, Campus Box 7290, Chapel Hill, North Carolina 27599-7290
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, Florida 32610-0245
ABSTRACT
Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.
INTRODUCTION
The proteins that make up the retroviral core particle are translated as part of polyprotein precursors (13, 32). Processing of these precursors is accomplished by a viral protease (PR) that is contained within one of the precursors (9). During virus assembly, the processing sites within the retroviral precursor proteins are cleaved by the viral PR (9, 13, 45). Accurate and precise PR-mediated precursor processing is an absolute requirement for the production of fully infectious viral particles; mutations that produce imprecise cleavage at individual sites or that alter the order in which sites are cleaved result in the elaboration of aberrantly assembled virions that are markedly less infectious (5, 20, 22, 36, 42, 54, 57).
In the case of the human immunodeficiency virus type 1 (HIV-1), the structural proteins of the core particle are contained within the Gag precursor (Fig. 1) (13). From the amino terminus, these are the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 (17, 18). There is a small spacer peptide, p2, between CA and NC; an additional spacer peptide, p1, resides between NC and p6 (17, 18, 36, 51, 56). The 5' end of the pol precursor reading frame overlaps with gag; Pol is translated as a fusion protein with Gag (GagPol) as the result of a –1 frameshift, producing a Gag/GagPol ratio of 20:1 (19, 39). Pol contains several enzymes, including the PR itself as well as the reverse transcriptase (RT) and integrase (IN) (21, 27, 50). There is an additional PR cleavage site within RT. Processing at this site has been estimated to occur in about 50% of the GagPol molecules and produces a truncated, 51-kDa form of the 66-kDa full-length RT that is missing the carboxy-terminal RNase H domain (12, 26, 30, 47, 50, 52).
The PR becomes active only as a homodimer; the enzyme active-site cleft is comprised of residues from two monomers (6, 53). Numerous crystal structures of the mature, 99-amino-acid PR have been described, and several groups have conducted exhaustive genetic and biophysical analyses of the mature dimer (10, 27, 31, 44, 55). It has been estimated that approximately 50% of the interactions that maintain the mature dimer are found in a four-stranded antiparallel -sheet; the first four residues of each of the protease monomers form the two outer strands of the -sheet, and the last four residues (residues 96 to 99) of each monomer form the two inner strands (53).
The location of the PR within a large precursor combined with the requirement that the enzyme must dimerize to become active dictates that the PR domains of two GagPol precursors, along with the precursors themselves, must dimerize. The first processing events are carried out by the embedded PR, before it is released from the GagPol precursor. The initial cleavages in GagPol are intramolecular; the activated PR dimer on one pair of GagPol molecules cleaves these initial sites on those same molecules (33).
Several studies have examined the order of cleavage for the different sites within the HIV-1 Gag precursor, and there is general agreement that the first cleavage occurs between the p2 spacer peptide and the NC protein (11, 15, 23, 28, 35, 36, 54). Intermediate cleavages occur at the MA/CA and p1/p6 sites (36, 54). The relative rate of cleavage at different sites within the precursors has also been evaluated. Using a system in which the Gag precursor is processed by the addition of purified PR in trans, the rates of cleavage were determined to vary by as much as 400-fold between sites (35, 36). Of note, this difference is much greater than that reported for the cleavage of peptides designed to reflect the sequence at the different sites (3, 7, 23, 29, 38, 40, 48).
Despite the critical role played by the orderly processing of the Gag and GagPol precursors in virus assembly and replication, little is known about the factors that determine the order in which different sites are cleaved. The initial steps in the processing of the GagPol precursor produced in vitro are intramolecular cleavages that are carried out by the embedded PR in cis (33). Although it seems likely that the context of the PR domain within GagPol influences the early events in precursor processing, the behavior of the PR in cis versus trans has not been extensively examined. Using an in vitro expression system in which full-length GagPol is cleaved by its embedded PR, we sought to define the effect of the context of the PR (precursor-embedded or mature, free PR) on precursor processing. We demonstrate that the embedded PR is constrained to make a limited number of cleavages by its location within GagPol. Further, substitutions at the first amino acid in the PR, a proline, lift this constraint and free the PR domain within GagPol to cleave additional native processing sites in the precursor not cleaved by the wild-type (WT) embedded PR. Our data demonstrate that the context of the PR within GagPol influences the order of precursor processing and indicate that there are structural determinants within the PR, outside of the active site, that help regulate the ordered selection of cleavage sites during precursor processing. Overall, these findings further our understanding of the regulation of the initial events in HIV-1 maturation and suggest that the ordered pattern of precursor cleavage is defined, at least in part, by the structure of GagPol itself.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. The construction of pGPfs and pGPfs-PR was previously described (34). Briefly, pGPfs contains the full-length GagPol open reading frame downstream of the bacteriophage T7 promoter. A contiguous GagPol open reading frame was constructed by site-directed mutagenesis and exactly produces the major GagPol product (the same amino acid sequence) found in virions (pr160) (14, 19). The pGPfs-PR plasmid contains an additional D25A mutation of the catalytic aspartate with the PR domain and thus lacks intrinsic PR activity. Site-directed mutagenesis was performed as described previously (1, 24), and mutations were confirmed by chain termination sequencing (41).
In vitro assays for the proteolytic processing of GagPol. Expression of GagPol in vitro and cis-processing reactions were performed as previously described (33, 34). Briefly, the GagPol constructs were translated using a coupled transcription/translation system (TNT system; Promega) in a rabbit reticulocyte lysate (RRL). The reaction was carried out in 50-μl total volume with 20 μCi of [35S]cysteine (>1,000 Ci/mM; Amersham Pharmacia Biotech). trans PR processing reactions were performed as described previously (33, 34) in 50-μl reaction volumes containing approximately 160 pM GagPol (final concentration) and purified protease in a buffer previously described (34). Either purified WT or P1A mutant protease was added to initiate the reactions, and aliquots were removed at specific times for evaluation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (33). Competitive inhibitions of cis- and trans-processing reactions by ritonavir, sequinavir, or indinavir were performed as described previously (33). Reactions containing WT or P1A mutant PR contained equivalent amounts of protease as determined by specific activity on GagPol substrate.
Expression, titration, and purification of HIV PR. WT and P1A mutant protease was purified as described previously (16, 34). For peptide studies, the two protease preparations were titrated to determine the percentage of active sites determined by inhibition with tight binding inhibitor (46).
Determination of Michaelis-Menten constants. The Michaelis-Menten constants kcat, Km, and kcat/Km and Ki values were determined for each variant as previously described (4). The chromogenic substrate K-A-R-V-LNph-E-A-nL-G, which mimics the CA-p2 cleavage site, was used to determine the inhibitor binding constants of each variant at 37°C in sodium acetate buffer (0.05 M sodium acetate, 0.15 M NaCl, 0.002 M EDTA, 0.001 M dithiothreitol, pH 4.7). Cleavage of the substrate was monitored using a Hewlett-Packard 8452A spectrophotometer equipped with a seven-cell sample handling system as described by Dunn et al. (8). The inhibition constants Ki were determined by monitoring the inhibition of hydrolysis of the chromogenic substrate as described by Bhatt and Dunn (2). Analysis of the fluorescent substrate R-E(EDANS)-R-K-V-LF-L-D-G-K(DABCYL)-R was done in the same buffer and temperature using a CytoFluor Multi-Cell plate reader from PerSeptive Biosystems.
RESULTS
Substitution of the amino-terminal proline in the embedded PR with alanine relieves the block to PR-mediated cleavage at the processing site between the RNase H domain of RT and IN. Expression of full-length WT GagPol in an in vitro RRL transcription/translation system results in PR activation and ordered processing of the precursor at two cleavage sites; an initial cleavage is observed between the NC and p2 proteins, and a second cleavage occurs at a site within the transframe region (TF) between amino acids 440 and 441 (34). These intramolecular processing events produce processing intermediates of 120, 113, and 41 kDa, corresponding to the NC-IN, TF (L441)-IN, and MA-p2 fragments, respectively (Fig. 1 and 2A) (33, 35). Of note, we do not observe additional cleavages in the precursor despite prolonged incubation (data not shown). It seems most likely that the lack of additional processing events is due to the low concentrations of the GagPol precursors in the RRL. We have estimated that the concentration of the precursor produced in this system is 1 nM, which is close to the reported Kd of the mature, full processed PR (34).
We used this system to examine the processing of GagPol by the embedded PR. We were particularly interested in characterizing the structural determinants that define the order in which different GagPol sites are cleaved. Previously, we demonstrated that substitution of the proline at position 1 of the embedded PR with any one of several amino acids (e.g., Ala, Gly, Leu, or Phe) results in additional cleavages in the GagPol precursor (Fig. 2A) (35). The sizes of these additional cleavage products (67 kDa, 66 kDa, and 62 kDa) were consistent with enhanced processing at three sites within GagPol: the amino terminus of the PR, between the RT and RNase H, and between the RNase H domain of RT and IN. Cleavages at these sites would produce three additional processing intermediates, TF-RT (67 kDa), RT-RH (66 kDa), and PR-RT (62 kDa).
We identified the precise locations of these cleavage sites by introducing blocking mutations at the known RNase H/IN processing site. Others have noted that substitution of an Ile for the P1 (N-terminal) amino acid of the scissile bond inhibits PR-mediated processing for a majority of the HIV-1 processing sites (3, 37, 40, 49). A GagPol construct containing an Ile blocking mutation at the RNase H/IN processing site and a Pro-to-Ala substitution at position 1 of the PR was generated (pGPfs-P1A RH/IN). The 67-, 66-, and 62-kDa fragments observed upon expression of GagPolP1A are not seen in the presence of the Ile blocking substitution, indicating that they arise from cleavage at this site. In addition to the abrogated proteolytic cleavage at the RT-IN junction, inhibition of cleavage of the RT/RH site was also noted (Fig. 2A, P1A-RH/IN, 67- and 62-kDa products). These observations are consistent with the findings of Sluis-Cremer et al. (43), who suggested that formation of the p66/p66 homodimer via removal of IN is an obligatory step in the processing pathway to the p66/p51 heterodimer. These data indicate that the P1A substitution allows the embedded PR to make additional cleavages at native processing sites that are in the C-terminal portion of the precursor (Fig. 2B).
Purified mature P1A PR added in trans to GagPol produces a cleavage pattern that is indistinguishable from the trans-processing pattern observed with purified mature WT PR. We reasoned that the altered processing pattern observed with the embedded PR containing the P1A substitution might be dependent on the context of the mutant PR within GagPol. To assess the effect of the P1A mutant on the 99-amino-acid mature PR, we examined the trans processing of a full-length GagPol substrate containing an inactivating mutation in its PR domain; the substitution of an alanine for the aspartic acid present at position 25 (the active site) of the embedded PR (GagPolPR-) renders the embedded enzyme inactive. For these experiments, purified WT or P1A PR was provided in trans to full-length GagPolPR- and processing was monitored over time.
As noted above (Fig. 2A), the processing of GagPol by the embedded mutant P1A PR produced a pattern of cleavages that was markedly different than the processing intermediates observed with the embedded WT PR. In contrast, the purified WT and P1A PRs produced identical cleavage patterns when added to GagPolPR- in trans (Fig. 3). For both the purified WT and P1A PRs, processing occurred in an order similar to that seen with the WT embedded PR; cleavage at the p2/NC site yields 120- and 42-kDa intermediates and is followed by processing at the site within the precursor (Fig. 3). In particular, we observed that the generation of the 67-, 66-, and 62-kDa products occurred at similar time points for both WT and P1A trans protease (Fig. 3).
To determine whether the altered processing pattern seen with the embedded P1A PR was due to an increase in affinity of the P1A mutant for the RNase H/IN cleavage site, we measured the Km of the P1A mutant for a peptide corresponding to this site [R-E(EDANS)-R-K-V-LF-L-D-G-K(DABCYL)-R]. As shown in Table 1, the Km of the purified P1A mutant PR for this peptide is very similar to that observed with the WT enzyme and we observed a 3.7-fold decrease in kcat/Km by P1A compared to WT protease.
The embedded P1A PR, but not the purified mature P1A, is more sensitive than WT enzyme to inhibition by PR inhibitors. Previously, we demonstrated that purified mature WT PR added to full-length GagPolPR- in trans is dramatically more sensitive to inhibition by an active-site PR inhibitor (PI) than the WT embedded PR (33). Specifically, we established that approximately 10,000-fold more ritonavir is required to inhibit the embedded PR from processing the precursor than is required to inhibit purified PR added in trans. This large difference in sensitivity to inhibition is consistent with our observation that the initial cleavages by the embedded PR are intramolecular processing events (33). Given the altered GagPol processing patterns produced by the embedded, but not the purified, P1A PR, we determined the sensitivity of the mutant enzyme to inhibition by several of the available PR inhibitors, including ritonavir, saquinavir, and indinavir.
Higher concentrations of ritonavir were required to inhibit the embedded WT PR from cleaving the p2/NC site than was required to inhibit the P1A PR from cleaving the same site. As summarized in Table 2, we found that the embedded P1A PR is also more sensitive than the WT PR to inhibition by indinavir for cleavage at the p2/NC site. The increase in sensitivity ranges from a 4.0-fold decrease in the 50% inhibitory concentration (IC50) for ritonavir to a 6.4-fold decrease for indinavir. The decrease in IC50 for the P1A protease, while small, was consistent across multiple experiments. Thus, P1A protease as a component of GagPol has a modest increase in sensitivity to ritonavir and indinavir. In contrast, the concentration of protease inhibitor (PI) required to inhibit either the purified P1A or WT protease from processing the full-length GagPolPR- in trans was quite similar (Table 2).
We also determined the Ki for the purified P1A and WT enzymes in trans for each of the PIs (Table 3). The sensitivities of the purified P1A PR to inhibition by the three drugs were similar to those observed with the WT enzyme.
DISCUSSION
The initial steps in retroviral precursor processing and viral assembly include the interaction of GagPol precursors and PR activation. Our previous studies have demonstrated that extra-PR regions in GagPol promote PR activation and dimerization (33). Following the dimerization of the GagPol precursors and their embedded PR domains, the activated enzyme makes its initial intramolecular cleavages. Therefore, the dimerization of the PR domains within the two GagPol precursors, the concomitant activation of the embedded enzyme homodimer, and the initial processing events are inextricably linked.
Published crystallographic studies of the mature 99-amino-acid PR have established that the two monomers interact in large part through a dimer interface in which the amino-terminal four amino acids (Pro-Gln-Ile-Thr) form the outer strands and the carboxy-terminal amino acids (Thr-Leu-Asn-Phe) comprise the inner strands of a four-stranded antiparallel -sheet (53). Despite the extensive structural studies of the mature PR, very limited information is available regarding the structure of the embedded PR within GagPol. This, in turn, has limited our ability to draw definitive conclusions about the influence of the context of surrounding GagPol sequences on ordered precursor processing by the embedded PR.
Using a system in which the full-length GagPol precursor is processed by its embedded PR, we sought to examine the extent to which the initial precursor processing events are influenced by the context of the PR within GagPol. Our studies reveal several new insights into the role that the PR dimer interface plays in directing the order of GagPol cleavage. First, these data indicate that the rigid, amino-terminal Pro constrains the embedded PR from processing cleavage sites downstream of the PR domain in GagPol. A role in defining the order of precursor processing appears to be limited to this position; as we have reported previously, substitutions at other positions in the dimer interface do not alter the pattern of precursor processing at these downstream sites (34). This conclusion is also strengthened by the observation that similar results are obtained when the amino-terminal Pro is replaced by Leu, Phe, or Gly (34). Second, we demonstrate that the context of the PR within GagPol influences the order in which sites within the precursor are processed; the purified enzyme containing a P1A substitution did not display the altered processing phenotype and did not display an appreciably altered Km for the RNase H/IN site (Table 1). Finally, the embedded PR with the P1A substitution was more sensitive to inhibition by two of the three active-site PR inhibitors than the WT PR. Of note, the purified P1A PR did not share this enhanced susceptibility (Table 2).
We were surprised to find that the embedded P1A PR was more sensitive to inhibition by two of the PR active-site inhibitors tested. We believe that this reflects the altered context of the PR domain embedded within GagPol. As noted above, relieving the constraints of the rigid Pro at position 1 of the PR domain allows cleavage of the additional downstream sites. This is consistent with the interpretation that the PR domain of the precursor with the P1A substitution is more flexible and thus able to access these additional sites. It seems likely that the increased flexibility that allows the embedded PR to cleave the downstream sites also increases the accessibility of the enzyme active site. Therefore, the enhanced susceptibility of the embedded P1A PR may be explained by an increased accessibility of the enzyme active site to the inhibitor. Alternatively, the enhanced susceptibility of the PR with the P1A substitution may be explained by unanticipated changes in the enzyme active site. As we do not observe the enhanced susceptibility in the purified mutant PR, this latter possibility seems less likely. However, in the absence of structural information for the precursor, we cannot definitively distinguish between these two interpretations.
In summary, these results demonstrate that substitutions outside of the PR active site influence the selection of processing sites for cleavage. Further, we have identified the initial amino acid in the GagPol PR domain as playing a critical role in constraining the order of cleavage in the WT precursor. Taken together, our data are consistent with a model in which two factors have evolved that define the ordered processing of the cleavage sites within the HIV-1 GagPol precursor. First, the order of cleavage is influenced by the well-described characteristics of the processing sites themselves as reflected in peptide-based studies (3, 7, 23, 29, 38, 40, 48). Second, our data also support the notion that structural constraints act on the PR domain within the context of the GagPol precursor and that the structure of the precursor helps define the order of the initial cleavage events.
ACKNOWLEDGMENTS
We gratefully acknowledge helpful discussions with G. Schatz and V. M. Vogt. Ritonavir, saquinavir, and indinavir were obtained from the NIH AIDS Research and Reference Reagent Program.
This work was supported by NIH grants RO1 GM GM66681-01 and K24 MH071191-01 to A.H.K. and the UNC Center for AIDS Research.
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Lineberger Comprehensive Cancer Center
Center for Infectious Diseases, University of North Carolina School of Medicine, Campus Box 7290, Chapel Hill, North Carolina 27599-7290
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, Florida 32610-0245
ABSTRACT
Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.
INTRODUCTION
The proteins that make up the retroviral core particle are translated as part of polyprotein precursors (13, 32). Processing of these precursors is accomplished by a viral protease (PR) that is contained within one of the precursors (9). During virus assembly, the processing sites within the retroviral precursor proteins are cleaved by the viral PR (9, 13, 45). Accurate and precise PR-mediated precursor processing is an absolute requirement for the production of fully infectious viral particles; mutations that produce imprecise cleavage at individual sites or that alter the order in which sites are cleaved result in the elaboration of aberrantly assembled virions that are markedly less infectious (5, 20, 22, 36, 42, 54, 57).
In the case of the human immunodeficiency virus type 1 (HIV-1), the structural proteins of the core particle are contained within the Gag precursor (Fig. 1) (13). From the amino terminus, these are the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 (17, 18). There is a small spacer peptide, p2, between CA and NC; an additional spacer peptide, p1, resides between NC and p6 (17, 18, 36, 51, 56). The 5' end of the pol precursor reading frame overlaps with gag; Pol is translated as a fusion protein with Gag (GagPol) as the result of a –1 frameshift, producing a Gag/GagPol ratio of 20:1 (19, 39). Pol contains several enzymes, including the PR itself as well as the reverse transcriptase (RT) and integrase (IN) (21, 27, 50). There is an additional PR cleavage site within RT. Processing at this site has been estimated to occur in about 50% of the GagPol molecules and produces a truncated, 51-kDa form of the 66-kDa full-length RT that is missing the carboxy-terminal RNase H domain (12, 26, 30, 47, 50, 52).
The PR becomes active only as a homodimer; the enzyme active-site cleft is comprised of residues from two monomers (6, 53). Numerous crystal structures of the mature, 99-amino-acid PR have been described, and several groups have conducted exhaustive genetic and biophysical analyses of the mature dimer (10, 27, 31, 44, 55). It has been estimated that approximately 50% of the interactions that maintain the mature dimer are found in a four-stranded antiparallel -sheet; the first four residues of each of the protease monomers form the two outer strands of the -sheet, and the last four residues (residues 96 to 99) of each monomer form the two inner strands (53).
The location of the PR within a large precursor combined with the requirement that the enzyme must dimerize to become active dictates that the PR domains of two GagPol precursors, along with the precursors themselves, must dimerize. The first processing events are carried out by the embedded PR, before it is released from the GagPol precursor. The initial cleavages in GagPol are intramolecular; the activated PR dimer on one pair of GagPol molecules cleaves these initial sites on those same molecules (33).
Several studies have examined the order of cleavage for the different sites within the HIV-1 Gag precursor, and there is general agreement that the first cleavage occurs between the p2 spacer peptide and the NC protein (11, 15, 23, 28, 35, 36, 54). Intermediate cleavages occur at the MA/CA and p1/p6 sites (36, 54). The relative rate of cleavage at different sites within the precursors has also been evaluated. Using a system in which the Gag precursor is processed by the addition of purified PR in trans, the rates of cleavage were determined to vary by as much as 400-fold between sites (35, 36). Of note, this difference is much greater than that reported for the cleavage of peptides designed to reflect the sequence at the different sites (3, 7, 23, 29, 38, 40, 48).
Despite the critical role played by the orderly processing of the Gag and GagPol precursors in virus assembly and replication, little is known about the factors that determine the order in which different sites are cleaved. The initial steps in the processing of the GagPol precursor produced in vitro are intramolecular cleavages that are carried out by the embedded PR in cis (33). Although it seems likely that the context of the PR domain within GagPol influences the early events in precursor processing, the behavior of the PR in cis versus trans has not been extensively examined. Using an in vitro expression system in which full-length GagPol is cleaved by its embedded PR, we sought to define the effect of the context of the PR (precursor-embedded or mature, free PR) on precursor processing. We demonstrate that the embedded PR is constrained to make a limited number of cleavages by its location within GagPol. Further, substitutions at the first amino acid in the PR, a proline, lift this constraint and free the PR domain within GagPol to cleave additional native processing sites in the precursor not cleaved by the wild-type (WT) embedded PR. Our data demonstrate that the context of the PR within GagPol influences the order of precursor processing and indicate that there are structural determinants within the PR, outside of the active site, that help regulate the ordered selection of cleavage sites during precursor processing. Overall, these findings further our understanding of the regulation of the initial events in HIV-1 maturation and suggest that the ordered pattern of precursor cleavage is defined, at least in part, by the structure of GagPol itself.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. The construction of pGPfs and pGPfs-PR was previously described (34). Briefly, pGPfs contains the full-length GagPol open reading frame downstream of the bacteriophage T7 promoter. A contiguous GagPol open reading frame was constructed by site-directed mutagenesis and exactly produces the major GagPol product (the same amino acid sequence) found in virions (pr160) (14, 19). The pGPfs-PR plasmid contains an additional D25A mutation of the catalytic aspartate with the PR domain and thus lacks intrinsic PR activity. Site-directed mutagenesis was performed as described previously (1, 24), and mutations were confirmed by chain termination sequencing (41).
In vitro assays for the proteolytic processing of GagPol. Expression of GagPol in vitro and cis-processing reactions were performed as previously described (33, 34). Briefly, the GagPol constructs were translated using a coupled transcription/translation system (TNT system; Promega) in a rabbit reticulocyte lysate (RRL). The reaction was carried out in 50-μl total volume with 20 μCi of [35S]cysteine (>1,000 Ci/mM; Amersham Pharmacia Biotech). trans PR processing reactions were performed as described previously (33, 34) in 50-μl reaction volumes containing approximately 160 pM GagPol (final concentration) and purified protease in a buffer previously described (34). Either purified WT or P1A mutant protease was added to initiate the reactions, and aliquots were removed at specific times for evaluation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (33). Competitive inhibitions of cis- and trans-processing reactions by ritonavir, sequinavir, or indinavir were performed as described previously (33). Reactions containing WT or P1A mutant PR contained equivalent amounts of protease as determined by specific activity on GagPol substrate.
Expression, titration, and purification of HIV PR. WT and P1A mutant protease was purified as described previously (16, 34). For peptide studies, the two protease preparations were titrated to determine the percentage of active sites determined by inhibition with tight binding inhibitor (46).
Determination of Michaelis-Menten constants. The Michaelis-Menten constants kcat, Km, and kcat/Km and Ki values were determined for each variant as previously described (4). The chromogenic substrate K-A-R-V-LNph-E-A-nL-G, which mimics the CA-p2 cleavage site, was used to determine the inhibitor binding constants of each variant at 37°C in sodium acetate buffer (0.05 M sodium acetate, 0.15 M NaCl, 0.002 M EDTA, 0.001 M dithiothreitol, pH 4.7). Cleavage of the substrate was monitored using a Hewlett-Packard 8452A spectrophotometer equipped with a seven-cell sample handling system as described by Dunn et al. (8). The inhibition constants Ki were determined by monitoring the inhibition of hydrolysis of the chromogenic substrate as described by Bhatt and Dunn (2). Analysis of the fluorescent substrate R-E(EDANS)-R-K-V-LF-L-D-G-K(DABCYL)-R was done in the same buffer and temperature using a CytoFluor Multi-Cell plate reader from PerSeptive Biosystems.
RESULTS
Substitution of the amino-terminal proline in the embedded PR with alanine relieves the block to PR-mediated cleavage at the processing site between the RNase H domain of RT and IN. Expression of full-length WT GagPol in an in vitro RRL transcription/translation system results in PR activation and ordered processing of the precursor at two cleavage sites; an initial cleavage is observed between the NC and p2 proteins, and a second cleavage occurs at a site within the transframe region (TF) between amino acids 440 and 441 (34). These intramolecular processing events produce processing intermediates of 120, 113, and 41 kDa, corresponding to the NC-IN, TF (L441)-IN, and MA-p2 fragments, respectively (Fig. 1 and 2A) (33, 35). Of note, we do not observe additional cleavages in the precursor despite prolonged incubation (data not shown). It seems most likely that the lack of additional processing events is due to the low concentrations of the GagPol precursors in the RRL. We have estimated that the concentration of the precursor produced in this system is 1 nM, which is close to the reported Kd of the mature, full processed PR (34).
We used this system to examine the processing of GagPol by the embedded PR. We were particularly interested in characterizing the structural determinants that define the order in which different GagPol sites are cleaved. Previously, we demonstrated that substitution of the proline at position 1 of the embedded PR with any one of several amino acids (e.g., Ala, Gly, Leu, or Phe) results in additional cleavages in the GagPol precursor (Fig. 2A) (35). The sizes of these additional cleavage products (67 kDa, 66 kDa, and 62 kDa) were consistent with enhanced processing at three sites within GagPol: the amino terminus of the PR, between the RT and RNase H, and between the RNase H domain of RT and IN. Cleavages at these sites would produce three additional processing intermediates, TF-RT (67 kDa), RT-RH (66 kDa), and PR-RT (62 kDa).
We identified the precise locations of these cleavage sites by introducing blocking mutations at the known RNase H/IN processing site. Others have noted that substitution of an Ile for the P1 (N-terminal) amino acid of the scissile bond inhibits PR-mediated processing for a majority of the HIV-1 processing sites (3, 37, 40, 49). A GagPol construct containing an Ile blocking mutation at the RNase H/IN processing site and a Pro-to-Ala substitution at position 1 of the PR was generated (pGPfs-P1A RH/IN). The 67-, 66-, and 62-kDa fragments observed upon expression of GagPolP1A are not seen in the presence of the Ile blocking substitution, indicating that they arise from cleavage at this site. In addition to the abrogated proteolytic cleavage at the RT-IN junction, inhibition of cleavage of the RT/RH site was also noted (Fig. 2A, P1A-RH/IN, 67- and 62-kDa products). These observations are consistent with the findings of Sluis-Cremer et al. (43), who suggested that formation of the p66/p66 homodimer via removal of IN is an obligatory step in the processing pathway to the p66/p51 heterodimer. These data indicate that the P1A substitution allows the embedded PR to make additional cleavages at native processing sites that are in the C-terminal portion of the precursor (Fig. 2B).
Purified mature P1A PR added in trans to GagPol produces a cleavage pattern that is indistinguishable from the trans-processing pattern observed with purified mature WT PR. We reasoned that the altered processing pattern observed with the embedded PR containing the P1A substitution might be dependent on the context of the mutant PR within GagPol. To assess the effect of the P1A mutant on the 99-amino-acid mature PR, we examined the trans processing of a full-length GagPol substrate containing an inactivating mutation in its PR domain; the substitution of an alanine for the aspartic acid present at position 25 (the active site) of the embedded PR (GagPolPR-) renders the embedded enzyme inactive. For these experiments, purified WT or P1A PR was provided in trans to full-length GagPolPR- and processing was monitored over time.
As noted above (Fig. 2A), the processing of GagPol by the embedded mutant P1A PR produced a pattern of cleavages that was markedly different than the processing intermediates observed with the embedded WT PR. In contrast, the purified WT and P1A PRs produced identical cleavage patterns when added to GagPolPR- in trans (Fig. 3). For both the purified WT and P1A PRs, processing occurred in an order similar to that seen with the WT embedded PR; cleavage at the p2/NC site yields 120- and 42-kDa intermediates and is followed by processing at the site within the precursor (Fig. 3). In particular, we observed that the generation of the 67-, 66-, and 62-kDa products occurred at similar time points for both WT and P1A trans protease (Fig. 3).
To determine whether the altered processing pattern seen with the embedded P1A PR was due to an increase in affinity of the P1A mutant for the RNase H/IN cleavage site, we measured the Km of the P1A mutant for a peptide corresponding to this site [R-E(EDANS)-R-K-V-LF-L-D-G-K(DABCYL)-R]. As shown in Table 1, the Km of the purified P1A mutant PR for this peptide is very similar to that observed with the WT enzyme and we observed a 3.7-fold decrease in kcat/Km by P1A compared to WT protease.
The embedded P1A PR, but not the purified mature P1A, is more sensitive than WT enzyme to inhibition by PR inhibitors. Previously, we demonstrated that purified mature WT PR added to full-length GagPolPR- in trans is dramatically more sensitive to inhibition by an active-site PR inhibitor (PI) than the WT embedded PR (33). Specifically, we established that approximately 10,000-fold more ritonavir is required to inhibit the embedded PR from processing the precursor than is required to inhibit purified PR added in trans. This large difference in sensitivity to inhibition is consistent with our observation that the initial cleavages by the embedded PR are intramolecular processing events (33). Given the altered GagPol processing patterns produced by the embedded, but not the purified, P1A PR, we determined the sensitivity of the mutant enzyme to inhibition by several of the available PR inhibitors, including ritonavir, saquinavir, and indinavir.
Higher concentrations of ritonavir were required to inhibit the embedded WT PR from cleaving the p2/NC site than was required to inhibit the P1A PR from cleaving the same site. As summarized in Table 2, we found that the embedded P1A PR is also more sensitive than the WT PR to inhibition by indinavir for cleavage at the p2/NC site. The increase in sensitivity ranges from a 4.0-fold decrease in the 50% inhibitory concentration (IC50) for ritonavir to a 6.4-fold decrease for indinavir. The decrease in IC50 for the P1A protease, while small, was consistent across multiple experiments. Thus, P1A protease as a component of GagPol has a modest increase in sensitivity to ritonavir and indinavir. In contrast, the concentration of protease inhibitor (PI) required to inhibit either the purified P1A or WT protease from processing the full-length GagPolPR- in trans was quite similar (Table 2).
We also determined the Ki for the purified P1A and WT enzymes in trans for each of the PIs (Table 3). The sensitivities of the purified P1A PR to inhibition by the three drugs were similar to those observed with the WT enzyme.
DISCUSSION
The initial steps in retroviral precursor processing and viral assembly include the interaction of GagPol precursors and PR activation. Our previous studies have demonstrated that extra-PR regions in GagPol promote PR activation and dimerization (33). Following the dimerization of the GagPol precursors and their embedded PR domains, the activated enzyme makes its initial intramolecular cleavages. Therefore, the dimerization of the PR domains within the two GagPol precursors, the concomitant activation of the embedded enzyme homodimer, and the initial processing events are inextricably linked.
Published crystallographic studies of the mature 99-amino-acid PR have established that the two monomers interact in large part through a dimer interface in which the amino-terminal four amino acids (Pro-Gln-Ile-Thr) form the outer strands and the carboxy-terminal amino acids (Thr-Leu-Asn-Phe) comprise the inner strands of a four-stranded antiparallel -sheet (53). Despite the extensive structural studies of the mature PR, very limited information is available regarding the structure of the embedded PR within GagPol. This, in turn, has limited our ability to draw definitive conclusions about the influence of the context of surrounding GagPol sequences on ordered precursor processing by the embedded PR.
Using a system in which the full-length GagPol precursor is processed by its embedded PR, we sought to examine the extent to which the initial precursor processing events are influenced by the context of the PR within GagPol. Our studies reveal several new insights into the role that the PR dimer interface plays in directing the order of GagPol cleavage. First, these data indicate that the rigid, amino-terminal Pro constrains the embedded PR from processing cleavage sites downstream of the PR domain in GagPol. A role in defining the order of precursor processing appears to be limited to this position; as we have reported previously, substitutions at other positions in the dimer interface do not alter the pattern of precursor processing at these downstream sites (34). This conclusion is also strengthened by the observation that similar results are obtained when the amino-terminal Pro is replaced by Leu, Phe, or Gly (34). Second, we demonstrate that the context of the PR within GagPol influences the order in which sites within the precursor are processed; the purified enzyme containing a P1A substitution did not display the altered processing phenotype and did not display an appreciably altered Km for the RNase H/IN site (Table 1). Finally, the embedded PR with the P1A substitution was more sensitive to inhibition by two of the three active-site PR inhibitors than the WT PR. Of note, the purified P1A PR did not share this enhanced susceptibility (Table 2).
We were surprised to find that the embedded P1A PR was more sensitive to inhibition by two of the PR active-site inhibitors tested. We believe that this reflects the altered context of the PR domain embedded within GagPol. As noted above, relieving the constraints of the rigid Pro at position 1 of the PR domain allows cleavage of the additional downstream sites. This is consistent with the interpretation that the PR domain of the precursor with the P1A substitution is more flexible and thus able to access these additional sites. It seems likely that the increased flexibility that allows the embedded PR to cleave the downstream sites also increases the accessibility of the enzyme active site. Therefore, the enhanced susceptibility of the embedded P1A PR may be explained by an increased accessibility of the enzyme active site to the inhibitor. Alternatively, the enhanced susceptibility of the PR with the P1A substitution may be explained by unanticipated changes in the enzyme active site. As we do not observe the enhanced susceptibility in the purified mutant PR, this latter possibility seems less likely. However, in the absence of structural information for the precursor, we cannot definitively distinguish between these two interpretations.
In summary, these results demonstrate that substitutions outside of the PR active site influence the selection of processing sites for cleavage. Further, we have identified the initial amino acid in the GagPol PR domain as playing a critical role in constraining the order of cleavage in the WT precursor. Taken together, our data are consistent with a model in which two factors have evolved that define the ordered processing of the cleavage sites within the HIV-1 GagPol precursor. First, the order of cleavage is influenced by the well-described characteristics of the processing sites themselves as reflected in peptide-based studies (3, 7, 23, 29, 38, 40, 48). Second, our data also support the notion that structural constraints act on the PR domain within the context of the GagPol precursor and that the structure of the precursor helps define the order of the initial cleavage events.
ACKNOWLEDGMENTS
We gratefully acknowledge helpful discussions with G. Schatz and V. M. Vogt. Ritonavir, saquinavir, and indinavir were obtained from the NIH AIDS Research and Reference Reagent Program.
This work was supported by NIH grants RO1 GM GM66681-01 and K24 MH071191-01 to A.H.K. and the UNC Center for AIDS Research.
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