This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakalian, M.
Right arrow Articles by Salzwedel, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakalian, M.
Right arrow Articles by Salzwedel, K.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

 Previous Article  |  Next Article 

Journal of Virology, June 2006, p. 5716-5722, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02743-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

3-O-(3',3'-Dimethysuccinyl) Betulinic Acid Inhibits Maturation of the Human Immunodeficiency Virus Type 1 Gag Precursor Assembled In Vitro

Michael Sakalian,1* Curtis P. McMurtrey,1 Frederick J. Deeg,1 Christopher W. Maloy,1 Feng Li,2 Carl T. Wild,2,{dagger} and Karl Salzwedel2

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, Oklahoma 73104,1 Panacos Pharmaceuticals, Inc., 209 Perry Parkway, Gaithersburg, Maryland 208772

Received 30 December 2005/ Accepted 23 March 2006


arrow
ABSTRACT
 
3-O-(3',3'-Dimethysuccinyl) betulinic acid (PA-457) has been shown to potently inhibit human immunodeficiency virus (HIV) replication in culture. In contrast to inhibitors that act upon the viral proteinase, PA-457 appears to block only the final maturational cleavage of p25CA-p2 to p24CA. However, attempts to replicate this effect in vitro using recombinant Gag have failed, leading to the hypothesis that activity is dependent upon the assembly state of Gag. Using a synthesis/assembly system for chimeric HIV type 1 Gag proteins, we have replicated the activity of PA-457 in vitro. The processing of assembled chimeric Gag can be inhibited by the addition of drug with only the final cleavage of p25CA-p2 to p24CA blocked. Consistent with our hypothesis and with previous findings, inhibition appears specific to Gag assembled into an immature capsid-like structure, since synthetic Gag that remains unassembled is properly processed in the presence of the compound. To further analyze the authenticity of the assay, PA-457 was tested in parallel with its inactive parental compound, betulinic acid. Betulinic acid had no effect upon p25 processing in this system. Analysis of a PA-457-resistant mutant, A1V, in this system pointed to more rapid cleavage as a possible mechanism for resistance. However, characterization of additional mutations at the cleavage site and in p2 suggests that resistance does not strictly correlate with the rate of cleavage. With the establishment of an in vitro assay for the detection of PA-457 activity, a more detailed characterization of its mechanism of action will be possible.


arrow
INTRODUCTION
 
Human immunodeficiency virus type 1 (HIV-1) assembly is a complex, multistage process in which polyprotein precursors coalesce into an immature particle that is released from the cell and is proteolytically processed into a mature infectious particle. Chief among the polyproteins responsible for assembly is the Gag precursor Pr55gag, which on its own can produce immature particles from transfected cells (reviewed in reference 16). In recent years, the Gag precursor and its major cleavage product, capsid (CA) (p24), have become attractive targets for the development of antiviral therapeutics (reviewed in reference 10). This stems in part from the clinical success of protease inhibitors, which block the processing of Gag and thereby the many downstream functions of the mature CA protein in core assembly (5, 18), uncoating (3), and reverse transcription (3, 17).

The central role for Gag and its cleavage product, CA, in viral infectivity has been highlighted recently by the emergence of a potential new class of HIV therapeutic agents, the maturation inhibitors. This new class is typified by the compound 3-O-(3',3'-dimethysuccinyl) betulinic acid, known alternatively as PA-457 (8), DSB (1, 21), or YK-FH312 (6). PA-457 appears to inhibit replication by blocking the very last step in Gag processing, preventing the final product, p24CA, from being liberated by the cleavage of p25CA-p2 (8, 21). The inhibition of only one of the five major cleavage sites in HIV Gag by this compound indicates that it acts not as a traditional enzyme competitive inhibitor but rather likely by direct interaction with the substrate Pr55gag. Evidence to support this unique mechanism of action has come from analyses of resistance mutations that map to the CA-p2 cleavage site (8) and from mutational studies where sensitivity to PA-457 was conferred by the substitution of the cleavage site region sequence from HIV into that of simian immunodeficiency virus (9, 19), which would otherwise be resistant. Uniquely, it appeared that PA-457 activity was dependent not merely upon the cleavage site sequence but also upon the higher-order structure of the molecule, since recombinant Gag protein in solution proved to be resistant to PA-457 in an in vitro processing assay (8).

Utilizing a system whereby the Gag protein is properly assembled into immature particle structures, we have developed an in vitro assay for PA-457 activity, confirmed the requirement for a higher-order Gag structure, and shown the assay to be specific for the active form of the drug. In addition, we have also used this system to evaluate a panel of Gag mutants that have been shown to display a range of resistance in culture assays. Previous analysis of one mutant, A1V, indicated a significantly increased rate of CA-p2 cleavage (9). However, our results from the in vitro analysis indicate that resistance to PA-457 does not precisely correlate with the inherent cleavage rate of the mutant sequence.


arrow
MATERIALS AND METHODS
 
DNA constructs. Plasmids pDABCh3, pDABCh4, and pDABCh4.M185A have been described previously (13). Briefly, these plasmids contain HIV/Mason-Pfizer monkey virus (M-PMV) chimeric gag genes driven by the bacteriophage T7 promoter. They also all contain a mutation to prevent the normal ribosomal frameshift into the pro sequence. Plasmids pDABCh3.A1V and pDABCh4.A1V were constructed by subcloning the 499-bp SpeI-to-ApaI fragment from pDAB72-A1V into the pDABCh3 and pDABCh4 backgrounds. Plasmids pDABCh3.L231C, pDABCh3.T8{Delta}, pDABCh3.I13{Delta}, and pDABCh3.M14{Delta} were all similarly constructed by subcloning the same SpeI-to-ApaI fragment from pNL4-3/L231C, pNL4-3/{Delta}T8, pNL4-3/{Delta}I13, and pNL4-3/{Delta}M14, respectively.

Drugs. PA-457 (PA103001), also known as DSB (21), was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. Serial dilutions of this stock in DMSO were made to the appropriate concentration such that the addition of 1 µl to a processing reaction mixture would yield the indicated final drug concentration. Indinavir was similarly dissolved in DMSO and diluted for use.

In vitro production of assembled Gag. Transcription and translation were performed sequentially using the Single Tube Protein System 3 (Novagen) in the presence of [35S]methionine and programmed with the pDABCh series plasmids. Products of these synthesis reactions were analyzed on sucrose gradients. Reaction mixtures (200 µl) were loaded onto 2.2-ml continuous 30 to 55% (wt/wt) sucrose gradients in buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100. Gradients were centrifuged in a TLS-55 rotor (Beckman Instruments) for 2 h at 55,000 rpm. Fractions of approximately 200 µl were taken by hand with a Pipetman (Gilson) from the top of the gradient. The pellet was resuspended in 200 µl of 55% (wt/wt) sucrose in gradient buffer. Aliquots (10 µl) of each fraction were dissolved in sodium dodecyl sulfate (SDS) sample buffer and then loaded onto an SDS-10% polyacrylamide gel. After electrophoresis, radioactive bands were visualized on a Perkin-Elmer Cyclone Storage Phosphor system. Peak fractions from the gradient containing assembled Gag were pooled for use in processing experiments. Samples of pooled, assembled Gag and unassembled Gag were then run on SDS-15% polyacrylamide gels and quantitated by phosphorimager analysis to measure radioactive units of Gag per microliter in each sample.

Processing of Gag in vitro. Processing of Gag in vitro was performed similarly to the procedure described previously by Pettit et al. (11). Briefly, equivalent amounts of Gag as measured by phoshorimager analysis (typically approximately 10 µl) were diluted into phosphate-buffered saline (pH 7.4) to a final volume of 50 µl in the presence or absence of drug. A varied amount of recombinant HIV-1 protease (Bachem) was added to each reaction mixture, and processing was allowed to proceed for the times indicated at 30°C. After incubation, a 10-µl aliquot of the reaction mixture was removed, dissolved in SDS-polyacrylamide gel electrophoresis (PAGE) protein loading buffer, and frozen on dry ice. Since the initial cleavage of Gag is so rapid, the "time-zero" aliquot was removed before the addition of the protease. Following the time course experiments, a standardized protocol was established using 0.48 µg of protease for 3 h of incubation. Samples were boiled for 5 min and loaded onto SDS-15% PAGE gels. Fixed and dried gels were then exposed to phosphor plates, and the resulting images were analyzed with OptiQuant software (Perkin-Elmer). For inhibition experiments, radioactivity in the p24 and p25 bands was quantified and adjusted for the number of methionines in each protein. The extent of processing in each reaction was compared to the maximum seen in the control no-drug reaction and then represented as the percentage of that maximum.


arrow
RESULTS
 
In vitro processing of synthesized and assembled Gag. We have previously reported on a system, based on in vitro translation in a rabbit reticulocyte lysate, whereby the HIV Gag polyprotein precursor can be assembled into immature capsid-like structures. This system was initially described for Mason-Pfizer monkey virus (15) and later adapted to HIV (13). HIV Gag, which normally does not assemble in this system, was adapted by construction of a chimera containing the M-PMV Gag p12 region. M-PMV p12 contains an internal scaffold domain that increases the efficiency of Gag assembly (14). Among the chimeric proteins that are assembly competent, the most complete one for HIV content is chimera 4 (Fig. 1), which consists of the entire HIV Gag protein with p12 fused to the C terminus. The products of chimera 4 synthesis can be examined on a sucrose gradient to detect the formation of particulate material indicative of assembled Gag (Fig. 2), or completed reactions can be processed for thin-section electron microscopy, where structures morphologically identical to immature retrovirus capsids can be identified (data not shown) (see reference 13). For studies of Gag processing and its inhibition in vitro, assembled chimera 4 Gag was partially purified on sucrose gradients, and the peak fractions were pooled, for example, fractions 6 and 7 in Fig. 2. In contrast to the assembled full-length chimera 4, an approximately 40-kDa product does not assemble and remains at the top of the gradient. This smaller protein is likely the product of internal translation initiation at the second gag methionine codon located in the CA coding sequence.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. Schematics of Gag polyprotein precursors used in this study. Shown are the Gag proteins of M-PMV, HIV-1, chimera 4 (Ch4), and chimera 3 (Ch3). The shaded regions are the p12 domain, which contains the internal scaffold domain of M-PMV Gag. Domain designations are given by the convention where protein, "p," or phosphoprotein, "pp," is followed by its apparent molecular weight (in thousands) by SDS-PAGE, i.e., "p12" or "pp24." Letter designations conform to the standardized nomenclature for retroviral proteins where MA is the matrix, CA is the capsid, and NC is the nucleocapsid domain. Numbers above each diagram give the amino acid residue number for the start of each domain or the end of the polyprotein.


Figure 2
View larger version (51K):
[in this window]
[in a new window]
  		FIG. 2. Generation of assembled substrate for processing experiments. Gag proteins were synthesized in a coupled transcription and translation system and incubated for 24 h to allow assembly to occur. (A) Sucrose gradient analysis of chimera 4 assembly. Assembled Gag was fractionated on a 30 to 55% (wt/wt) sucrose gradient, and aliquots of each fraction were electrophoresed on SDS-10% polyacrylamide gels. Gag is marked by its molecular mass of 65 kDa. An approximately 40-kDa internal initiation product is also indicated. Lanes are marked for fraction numbers starting at the top of the gradient (lane 1) to the pellet (lane P). Lane L is an equal representative fraction of the total before loading onto the gradient. (B) Quantitative representation of the gel data. Radioactivities of Gag and the assembly-incompetent 40-kDa protein were quantified by phosphorimager analysis and are represented as a percentage of the total in lanes 1 to 12, plus P.

Before examining the potential effect of PA-457 on maturation in vitro, we first examined whether assembled chimera 4 Gag would be processed properly to p24CA by the addition of recombinant protease. Time course analysis of chimera 4 and HIV Gag proteins showed that both proteins were quickly processed to p25CA-p2 but that significant final processing to p24CA required incubation for three or more hours, with cleavage completed by 24 h (Fig. 3). Chimera 4, therefore, can be processed properly to CA in vitro and is cleaved with kinetics approximately equivalent to those for wild-type (wt) HIV Gag. Further experiments were conducted to determine the optimal conditions for measuring the inhibition of final processing. These were determined to be the point at which cleavage of p25CA-p2 to p24CA is nearly but not yet completed so that small differences in the extent of cleavage will be evident. These standardized conditions (see Materials and Methods) were used in all subsequent processing experiments.


Figure 3
View larger version (66K):
[in this window]
[in a new window]
  		FIG. 3. Processing time course of in vitro-synthesized Gag proteins. HIV and chimera 4 Gag proteins were processed in parallel by recombinant HIV-1 protease. The positions of full-length HIV and chimera 4 Gag proteins are indicated by Pr55gag and Pr65gag, respectively. The final matured p24CA and its immediate precursor, p25CA-p2, are indicated. Apparent molecular mass markers (kilodaltons) are given at the left, and times of incubation are given at the tops of the gels.

Inhibition of processing by PA-457 requires assembled Gag. To examine the potential for PA-457 to inhibit the final processing of Gag in vitro, a titration experiment was performed with chimera 4 and HIV Gag proteins. HIV Gag, which does not assemble in our in vitro system (13, 15), was obtained from simple transcription/translation reactions and not partly purified by gradient sedimentation. The concentration of PA-457 was varied over 4 orders of magnitude from 0.01 to 10 µM in the cleavage reactions (Fig. 4). Conversion of p25CA-p2 to p24CA appeared to be reduced for chimera 4 but not for HIV Gag as the concentration of PA-457 was increased. This is especially evident by a comparison of the 10 µM lane to the no-drug control lane, where the ratio of p25CA-p2 to p24CA is clearly less (Fig. 4A). Quantitation confirmed the visual observation, with the highest concentration of PA-457 producing only a modest reduction in cleavage of the wt compared to a dramatic (approximately 50%) reduction for assembled chimera 4 (Fig. 4B). The measurement of further inhibition of p25CA-p2 cleavage in this assay was complicated by the fact that higher concentrations (≥100 µM) of PA-457 had nonspecific global effects on processing by protease at all Gag cleavage sites (data not shown).


Figure 4
View larger version (48K):
[in this window]
[in a new window]
  		FIG. 4. Processing inhibition by PA-457. HIV and assembled chimera 4 (Ch4) Gag proteins were processed in parallel with increasing concentrations of PA-457. (A) The regions of the gels containing p24 and p25 are shown. The micromolar concentration of PA-457 in each reaction is given above the corresponding gel lanes. (B) Quantitative representation of the gel data normalized to the maximum amount of processing seen in the no-drug control.

The activity of PA-457 against chimera 4 but not HIV Gag is consistent with the hypothesis that the drug is recognizing or binding to a higher-order structure in the immature assembled particle; however, chimera 4 contains M-PMV p12 in addition to the complete HIV Gag sequence. To eliminate the possibility that p12 was somehow conferring drug susceptibility upon HIV Gag, we examined a version of chimera 4 containing the assembly-defective mutation M185A, which lies within the critical dimer interface of the C-terminal domain of CA (4). A previous analysis of this mutation in the context of chimera 4 confirmed that it is likewise assembly defective in the in vitro system (13).

Assembled chimera 4, unassembled chimera 4-M185A, and wild-type HIV Gag proteins were compared in parallel (Fig. 5A). Again, the drug appears to have no effect on HIV Gag and, more significantly, no effect upon unassembled chimera 4-M185A, while the proportion of p24CA cleaved from the assembled chimera 4 was reduced in a dose-dependent manner. Quantitation confirmed that the mutant chimera 4 and wild-type HIV Gag proteins are equivalently unaffected by the drug (Fig. 5B). Thus, the ability of PA-457 to block cleavage of p25CA-p2 correlated with the assembly state of Gag rather than with the presence of p12 in the polyprotein.


Figure 5
View larger version (41K):
[in this window]
[in a new window]
  		FIG. 5. Comparison of processing inhibition for assembled chimera 4 (Ch4) Gag and unassembled HIV and mutant chimera 4 M185A Gag proteins. (A) Portions of gels showing p24 and p25. The concentration of PA-457 is given at the top. (B) Quantitative representation of gel data. Data were analyzed as described in the legend of Fig. 4.

Inhibition of processing is specific to the active form of the drug. As a check for specificity to PA-457 in this assay, we compared its activity to that of the parental molecule, betulinic acid. Betulinic acid and PA-457 share the same five-membered polycyclic structure, except that PA-457 is modified with the addition of a dimethylsuccinyl side chain at position 3 (6). PA-457 blocks p25CA-p2 in p24CA processing, whereas betulinic acid does not (F. Li, K. Salzwedel, and C. T. Wild, unpublished data). Titrations of PA-457 and betulinic acid were performed in parallel, and again, the reduction in p24CA production with PA-457 was evident. In contrast, betulinic acid had little effect at all on the cleavage of p25CA-p2 to p24CA (Fig. 6). These results demonstrate that the inhibition of p25CA-p2 in p24CA processing was specific to the structure of PA-457.


Figure 6
View larger version (38K):
[in this window]
[in a new window]
  		FIG. 6. Comparison of processing inhibition by PA-457 and the related compound betulinic acid. (A) Portions of gels showing p24 and p25. The concentration of the drug is given at the top. (B) Quantitative representation of gel data. Data were analyzed as described in the legend of Fig. 4.

PA-457 activity does not require the p6 domain of HIV-1 Gag. During the development of the in vitro assembly system, several chimeras that have the capacity to assemble were constructed (13). One of these, chimera 3, is similar in domain arrangement to chimera 4 but is deleted of HIV p6 (Fig. 1). The utilization of chimera 3 provided two advantages: first, this chimeric Gag is more consistent than chimera 4 for producing assembled material across different lots of reticulocyte lysate (not shown); second, it provided a convenient way to eliminate the potential involvement of the p6 domain in drug activity that, although unlikely, was still formally possible. Assembled chimera 3 was generated and partially purified on sucrose gradients as described above for chimera 4. Both chimeric Gag proteins were then used in an experiment with PA-457 and betulinic acid in parallel. The results were equivalent for the two substrate polyproteins: final processing was inhibited by PA-457, whereas betulinic acid had no discernible effect (data not shown). Quantitation confirmed the visual interpretation of the gel (Fig. 7), and therefore, the HIV-1 p6 domain is dispensable for PA-457 activity.


Figure 7
View larger version (7K):
[in this window]
[in a new window]
  		FIG. 7. Comparison of processing inhibition by PA-457 and betulinic acid for chimeras 3 and 4. Gels and data were analyzed as described in the legend of Fig. 4.

A resistance mutant derived from culture is similarly resistant in vitro. Resistance mutants to PA-457 that were developed by cultures of viruses in the presence of the drug have been described previously (8, 21). Consistent with a model in which PA-457 interacts directly with the Gag protein to block the protease, each of these mutants maps to the CA-p2 cleavage site (8, 19, 21). To assess the ability of our in vitro system to replicate results seen with the tissue culture virus assays, we introduced one of these mutants, A1V (8), into our chimeras as well as several other synthetic mutations of interest: the single-substitution mutant L231C and the single-residue deletions {Delta}T8, {Delta}I13, and {Delta}M14 (Fig. 8A). In virus replication assays, the deletion mutants displayed a range of sensitivity to PA-457, with {Delta}I13 being sensitive, {Delta}T8 being partially resistant, and {Delta}M14 being completely resistant (9). In contrast, substitution mutant L231C could not be examined in culture assays since it is defective for particle production (9). This mutant was of particular interest in our analysis of the PA-457 mechanism of action not only because it cannot be assayed in a culture system but also because, in contrast to the resistance mutation A1V, which increases the rate of cleavage, this mutation is known to slow cleavage of p2 from CA in an in vitro processing assay (11).


Figure 8
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 8. Analysis of CA-p2 region mutants. (A) Diagram showing the amino acid sequence of the cleavage site and p2 with the mutants indicated. The symbol {Delta} denotes a deletion. (B) Portions of gels from representative experiments showing p24 and p25. The concentrations of PA-457 are given at the top of gel panes. (C) Extent of cleavage achieved by standard reaction conditions in the absence of drug. Error bars indicate standard deviations for three replicate experiments for Ch3.{Delta}T8; four experiments for Ch3.L231C, Ch3.A1V, and Ch3.{Delta}M14; and six experiments for Ch3 and Ch3.{Delta}I13. (D) Quantitative representation of inhibition by PA-457 for each mutant. Error bars are standard deviations for replicates as given in panel C.

Mutant A1V was introduced into both chimera 3 (Fig. 8B) and chimera 4 (data not shown). The results were identical for each chimera. The remaining mutants were analyzed only in the context of chimera 3. Each mutant was tested using the standard processing assay with and without PA-457. The extent of cleavage for most of the mutants was increased compared to that of the wt sequence (Fig. 8C), with Ch3.A1V and Ch3.{Delta}M14 being processed completely to p24CA (Fig. 8C). Ch3.{Delta}T8 and Ch3.{Delta}I13 were processed to an intermediate extent, while Ch3.L231C was processed at only approximately half the rate of the wt (Fig. 8C).

With respect to their resistance to PA-457 in the in vitro assay, the mutants could be classified into two groups compared to the wt sequence: those that were completely resistant (A1V and {Delta}M14) and those that were partially resistant (L231C, {Delta}T8, and {Delta}I13). These results are in rough agreement with those from the culture assays, again with one significant exception. L231C could be assessed only in vitro, and it was partially resistant to PA-457 even though it was processed more slowly. Thus, it appears that resistance to PA-457 does not strictly correlate with the intrinsic rate of cleavage of the mutant Gag.


arrow
DISCUSSION
 
The development of an in vitro assay represents a significant step towards an understanding of the mechanism of HIV-1 maturation inhibitors. The in vitro system based on synthesized radiolabeled Gag in particular provides for a more quantitative readout than methods that might be based on particles produced by tissue culture. Previous failed attempts to reproduce the activity of PA-457 in vitro with a recombinant Gag precursor led to the hypothesis that the drug recognizes a structure present only in the protein when assembled in a particle (8). With this assay, we now provide a direct demonstration that Gag must be assembled into an immature particle-like structure for drug activity.

In addition to the assembly state of Gag, the assay proved valuable in assessing the relative extent of resistance to the drug by several mutations both developed through virus culture and created through site-directed mutagenesis. Beyond establishing that the in vitro assay reproduced the resistance seen in cultures with these mutants, it also provided insight into the drug mechanism of action. Most of these mutants significantly increased the inherent rate of cleavage at the CA-p2 junction. One mutant, A1V, changes a residue in the P1' position of the cleavage site itself, but two others, {Delta}I13 and {Delta}M14, are at the opposite end of p2. How can these two mutations affect cleavage 13 or 14 residues N-terminal to the deletion? The rate of cleavage at the CA-p2 site is apparently regulated by cleavage at the p2-NC site such that blocking cleavage at the downstream site increases the rate at the upstream site by 20-fold (12). Furthermore, the substitution of an isoleucine for the methionine at P1 of the p2-NC cleavage site reduces cleavage there to 35% of that of the wild-type sequence (11). Deletion of M14 results in the placement of I13 at P1 of the p2-NC site, thus partially blocking cleavage and thereby increasing cleavage at CA-p2. How cleavage at p2-NC can affect the rate of cleavage at CA-p2 is not well understood, but this observation provides a plausible argument for the behavior of mutant {Delta}M14 as well as mutant {Delta}I13, which effectively changes the residue at P2 and which is cleaved more rapidly than the wt. Mutant {Delta}T8 also, but less significantly, increases cleavage at CA-p2. Since this deletion is positioned in the center of p2, there is no obvious explanation for its effect upon CA-p2 cleavage other than to say that perhaps it has a modest negative effect on cleavage at p2-NC.

The increase in the cleavage for each of the above-described mutants was roughly proportional to the extent of cleavage in the presence of PA-457, suggesting that the mechanism of resistance is through a more rapid inherent rate at the cleavage site. However, there was one exception. Mutant L231C cleaved only to about 50% of the level of the wt in the absence of the drug, yet it fell into the class of mutants displaying intermediate resistance. This mutant, which could only be tested in our in vitro system due to a defect in particle release, demonstrates that resistance does not correlate precisely with the cleavage rate and provides evidence that would be consistent with a direct drug-Gag interaction model for the mechanism of PA-457.

PA-457 produced a detectable change in the extent of p25CA-p2 cleavage over the range of 0.1 to 10 µM with a 50% inhibitory concentration (IC50) between 1 and 10 µM. These numbers are in apparent contradiction with the previously reported IC50 of approximately 10 nM in tissue culture assays, which is a 100- to 1,000-fold difference. The discrepancy arises from the different endpoints of the assays. The in vitro assay measures the extent of CA-p2 cleavage, while the culture assays measured cell killing as a gauge of virus replication and were therefore an indirect measurement of infectivity. Indeed, if the processing of p25CA-p2 to p24CA is used as an endpoint in the tissue culture system, the activity of PA-457 in cells should be similar to that seen in our in vitro system (9). Since the virus replication IC50 is below the detectable range of activity in the in vitro cleavage assay, the logical conclusion is that the virus is either extremely sensitive to small changes in the rate of processing or that only a very small number of p25CA-p2 molecules is sufficient to poison the virion. Either of these predictions is surprising given that only approximately one-half of the CA protein in a virus particle is assembled into the mature core structure (2, 7). By the first mechanism, one must invoke a rather delicate process of core condensation that requires precise timing to successfully produce an infectious virion. By the second mechanism, two equally plausible theories can be invoked, whereby (i) incorporation of one or more p25CA-p2 molecules can throw core assembly off pathway or (ii) p25CA-p2 cannot coassemble with p24CA into the core but nonetheless poisons an as-yet-cryptic but necessary function of either CA or p2 in infectivity.

As the paper was in final preparation, Zhou et al. (20) reported on the incorporation of PA-457 into immature particles and on an assay using such released particles to reproduce the dose-dependent activity of PA-457 in blocking the processing of p25CA-p2 in vitro. Using 3H-labeled drug and an enzyme-linked immunosorbent assay to quantify CA, those authors estimated the stoichiometry of drug incorporation into particles to be approximately 1:1 with respect to the Gag monomer, while no drug was found within similarly prepared resistant mutant particles. Consistent with these data, our findings provide significant additional evidence for a mechanism of PA-457 whereby the drug interacts directly with the Gag substrate. Moreover, our in vitro assay, by the synthesis of high-specific-activity radiolabeled Gag substrates, provides a more facile approach to a quantitative comparison of additional potential maturation inhibitors and of Gag mutants, including those that may be defective for particle production from cells.


arrow
ACKNOWLEDGMENTS
 
This work was supported by the University of Oklahoma Health Sciences Center.

We thank Eric Freed for critical review of the manuscript.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, BMSB 1019, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104. Phone: (405) 271-2133. Fax: (405) 271-3117. E-mail: mike-sakalian{at}ouhsc.edu. Back

{dagger} Present address: 19008 Oxcart Place, Gaithersburg, MD 20886. Back


arrow
REFERENCES
 
    1
  1. Aiken, C., and C. H. Chen. 2005. Betulinic acid derivatives as HIV-1 antivirals. Trends Mol. Med. 11:31-36.[CrossRef][Medline]
    2. 2
  2. Briggs, J. A., M. N. Simon, I. Gross, H. G. Krausslich, S. D. Fuller, V. M. Vogt, and M. C. Johnson. 2004. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 11:672-675.[CrossRef][Medline]
    3. 3
  3. Forshey, B. M., U. K. von Schwedler, W. I. Sundquist, and C. Aiken. 2002. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J. Virol. 76:5667-5677.[Abstract/Free Full Text]
    4. 4
  4. Gamble, T. R., S. Yoo, F. F. Vajdos, U. K. von Schwedler, D. K. Worthylake, H. Wang, J. P. McCutcheon, W. I. Sundquist, and C. P. Hill. 1997. Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 278:849-853.[Abstract/Free Full Text]
    5. 5
  5. Ganser, B. K., S. Li, V. Y. Klishko, J. T. Finch, and W. I. Sundquist. 1999. Assembly and analysis of conical models for the HIV-1 core. Science 283:80-83.[Abstract/Free Full Text]
    6. 6
  6. Kanamoto, T., Y. Kashiwada, K. Kanbara, K. Gotoh, M. Yoshimori, T. Goto, K. Sano, and H. Nakashima. 2001. Anti-human immunodeficiency virus activity of YK-FH312 (a betulinic acid derivative), a novel compound blocking viral maturation. Antimicrob. Agents Chemother. 45:1225-1230.[Abstract/Free Full Text]
    7. 7
  7. Lanman, J., T. T. Lam, M. R. Emmett, A. G. Marshall, M. Sakalian, and P. E. Prevelige, Jr. 2004. Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange. Nat. Struct. Mol. Biol. 11:676-677.[CrossRef][Medline]
    8. 8
  8. Li, F., R. Goila-Gaur, K. Salzwedel, N. R. Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, D. E. Martin, J. M. Orenstein, G. P. Allaway, E. O. Freed, and C. T. Wild. 2003. PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc. Natl. Acad. Sci. USA 100:13555-13560.[Abstract/Free Full Text]
    9. 9
  9. Li, F., R. Goila-Gaur, K. Salzwedel, N. R. Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis, D. E. Martin, J. M. Orenstein, G. P. Allaway, E. O. Freed, and C. T. Wild. Unpublished results.
    10. 10
  10. Li, F., and C. Wild. 2005. HIV-1 assembly and budding as targets for drug discovery. Curr. Opin. Investig. Drugs 6:148-154.[Medline]
    11. 11
  11. Pettit, S. C., G. J. Henderson, C. A. Schiffer, and R. Swanstrom. 2002. 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. J. Virol. 76:10226-10233.[Abstract/Free Full Text]
    12. 12
  12. Pettit, S. C., M. D. Moody, R. S. Wehbie, A. H. Kaplan, P. V. Nantermet, C. A. Klein, and R. Swanstrom. 1994. The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions. J. Virol. 68:8017-8027.[Abstract/Free Full Text]
    13. 13
  13. Sakalian, M., S. S. Dittmer, A. D. Gandy, N. D. Rapp, A. Zabransky, and E. Hunter. 2002. The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag. J. Virol. 76:10811-10820.[Abstract/Free Full Text]
    14. 14
  14. Sakalian, M., and E. Hunter. 1999. Separate assembly and transport domains within the Gag precursor of Mason-Pfizer monkey virus. J. Virol. 73:8073-8082.[Abstract/Free Full Text]
    15. 15
  15. Sakalian, M., S. D. Parker, R. A. Weldon, Jr., and E. Hunter. 1996. Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro. J. Virol. 70:3706-3715.[Abstract]
    16. 16
  16. Swanstrom, R., and J. W. Wills. 1997. Synthesis, assembly, and processing of viral proteins, p. 263-334. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    17. 17
  17. Tang, S., T. Murakami, B. E. Agresta, S. Campbell, E. O. Freed, and J. G. Levin. 2001. Human immunodeficiency virus type 1 N-terminal capsid mutants that exhibit aberrant core morphology and are blocked in initiation of reverse transcription in infected cells. J. Virol. 75:9357-9366.[Abstract/Free Full Text]
    18. 18
  18. von Schwedler, U. K., K. M. Stray, J. E. Garrus, and W. I. Sundquist. 2003. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J. Virol. 77:5439-5450.[Abstract/Free Full Text]
    19. 19
  19. Zhou, J., C. H. Chen, and C. Aiken. 2004. The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid. Retrovirology 1:15.[CrossRef][Medline]
    20. 20
  20. Zhou, J., L. Huang, D. L. Hachey, C. H. Chen, and C. Aiken. 2005. Inhibition of HIV-1 maturation via drug association with the viral Gag protein in immature HIV-1 particles. J. Biol. Chem. 280:42149-42155.[Abstract/Free Full Text]
    21. 21
  21. Zhou, J., X. Yuan, D. Dismuke, B. M. Forshey, C. Lundquist, K.-H. Lee, C. Aiken, and C. H. Chen. 2004. Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation. J. Virol. 78:922-929.[Abstract/Free Full Text]

Journal of Virology, June 2006, p. 5716-5722, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02743-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Adamson, C. S., Waki, K., Ablan, S. D., Salzwedel, K., Freed, E. O. (2009). Impact of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors on Evolution of Resistance to the Maturation Inhibitor Bevirimat (PA-457). J. Virol. 83: 4884-4894 [Abstract] [Full Text]  
  • Van Baelen, K., Salzwedel, K., Rondelez, E., Van Eygen, V., De Vos, S., Verheyen, A., Steegen, K., Verlinden, Y., Allaway, G. P., Stuyver, L. J. (2009). Susceptibility of Human Immunodeficiency Virus Type 1 to the Maturation Inhibitor Bevirimat Is Modulated by Baseline Polymorphisms in Gag Spacer Peptide 1. Antimicrob. Agents Chemother. 53: 2185-2188 [Abstract] [Full Text]  
  • Keller, P. W., Johnson, M. C., Vogt, V. M. (2008). Mutations in the Spacer Peptide and Adjoining Sequences in Rous Sarcoma Virus Gag Lead to Tubular Budding. J. Virol. 82: 6788-6797 [Abstract] [Full Text]  
  • Purdy, J. G., Flanagan, J. M., Ropson, I. J., Rennoll-Bankert, K. E., Craven, R. C. (2008). Critical Role of Conserved Hydrophobic Residues within the Major Homology Region in Mature Retroviral Capsid Assembly. J. Virol. 82: 5951-5961 [Abstract] [Full Text]  
  • Zhou, J., Chen, C. H., Aiken, C. (2006). Human Immunodeficiency Virus Type 1 Resistance to the Small Molecule Maturation Inhibitor 3-O-(3',3'-Dimethylsuccinyl)-Betulinic Acid Is Conferred by a Variety of Single Amino Acid Substitutions at the CA-SP1 Cleavage Site in Gag. J. Virol. 80: 12095-12101 [Abstract] [Full Text]  
  • Adamson, C. S., Ablan, S. D., Boeras, I., Goila-Gaur, R., Soheilian, F., Nagashima, K., Li, F., Salzwedel, K., Sakalian, M., Wild, C. T., Freed, E. O. (2006). In Vitro Resistance to the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PA-457 (Bevirimat). J. Virol. 80: 10957-10971 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakalian, M.
Right arrow Articles by Salzwedel, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakalian, M.
Right arrow Articles by Salzwedel, K.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»