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J. Biol. Chem., Vol. 277, Issue 8, 5952-5961, February 22, 2002

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Amino Acid Substitutions in Gag Protein at Non-cleavage Sites Are Indispensable for the Development of a High Multitude of HIV-1 Resistance against Protease Inhibitors^*

Hiroyuki Gatanaga^§, Yasuhiro Suzuki, Hsinyi Tsang, Kazuhisa Yoshimura^¶, Mark F. Kavlick, Kunio Nagashima, Robert J. Gorelick^**, Sek Mardy, Chun Tang, Michael F. Summers, and Hiroaki Mitsuya^¶§§

From the  Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, the ^¶ Department of Internal Medicine II, Kumamoto University School of Medicine, Honjo 1-1-1 Kumamoto 860, Japan, the  Image Analysis Laboratory and ^** AIDS Vaccine Program, Science Applications International Corporation, NCI, Frederick, Maryland 21702, and the  Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland, Baltimore, Maryland 21250

Received for publication, August 20, 2001, and in revised form, November 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Combination antiretroviral therapy using reverse transcriptase inhibitors and protease inhibitors (PIs)¹ produces substantial suppression of viral replication in HIV-1-infected patients. However, the emergence of drug-resistant HIV-1 variants in such patients has limited the efficacy of combination chemotherapy. HIV-1 variants resistant to any of the currently available antiretroviral therapeutics have emerged both in vitro and in vivo (1). In particular, HIV-1 resistant to one PI is often cross-resistant to another PI, presenting formidable challenges in the therapy of HIV-1 infection. Indeed, HIV-1 protease has been shown to tolerate extensive sequence variations, remaining functional even with as many as 15 amino acid substitutions accumulated in a molecule composed of 99 amino acids (2, 3).

Amino acid substitutions in HIV-1 Gag precursor p7-p1 and p1-p6 cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy including PIs (4, 5). Those substitutions are believed to compensate for the enzymatic impairment of protease per se resulting from the acquisition of PI resistance-conferring amino acid substitutions within the protease-encoding region of the HIV-1 genome. However, a number of highly PI-resistant HIV-1 variants lack such cleavage site amino acid substitutions. Furthermore, recombinant HIV-1 clones to which PI resistance-conferring substitutions in the protease-encoding region were introduced are known to often fail to propagate in vitro (6). Therefore, we thought that as yet unidentified amino acid substitutions in the Gag-Pol polyprotein, the substrate for the enzyme, compensate for the altered enzymatic function caused by the acquisition of amino acid substitutions in the viral protease. To this end, we generated PI-resistant HIV-1 variants by exposing HIV-1 to four different PIs including amprenavir (APV) (7), JE-2147 (2), KNI-272 (8), and UIC-94003 (7), identified up to 23 amino acid substitutions in Gag and protease, generated a variety of infectious HIV-1 clones containing such amino acid substitutions, and characterized their replication profiles. We conclude that Gag amino acid substitutions such as H219Q and R409K, located outside the cleavage sites, contribute to the development of HIV-1 resistance to PIs and are essential for the replication of HIV-1 variants in the presence of PIs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Antiviral Agents-- MT-2 and H9 cells were grown in RPMI 1640-based culture medium supplemented with 10% fetal calf serum (HyClone, Logan, UT), 50 units/ml penicillin, and 50 µg/ml streptomycin. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were stimulated by phytohemagglutinin (PHA) in RPMI 1640-based medium containing interleukin-2 (5 ng/ml) (R & D Systems, Minneapolis, MN) for 2 days before HIV-1 exposure. APV was a kind gift from Glaxo Wellcome. JE-2147 and KNI-272 were synthesized as described previously (2, 8, 9). UIC-94003 was synthesized by Arun Ghosh as described previously (7, 10).

Generation of HIV-1 Resistant to PIs-- The wild type clonal HIV-1, HIV-1_NL4-3, obtained from COS-7 cells transfected with pHIV-1_NL4-3, was propagated in human CD4+ MT-2 cells in the presence of increasing concentrations of PIs as described previously (2, 7). Briefly, MT-2 cells (5 × 10⁵) were exposed to HIV-1_NL4-3 (500 50% tissue culture infectious dose (TCID₅₀)) and cultured in the presence of APV, KNI-272, or UIC-94003 at initial concentrations of 0.0005-0.03 µM. Viral replication was monitored by observation of the cytopathic effect (CPE) in MT-2 cells. The culture supernatant was harvested on day 7 of culture and used to infect fresh MT-2 cells for the next round of culture. When the virus began to propagate in the presence of the drug, the drug concentration was increased. This selection was carried out for a total of 27-62 passages. For the generation of JE-2147-resistant virus, HIV-1_NL4-3/I84V (2) was employed instead of HIV-1_NL4-3. Proviral DNAs from the lysates of infected cells from several passages were sequenced as indicated.

Determination of Nucleotide Sequences-- Molecular cloning and determination of nucleotide sequences of HIV-1 passaged in the presence or absence of antiretroviral agents was performed as described previously (2). In brief, high molecular weight DNA was extracted from HIV-1-infected MT-2 cells using High Pure Viral Nucleic Acid Kit (Roche Molecular Biochemicals) and the entire Gag- and protease-encoding regions of the HIV-1 genome was amplified with Taq DNA polymerase (PerkinElmer Life Sciences) using the following primer pair: the forward primer (LF3) 5'-TCT CGA CGC AGG ACT CGG CTT GCT GAA GCG CGC AC-3' and the reverse primer (XM1) 5'-GGC CAT CCA TCC CGG GCT TTA ATT TTA CTG-3' (Fig. 1). The forward primer had a BssHII site, whereas the SmaI site was introduced to the reverse primer with synonymous substitutions at nucleotides 2590 and 2593. The PCR products were purified with PCR Select III columns (5 Prime 3 Prime, Inc., Boulder, CO) and subjected to molecular cloning using Original TA Cloning Kit (Invitrogen), followed by sequence determination using an Applied Biosystems model 373 automated DNA sequencer.

Generation of Recombinant HIV-1 Clones-- The PCR products obtained as described above were digested with two of three enzymes BssHII, ApaI, and SmaI, and the obtained fragments were introduced into pHIV-1_NLSma designed to have a SmaI site by changing two nucleotides (2590 and 2593) of pHIV-1_NL4-3. To generate HIV-1 clones carrying the mutations, site-directed mutagenesis using the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) was performed, and the mutation containing genomic fragments were introduced to pHIV-1_NLSma. Determination of the nucleotide sequences of plasmids confirmed that each clone had the desired mutations but no unintended mutations. Each recombinant plasmid was transfected into COS-7 cells with GenePORTER Transfection Reagent (Gene Therapy Systems, San Diego, CA), and thus obtained infectious virions were harvested 48 h after transfection and stored at 80 °C until use.

MAGI Assay-- MAGI assay was employed to determine the infectivity of the stock HIV-1 preparations as described previously (11, 12). HeLa-CD4-LTR--gal cells (11) (10⁴/well) were plated in 96-well flat-bottomed microtiter culture plates. On the following day, the medium was aspirated, and the cells were exposed to HIV-1 in a total volume of 50 µl. After incubation at 37 °C for 2 h, fresh complete Dulbecco's modified Eagle's medium (50 µl) was added to each well. Forty eight hours after viral exposure, the total number of blue cells in each well was determined. All assays were performed in triplicate.

Replication Kinetics Assay-- MT-2 cells (10⁵) or PHA-stimulated PBMCs (PHA-PBMCs, 5 × 10⁶) were exposed to each infectious virus preparation (500 and 2000 blue cell-forming units defined in the MAGI assay for MT-2 cells and PBMCs, respectively) for 12 h, washed twice with PBS, and cultured in 5 ml of complete medium in the presence or absence of PI. Culture supernatants (200 µl) were harvested every other day, and p24 Gag amounts were determined using a commercially available radioimmunoassay kit (Dupont/NEN Research Products, Boston, MA). An enzyme-linked immunosorbent assay kit (Beckman Coulter, Inc., Fullerton, CA) was also used for the determination of p24 Gag amounts as needed.

MTT Assay-- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to determine the CPE of each stocked virus preparation. MT-2 cells (4 × 10³ in 200 µl complete medium per well) were plated onto 96-well flat-bottomed microplates, and each virus preparation (20 blue cell-forming units/well) was inoculated. In 10 days of culture, medium (100 µl) was removed, and MTT solution (10 µl, 7.5 mg/ml) was added to each well. The cells were incubated at 37 °C for 2 h and exposed to 100 µl of acidified isopropyl alcohol-containing 4% (v/v) Triton X-100 to dissolve the formazan crystals. The absorbance (wavelength, 570 nm) was then measured in a microplate reader. All assays were performed in triplicate.

Competitive HIV-1 Replication Assay (CHRA)-- Freshly prepared H9 cells (3 × 10⁵) were exposed to infectious clones to be examined for their replicative ability and cultured in the presence or absence of APV as described previously (12). To ensure that the infectivity of paired infectious clones was equal or comparable, a fixed amount (200 TCID₅₀) of one infectious clone was combined with three different amounts (100, 200, and 300 TCID₅₀) of the second infectious clone. On day 1 in culture, one-third of infected H9 cells were harvested and washed twice with PBS, and cellular DNA was extracted and subjected to nested PCR for amplification of the p7-p1-p6-encoding gag region plus the protease-encoding gene, and direct sequencing was performed. The first primer pair used was CM1 (5'-ATG TAA AAG ACA CCA AGG AAG C-3) and CM4.1 (5'-TAT TTT TTC TTC TGT CAA TGG C-3'). The second primer pair used was CM3 (5'-TGT AAA ACG ACG GCC AGT AAA TGA TGA CAG CAT GTC AGG G-3) and CM4 (5'-CAG GAA ACA GCT ATG ACC TAC TGG TAC AGT CTC AAT AGG A-3'), which included the M13 forward and reverse sequences, respectively (2). The HIV-1 coculture, which best approximated a 50:50 mixture on day 1, was further propagated, and the remaining cultures were discarded. Every 7 or 10 days, the supernatants of the virus coculture were transmitted to fresh uninfected H9 cells. The cells harvested at each passage were subjected to direct DNA sequencing, and viral population changes were determined. The persistence of the original amino acid substitutions was confirmed for all infectious clones used in this assay.

Western Blot Analysis-- To analyze whether HIV-1 polyproteins in various HIV-1 clones were cleaved by protease, Western blot analysis was conducted (13). It is of note that when the virus was propagated in the presence of high concentrations of PIs, the amounts of Pr55Gag, p41, and p24 varied vastly, and no normalization was possible. Therefore, by assuming that the transfection efficiency was comparable, the 48-h supernatants of the culture of COS-7 cells transfected with the same amount of each plasmid using the same conditions were directly subjected to Western blot analysis as described previously (13). Briefly, 48 h after transfection with plasmid preparations, the culture supernatant was centrifuged at 13,800 × g for 10 min and passed through a 0.45-µm pore-size filter to remove cellular debris. The filtrate was centrifuged at 20,380 × g for 4 h to pellet virions. The pelleted virions were lysed in lysis buffer (10 mM Tris (pH 7.4), 50 mM NaCl, 100 mM KCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethanesulfonyl fluoride). Transfected COS-7 cells were washed with PBS and lysed in lysis buffer at 4 °C for 30 min, followed by centrifugation at 13,800 × g to remove cell debris (13). Protein concentrations of the cell lysates were determined with the bicinchoninic acid protein assay kit (Pierce). Cell lysates (30 µg of protein) were subjected to electrophoresis on SDS-4-12% polyacrylamide gradient gels (Bio-Rad) under reducing conditions, followed by electroblotting onto nitrocellulose membranes. The HIV-1 Gag proteins were visualized by SuperSignal WestPico (Pierce) using anti-p24^Gag antiserum (Advanced Biotechnologies, Inc., Columbia, MD) or anti-p6^Gag antiserum (a kind gift from Louis E. Henderson). When needed, anti-cyclophilin A (CypA) antiserum was also used (Biomol Research Laboratories, Plymouth, PA).

Northern Blot Analysis-- To determine whether HIV-1 genomic RNA was properly packaged in virions, Northern blot analysis was performed as described previously (14). The above-mentioned pelleted virus preparations were lysed in Tris buffer (pH 7.4) containing 10 mM EDTA, 1% (v/v) SDS, 100 mM NaCl, 50 µg/ml tRNA, 100 µg/ml proteinase K for 4 h at 37 °C. Lysates were extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and then ethanol-precipitated overnight at 20 °C. The amount of lysates used for each clone was adjusted based on the p24 amount in the lysates for normalization. RNA precipitates were collected by centrifugation and fractionated on a denaturing formaldehyde/agarose gel. The fractionated RNAs were transferred to a nitrocellulose membrane, and the membrane was hybridized with ³²P-labeled 8088-bp AvaI fragments of pHIV-1_NL4-3 prepared using the DECAprime Kit (Ambion, Austin, TX). The filter was washed with 0.1% SDS in 0.1× SSC at 65 °C and exposed to x-ray film (14).

Determination of Virus Particle Numbers by Electron Microscopy-- To determine virus particle numbers in culture supernatants of H9 cells chronically infected with an infectious HIV-1 clone, the negative stain electron microscopy was performed as described previously by Palmer and Martin (15). Briefly, HIV-1-containing culture supernatants (25 µl) were mixed with an equal volume of 8% formaldehyde (Tousimis, Rockville, MD), and a portion (2 µl) of the mixture was placed on a Formvar carbon-coated grid. The grid was subsequently glow-discharged in a vacuum evaporator (Denton Vacuum Inc., Cherry Hill, NJ) prior to use (16). The grid was washed once with glass-distilled water, stained with 1% phosphotungstic acid (pH 7.0) (Fisher), and allowed to air-dry. The grid was examined and digitally photographed under electron microscopy (Hitachi H7000, Hitachi, Japan) operated at 75 kV. The number of virus particles in multiple (~10) areas of the grid square (60 × 60 mm) was determined, and the average numbers were generated for analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amino Acid Mutations Identified in Gag in PI-resistant HIV-1 Variants-- In an attempt to determine whether unidentified amino acid substitutions were involved in the development of HIV-1 resistance to various PIs, we generated HIV-1 variants highly resistant to APV and examined whether the resultant HIV-1 had amino acid substitutions in the Gag proteins. The wild type HIV-1_NL4-3 was propagated in MT-2 cells in the presence of increasing concentrations of APV. By passage 10 in culture where HIV-1 was propagating in the presence of 0.14 µM APV (HIV-1_P10/APV), four amino acid substitutions (L10F, V32I, M46I, and I84V) in the protease and three mutations (V35I, L75R, and H219Q) in the Gag protein emerged (Table I). Interestingly, 5 of 13 clones examined for HIV-1_P10/APV had L75R plus H219Q in Gag but no substitutions in its protease, showing that these two Gag substitutions emerged earlier than the substitutions in protease did in these five clones. By passage 20 (with 2.5 µM APV), the overall frequency of the 4 mutations in protease increased, and so did that of the two Gag mutations except V35I. In addition, two amino acid substitutions (R409K and E468K), which have not been previously reported, emerged in Gag together with the p1-p6 cleavage site mutation (L449F) reported by Doyon et al. (4). At passage 31 (with 10 µM APV), the addition of two mutations (I54M and A71V) in protease and two mutations (E12K and V390D) in Gag were seen in HIV-1 clones (Table I). The five mutations in Gag (L75R, H219Q, R409K, L449F, and E468K) seen at high percentages strongly suggest that these mutations played a significant role in the development of HIV-1 resistance against APV.

We next asked whether the mutations seen in APV-resistant HIV-1 (HIV-1_AR) described above also emerged in HIV-1 propagated in the presence of other PIs, JE-2147 (2), KNI-272 (8), and UIC-94003 (7, 10). HIV-1 variants resistant to each of the three PIs were generated under conditions similar to those when HIV-1_AR was generated. In these HIV-1 variants, different protease mutation profiles were seen compared with that of HIV-1_AR (Table I) (2, 7, 17). In HIV-1 propagated in the presence of JE-2147, one mutation (M46I) in protease and two mutations (H219Q and V390A) in Gag were detected at passage 6 (0.04 µM JE-2147). By passage 33 (2.2 µM JE-2147), the frequencies of these mutations increased and three additional mutations (V32I, I47V, and V82I) in protease and two additional mutations (R409K and L449F) in Gag emerged (Table I). In HIV-1 propagated in the presence of KNI-272 or UIC-94003, two mutations (H219Q and R409K) in Gag, which were seen in both HIV-1_AR and HIV-1_JR (JE-2147-resistant HIV-1), were identified at high frequencies (60-100%). These data suggest that although Gag mutation profiles may vary depending upon PI in use, certain Gag mutations such as H219Q and R409K develop in common and may facilitate the development of PI resistance. The locations where the amino acid substitutions occurred are illustrated in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Locations where six major amino acid substitutions occurred. The locations of six major amino acid substitutions identified in the present study are shown in bold with their codon numbers. The primer pair used for amplification of the gag and protease-encoding gene is also shown as a pair of arrows at the top. The forward primer (LF3) contains a BssHII site and the reverse primer (XM1) an SmaI site.

L75R and H219Q Mutations in Gag Confer Replication Advantages on HIV-1-- To analyze the effects of the above described three Gag mutations (L75R, H219Q, and V390D) in HIV-1_AR, we generated five infectious HIV-1 clones, which had all or a subset of the three mutations in addition to six protease mutations (L10F, V32I, M46I, I54M, A71V, and I84V) plus three other Gag mutations (R409K, L449F, and E468K) (see Table I and Fig. 1). As shown in Fig. 2A, when propagated in the absence of PIs, among the five, two clonal HIV-1s containing both L75R and H219Q substitutions (HIV-1_AR^{L75R/H219Q/V390} and HIV-1_AR^{L75R/H219Q/V390D}) replicated even at an greater level compared with the wild type HIV-1_NL4-3, whereas two clonal HIV-1s containing L75R or H219Q substitutions (HIV-1_AR^{L75R/H219/V390} and HIV-1_AR^{L75/H219Q/V390}) replicated comparably to HIV-1_NL4-3. The clone containing none of the three Gag mutations (HIV-1_AR^{L75/H219/V390}) replicated, but only poorly. In the presence of 2.5 µM APV, however, the two clones containing both L75R and H219Q (HIV-1_AR^{L75R/H219Q/V390} and HIV-1_AR^{L75R/H219Q/V390D}) propagated, whereas all other three clones failed to do so in the presence of APV (Fig. 2B). These data strongly suggest that for the replication of HIV-1_AR in the presence of APV, two Gag mutations together (L75R and H219Q) are critical, whereas V390D apparently does not play a significant role. It is not clear whether the E12K and V35I substitutions (not tested) in Gag play a role in the fitness of HIV-1_AR; however, their role may not be significant because their frequencies of emergence in HIV-1_AR were even less than that of V390D.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Replication kinetics of HIV-1 clones. MT-2 cells (A-E) or PHA-stimulated PBMCs (F) were exposed to each HIV-1 clone and cultured in the absence (A, C, and F, and solid lines in E) and presence of 2.5 µM APV (B and broken lines in E) or 0.5 µM JE-2147 (D). Virus replication was monitored by the amounts of p24 produced in the culture supernatants. HIV-1AR variants had six mutations in the viral protease (L10F, V32I, M46I, I54M, A71V, and I84V) and three mutations in Gag protein (R409K, L449F, and E468K). HIV-1JR variants had four mutations in the protease (M46I, I47V, V82I, and I84V) and one mutation (R409K) in Gag. The results shown are representative of three independent experiments (A-E). Standard deviations are shown for the profile of the wild type HIV-1NL4-3 (shown as HIV-1NL4-3L75/H219/V390 for the sake of clarity) replication in the absence of PI. Data in F represent the geometric means (±1 S.D.) of two independent experiments.

Because the Gag mutation H219Q was also seen in the HIV-1 variant selected against JE-2147 (HIV-1_JR), we examined the effects of H219Q on the replication of HIV-1_JR in the presence or absence of JE-2147. The H219Q-containing HIV-1_JR^{L75/H219Q/V390}, which also contained the JE-2147 resistance conferring mutations in protease (M46I, I47V, V82I, and I84V) and R409K in Gag, replicated even at a greater level than HIV-1_NL4-3 in the absence of JE-2147 (Fig. 2C). HIV-1_JR^{L75/H219Q/V390} also replicated well in the presence of JE-2147, whereas the H219Q-lacking HIV-1_JR^{L75/H219/V390} failed to replicate (Fig. 2D), suggesting that the H219Q substitution was required for the replication of HIV-1_JR in the presence of JE-2147.

Role of L75R and H219Q Mutations in HIV-1_NL4-3 Replication-- It has been reported that natural variations in Gag-Pol polyprotein of HIV-1 may occur in HIV-1-infected individuals receiving no PIs (18). We then asked whether any mutations in the Gag-Pol polyprotein occurred when HIV-1_NL4-3 was propagated in the absence of PIs. When HIV-1_NL4-3 was passaged 10 times in the absence of PIs, several mutations emerged sporadically in the polyprotein, whereas in one of three independent experiments, H219Q was identified in all 10 clones of HIV-1_NL4-3; however, L75R did not develop in any experiment (Table II). In the other two experiments, A224V and V218M emerged, but the H219Q substitution did not emerge. No other mutations occurred in common among the clones throughout entire gag gene (Table II). It is possible that the acquisition of the mutations illustrated in Table II including H219Q conferred on HIV-1_NL4-3 a greater replication capability. We therefore generated HIV-1_NL4-3 clones containing H219Q (HIV-1_NL^{L75/H219Q/V390}) and examined its replication profile in the presence or absence of APV. In the absence of APV, HIV-1_NL^{L75/H219Q/V390} readily outgrew the wild type HIV-1_NL4-3. An HIV-1 clone containing both H219Q and L75R (HIV-1_NL^{L75R/H219Q/V390}) replicated comparably to HIV-1_NL^{L75/H219Q/V390}. Another HIV-1 clone containing L75R (HIV-1_NL^{L75R/H219/V390}) had moderately greater replicative fitness compared with HIV-1_NL4-3. In the presence of APV, however, all these HIV-1_NL clones failed to propagate (Fig. 2E). These data indicate that both L75R and H219Q mutations confer replication advantages on HIV-1_NL4-3 in the absence of but not in the presence of PIs.

Role of L75R and H219Q Mutations in HIV-1 Replication in PBMCs-- To evaluate the possible biological relevance of the replication kinetics data shown above using immortalized and long term cultured MT-2 cells, we conducted similar experiments using PHA-PBMCs freshly prepared from two healthy donors. As shown in Fig. 2F, HIV-1_AR^{L75/H219/V390}, which replicated only poorly in MT-2 cells (Fig. 2A), moderately replicated compared with the wild type HIV-1_NL4-3 in the absence of PI. In contrast, HIV-1_AR^{L75R/H219Q/V390} quickly replicated compared with HIV-1_AR^{L75/H219/V390}. These data are in agreement with the data with MT-2 cells showing that the two amino acid substitutions, L75R and H219Q, recover the compromised replicative ability of HIV-1_AR^{L75/H219/V390}. It was noted, however, that HIV-1_AR^{L75R/H219Q/V390} did not outgrow HIV-1_NL4-3, a difference from the replication profiles shown in Fig. 2A. The reason for this difference is as yet unknown, but cell-specific difference seems to be involved, and this issue is a subject of future research.

Cytopathic Effect of Various HIV-1 Clones-- To define further the significance of the Gag mutations seen in this study, the MTT assay was performed using clonal HIV-1 preparations. MT-2 cells (4 × 10³/well) were exposed to viruses, and CPE of each HIV-1 clone was determined using the MTT assay (Fig. 3). Two clonal HIV-1_AR containing both L75R and H219Q in Gag (HIV-1_AR^{L75R/H219Q/V390} and HIV-1_AR^{L75R/H219Q/V390D}) exerted significant CPE in the presence and absence of APV, whereas two HIV-1 clones containing either L75R or H219Q (HIV-1_AR^{L75R/H219/V390} and HIV-1_AR^{L75/H219Q/V390}) showed CPE comparable with that of wild type HIV-1_NL4-3 (HIV-1_NL4-3^{L75/H219/V390}). The HIV-1_AR clone containing none of the three Gag mutations (HIV-1_AR^{L75/H219Q/V390}) failed to exert CPE in the presence and absence of drug. In contrast, the H219Q-containing HIV-1_JR (HIV-1_JR^{L75/H219Q/V390}) showed significant CPE in the presence and absence of JE-2147, whereas the H219Q lacking HIV-1_JR (HIV-1_JR^{L75/H219/V390}) showed only moderate CPE in the absence of JE-2147. Two HIV-1_NL clones carrying wild type protease and containing H219Q (HIV-1_NL^{L75R/H219Q/V390} and HIV-1_NL^{L75/H219Q/V390}) also showed potent significant CPE, whereas another clone containing only L75R (HIV-1_NL^{L75R/H219/V390}) showed moderate CPE in the absence of APV. In the presence of APV, however, all HIV-1_NL clones failed to infect cells, and no CPE were seen. These data corroborate the data of replication kinetics illustrated in Fig. 2, A-E.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Cytopathic effects of 11 HIV-1 clones. MT-2 cells were exposed to each clone and cultured for 10 days in the absence of PI (open bars) and presence of 2.5 µM APV (closed bars) or 0.5 µM JE-2147 (hatched bars). Cell survival was assessed using the MTT assay. The values shown are means (±1 S.D.) of three independent experiments.

HIV-1 Mutants That Recover Replicative Fitness with Gag Mutations Show No Recovery in Gag Processing-- To define the mechanism by which L75R and H219Q Gag mutations recover replication competence of HIV-1_AR and HIV-1_JR in the presence of APV and JE-2147, respectively, we examined Gag cleavage patterns using the supernatants and cell lysates of COS-7 cells transfected with each plasmid employing Western blot analysis. In this assay, assuming that the transfection efficiency was comparable, the 48-h supernatants of the culture of COS-7 cells transfected with the same amount of each plasmid using the same conditions were directly subjected to Western blot analysis as described previously (13). As expected, there was no significant difference in p24 amounts (Fig. 4A, lanes 1, 3, and 5-8). The pattern of HIV-1_NL4-3 showed distinct cleavage as described previously (13) (Fig. 4, A and B, lane 1), and 2.5 µM APV completely blocked the processing of p24 Gag (Fig. 4, A and B, lane 2). HIV-1_NL4-3 and HIV-1_NL^{L75R/H219Q/V390}, which replicated more efficiently than HIV-1_NL4-3 (Fig. 2E), gave comparable cleavage patterns in the presence and absence of 2.5 µM APV (Fig. 4, A and B, lanes 3 and 4). The replication-incompetent HIV-1_AR^{L75/H219/V390} gave a pattern with more immature p41 Gag protein (Fig. 4, A and B, lane 5), differing from the pattern of HIV-1_NL4-3. Unexpectedly, HIV-1_AR^{L75R/H219Q/V390}, which recovered its replicative fitness with L75R and H219Q substitutions (Fig. 2, A and B), gave the same profile as the replication-incompetent HIV-1_AR^{L75/H219/V390} (Fig. 4, A and B, lane 6). Even in the presence of APV (2.5 and 10 µM), there was no significant difference in cleavage pattern between HIV-1_AR^{L75/H219/V390} and HIV-1_AR^{L75R/H219Q/V390}, although the amounts of p24 Gag and p41 immature proteins were decreased, and the amount of Pr55^Gag polyprotein was increased in both viruses (Fig. 4C, lanes 1-4). HIV-1_JR^{L75/H219Q/V390}, which replicated more efficiently both in the presence and absence of JE-2147 than HIV-1_JR^{L75/H219/V390}, also produced profiles comparable with those of HIV-1_JR^{L75/H219/V390} (Fig. 4, A and B, lanes 7 and 8, and C, lanes 5-8).

View larger version (80K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of Gag protein cleavage profiles. Western blot analysis was performed using anti-p24Gag antiserum with virus pellets (A) and lysates of COS-7 cells transfected (B and C). A and B, COS-7 cells were transfected with pHIV-1NLL75/H219/V390 (lanes 1 and 2), pHIV 1NLL75R/H219Q/V390 (lanes 3 and 4), pHIV-1ARL75/H219/V390 (lane 5), pHIV-1ARL75R/H219Q/V390 (lane 6), pHIV-1JRL75/H219/V390 (lane 7), or pHIV-1JRL75/H219Q/V390 (lane 8). After 48 h of culture in the presence (lanes 2 and 4) or absence (other lanes) of 2.5 µM APV, the virions in the culture supernatant (A) and the lysates of COS-7 cells (B) were harvested. Note that no cleavage of Pr55Gag polyprotein was detected, and none or the least amount of p24 was seen in lanes 2 and 4, whereas p24 and p41 (p17 + p24) were seen in lanes 1 and 3 and 5-8. C, COS-7 cells were transfected with pHIV-1ARL75/H219/V390 (lanes 1 and 2), pHIV-1ARL75R/H219Q/V390 (lanes 3 and 4), pHIV-1JRL75/H219/V390 (lanes 5 and 6), or pHIV-1JRL75/H219Q/V390 (lanes 7 and 8). After 48 h of culture in the presence of 2.5 (lanes 1 and 3) or 10 µM (lanes 2 and 4) APV, 0.5 (lanes 5 and 7) or 2.5 µM (lanes 6 and 8) JE-2147, COS-7 cells were harvested and lysed, and the cell lysates were subjected to Western blot analysis.

Northern Blot Analysis of HIV-1 Viral Genome Packaging-- Because two Gag mutations L75R and H219Q did not significantly alter the Gag cleavage profiles as shown above (Fig. 4), we next asked whether viral RNA genomes were properly recognized and packaged into virion particles produced in transiently transfected COS-7 cells.

The wild type HIV-1_NL4-3 proved to contain a substantial amount of genomic RNA and showed a strong signal at 9.2 kb, which corresponds to the size of full genomic RNA (HIV-1_NL4-3 undiluted in Fig. 5). When the sample of HIV-1_NL4-3 was serially diluted (5-, 25-, and 125-fold), the signal decreased in a dose-response manner. Under the same conditions, virtually the same level of signal was seen at 9.2 kb among four viral preparations examined (HIV-1_AR^{L75/H219/V390}, HIV-1_AR^{L75R/H219Q/V390}, HIV-1_JR^{L75/H219/V390}, and HIV-1_JR^{L75/H219Q/V390}) regardless of the presence of L75R and/or H219Q mutations (Fig. 5). These data strongly suggest that neither mutations in protease nor those in Gag affected the recognition and packaging of HIV-1 RNA genome into virions.

View larger version (87K): [in this window] [in a new window]   Fig. 5.   Northern blot analysis of PI-resistant HIV-1 variants. After 48 h of post-transfection, viruses were harvested and lysed, and the viral RNA was purified and subjected to Northern blot analysis. Serially diluted (undiluted, 5-, 25-, and 125-fold) samples of the wild type HIV-1NL4-3 served as controls.

No Difference in RNA Copy Numbers or p24 Contents in Virions among HIV-1 Clones-- To corroborate the data of Northern blot analysis (Fig. 5), we propagated five infectious clones in H9 cells (HIV-1_AR^{L75/H219/V390}, HIV-1_AR^{L75R/H219Q/V390}, HIV-1_JR^{L75/H219/V390}, HIV-1_JR^{L75/H219Q/V390}, and HIV-1_NL4-3), determined viral particle numbers using electron microscopy (negative staining), counted viral RNA copies using RT-PCR, and measured p24 amounts in each culture (Table III). The culture supernatant of HIV-1_AR^{L75R/H219Q/V390} contained the greatest virion number, followed by that of HIV-1_JR^{L75/H219Q/V390}, in agreement with the replication kinetics data shown in Fig. 2. The remaining three cultures contained less but varying virion numbers. It should be noted that the variability in virion numbers determined with electron microscopy is up to 3-fold.² No significant morphological differences were noted among the five virus preparations examined under electron microscopy. The culture supernatant of HIV-1_AR^{L75R/H219Q/V390} contained the greatest p24 amount, but there was no significant difference (less than 3-fold difference) in p24 amounts among other culture supernatants. It is of note that there was a variability by 2-3-fold in RNA copies and p24 amounts as determined in supernatants of cultures prepared in multiple replicates (Fig. 2).³ When RNA copy number per virion was calculated, a difference by up to 8.3-fold (2.11 for HIV-1_AR^{L75R/H219Q/V390} versus 17.5 for HIV-1_JR^{L75/H219/V390}) was identified among the cultures, but considering the estimated variability in the determination methods for both virion numbers and RNA copies, it appears that there was no significant difference in RNA copy numbers per virion. We found that the same was true for p24 amounts per virion and RNA copy numbers per p24. Thus, we concluded that there was no difference in the incorporation of RNA genomes in virions in association with the mutations in the gag-pol gene we examined in this study.

Decreased Cyclophilin A Incorporation into HIV-1 with Gag Mutations-- Although L75R and H219Q mutations exerted positive effects on the replication of HIV-1_NL4-3 and its PI-resistant variants, no significant changes in Gag cleavage or genomic RNA packaging profiles were detected. Considering that His-219 is located in the cyclophilin A (CypA) binding loop (Fig. 1) and that the incorporation of CypA into HIV-1 particles is thought to be indispensable for efficient viral replication (20, 21), we asked whether the Gag mutations altered the incorporation of CypA into HIV-1 particles. HIV-1_AR^{L75/H219/V390}, which has mutant protease and wild type Gag protein, and HIV-1_AR^{L75R/H219Q/V390}, which has mutant protease plus L75R and H219Q mutations, were lysed, diluted, and subjected to Western blot analysis. With comparable amounts of Pr55^Gag, p41, and p24 detected (see lanes 3 and 6 in Fig. 6), a substantially less amount of CypA was found incorporated in HIV-1_AR^{L75R/H219Q/V390} than in HIV-1_AR^{L75/H219/V390}. These results suggest that the H219Q mutation might change the conformation of CypA binding loop and decrease CypA incorporation into virion particles.

View larger version (75K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of virion-incorporated CypA. Two HIV-1 clones were lysed, diluted (HIV-1ARL75/H219/V390: neat, 75, 50, and 25% in lanes 1-4, respectively, and HIV-1ARL75R/H219Q/V390: neat, 80, 60, and 40% in lanes 5-8, respectively), and subjected to Western blot analysis using anti-p24Gag and anti-CypA antisera. Note that with comparable amounts of Pr55Gag, p41, and p24 detected (see lanes 3 and 6), substantially less CypA was found in HIV-1ARL75R/H219Q/V390 than in HIV-1ARL75/H219/V390.

Effects of Three Gag Mutations (R409K, L449F, E468K) on the Replication Kinetics of HIV-1_AR-- We also examined the effect of R409K, L449F, and E468K seen in HIV-1_AR on the replicative fitness in MT-2 cells. To this end, we generated various HIV-1_AR clones containing all or a subset of three mutations in addition to three Gag mutations (L75R, H219Q, and V390D) and six protease mutations (L10F, V32I, M46I, I54M, A71V, and I84V) (see Table I and Fig. 1). When propagated in the absence of APV, a clone lacking the cleavage site amino acid substitution L449F (4) (HIV-1_AR^{R409K/L449/E468K}) failed to propagate, whereas the clonal HIV-1 with L449F (HIV-1_AR^{R409K/L449F/E468K}) showed a markedly improved fitness (Fig. 7A), indicating that this cleavage site mutation is essential for the fitness of HIV-1_AR. Two clones that lacked either R409K or E468K (HIV-1_AR^{R409/L449F/E468K} and HIV-1_AR^{R409K/L449F/E468}) also had a markedly improved replicative fitness, although the latter appeared to be less fit in the absence of APV (Fig. 7A).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Replication kinetics of HIV-1AR clones. MT-2 cells (A-C) or PBMCs (D) were exposed to various HIV-1AR clones and cultured in the absence (A, C, and D) or presence of 2.5 µM APV (B). Each HIV-1 clone contained three mutations, L75R, H219Q, and V390D in Gag. The results shown are representative of three independent experiments (A-C). Data shown in D represent geometric means (±1 S.D.) of two independent experiments.

When propagated in the presence of 2.5 µM APV, HIV-1_AR^{R409K/L449F/E468K}, as expected, predominated all other clones examined (Fig. 7B). We found that the clone lacking R409K (HIV-1_AR^{R409/L449F/E468K}), which exhibited the greatest level of replication in the absence of drug (Fig. 7A), had a moderate level of fitness in the presence of APV, whereas the replication pattern of HIV-1_AR ^{R409K/L449F/E468} was apparently comparable with that of HIV-1_AR^{R409K/L449F/E468K} (Fig. 7B). It was noted that in the presence of APV, the R409K mutation provided HIV-1_AR with a replication advantage compared with E468K.

It has been reported that when the cleavage site mutation L449F was introduced to HIV-1 carrying a wild type protease, its replication capability was significantly reduced (4). We therefore asked whether the replication profile of HIV-1_NL^{L75R/H219Q/V390D} containing the wild type protease was altered when R409K and E468K were introduced. In our assays, the replication of HIV-1_NL^{L75R/H219Q/V390D} exhibited a high level of replication regardless of the presence of these two mutations (Fig. 7C).

Furthermore, to evaluate the possible biological relevance of the replication kinetics data using MT-2 cells shown above, we also conducted experiments using PHA-PBMCs freshly prepared from two healthy donors. As shown in Fig. 7D, the replication profiles of four infectious clones, HIV-1_AR^{R409K/L449F/E468K}, HIV-1_AR^{R409/L449F/E468K}, HIV-1_AR^{R409K/L449F/E468}, HIV-1_AR^{R409K/L449/E468K}, were virtually the same as those shown in Fig. 7A.

Competitive HIV-1 Replication Assays for HIV-1_AR Clones-- To define further the significance of R409K and E468K, the viral fitness was compared among the HIV-1 clones described above in the presence and absence of 2.5 µM APV using CHRA (12). In the absence of APV, HIV-1_AR^{R409K/L449F/E468K} propagated comparably to HIV-1_AR^{R409/L449F/E468K} (Fig. 8A) but readily outgrew HIV-1_AR^{R409K/L449F/E468} (Fig. 8B). In the presence of APV, HIV-1_AR^{R409K/L449F/E468K} predominated HIV-1_AR^{R409/L449F/E468K} and HIV-1_AR^{R409K/L449F/E468}, corroborating that the presence of R409K and E468K in addition to the cleavage site mutation confers on HIV-1_AR a significant replication advantage in the presence of APV (Fig. 8, C and D). We also asked whether the cleavage of the Gag precursor polyprotein was impaired when the HIV-1_AR lacked either R409K or E468K. However, Western blot analyses using anti-p6 antiserum detected no difference in cleavage patterns among these three viruses (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Competitive HIV-1 replication assay for HIV-1AR. Replication profiles of HIV-1ARR409K/L449F/E468K, HIV-1ARR409/L449F/E468K, HIV-1ARR409K/L449F/E468, HIV-1NLR409K/L449/E468K, and HIV-1NLR409/L449/E468 were examined in CHRA. Each HIV-1 contained three mutations, L75R, H219Q, and V390D in Gag. Two infectious HIV-1 clones to be compared for their fitness were mixed 50:50 and used to infect H9 cells in the absence (A, B, and E) or presence (C and D) of 2.5 µM APV. The cell-free supernatant was transferred to fresh H9 cells every 7 or 10 days. High molecular weight DNA extracted from infected cells at the end of each passage was subjected to nucleotide sequencing, and the proportions of Arg and Lys at position 409 (A, C and E) in Gag and those of Glu and Lys at position 468 (B, D and E) were determined.

To delineate how these two mutations affect the replication kinetics of wild type protease-containing viruses, we also compared the fitness of two HIV-1 clones. HIV-1_NL^{R409K/L449/E468K} was slowly outgrown by HIV-1_NL^{R409/L449/E468} (Fig. 8E), suggesting that R409K/E468K mutations compromise the fitness of HIV-1_NL.

In this assay, it was possible that additional mutations were acquired in HIV-1 clones during the assays, and such mutations might have affected the replication profiles of HIV-1 clones examined. However, when we determined the nucleotide sequence of the entire gag and protease-encoding region, no additional mutations were identified in any of HIV-1 clones at the conclusion of CHRA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role and impact of amino acid substitutions in the gag gene of HIV-1 genome which emerge during therapy with protease inhibitors in patients with AIDS have been poorly understood. This is mainly due to the findings that most of the amino acid substitutions detected in the gag gene are not seen in common among clinical HIV-1 strains isolated from patients and even among HIV-1 clones generated from HIV-1 of a single patient. More problematic is that the functions and tertiary structures of HIV-1 Gag proteins remain largely to be determined. In the present study, we used a single HIV-1 clone obtained from newly transfected COS-7 cells, HIV-1_NL4-3, as a starting HIV-1 strain and propagated it in the presence of increasing concentrations of four different PIs, APV, JE-2147, KNI-272, and UIC-94003 over 27-62 passages; and we studied 14 mutations identified in Gag (Table I), among which 9 mutations were seen in more than two PI-exposed HIV-1 preparations. We focused on six major Gag amino acid substitutions, L75R, H219Q, V390D/V390A, R409K, L449F, and E468K in the present study (Table I).

When HIV-1 was passaged under APV pressure, L75R and H219Q mutations in the Gag protein emerged prior to the emergence of protease mutations, and we found that such Gag mutations were indispensable for the efficient replication of APV resistant HIV-1 (HIV-1_AR). We thought that Gag polyprotein containing L75R or H219Q would be more sensitive to the cleavage by mutant protease because such a protease has impaired enzymatic activity due to the accumulation of amino acid substitutions within itself. Indeed, HIV-1_AR whose mutant gag gene was substituted with a wild type gag gene (but with the mutant protease being intact) (HIV-1_AR^{L75/H219/V390}) failed to replicate (Fig. 2A), in agreement with prior studies (6, 7). However, Western blot analysis showed that the polyprotein cleavage pattern was comparable between the replication-incompetent clonal HIV-1 with wild type Gag (e.g. HIV-1_AR^{L75/H219/V390}) and the HIV-1 that recovered replication competence with amino acid substitutions in Gag (e.g. HIV-1_AR^{L75R/H219Q/V390}). It is possible that the difference in the Gag polyprotein processing is functionally significant but is not readily detected with Western blot analysis (8). It is also possible that such amino acid substitutions in Gag facilitate certain functions of Gag proteins such as assembly, packaging, and budding functions, and the recovery of polyprotein cleavage is not required.

There are several lines of evidence suggesting that trimerized matrix proteins (MAs) serve as a fundamental building block for the formation of the MA shell within the mature HIV-1 (19). The x-ray structural analysis of HIV-1 MA suggests that residues Pro-66 and Gly-71, which are highly conserved, function as "hinges" that allow a structural reorientation of MA at the trimer interface and that Ser-72 and Leu-75 have a tight interaction, and when the distance between these amino acids changes, the conformational interconversion of Gag occurs (22). Yu et al. (23) constructed a mutant HIV-1 with a wild type protease lacking 10 Gag amino acids (codons 68-77), which showed an impaired viral production, suggesting that that stretch of amino acids is essential for the replicative fitness of HIV-1. In the present study, the L75R substitution in Gag conferred replication advantages on HIV-1, particularly when combined with H219Q substitution. Taken together, one can speculate that the L75R substitution may alter the trimerization of MA and/or the interaction of MA with a lipid membrane of HIV-1 resulting in its "membrane anchoring" through altered electrostatic interactions. Such changes, with HIV-1_AR background, may ultimately confer a replication advantage on the virus.

The H219Q substitution in p24 was identified in all clones derived from four PI-resistant HIV-1 variants. In this regard, the amino acids 217-225 reportedly made a single exposed loop in the capsid protein that binds to the enzymatic active site of human CypA (24) whose incorporation into HIV-1 particles seems to be indispensable for the infectivity of HIV-1. In fact, HIV-1 replication is inhibited by cyclosporin A that binds to CypA and interferes with the virion association of CypA (20, 21). It has been noted that SIVmac capsid protein also has an exposed loop but has no affinity to CypA and is capable of replicating without CypA (25). Moreover, the transfer of the HIV-1 loop to the corresponding position in SIVmac results in the efficient incorporation of CypA and confers HIV-1-like sensitivity to cyclosporin A (25). Yoo et al. (26) generated a number of mutant p24 proteins, analyzed their binding affinity to CypA, and showed that the H219Q mutation in Gag of HIV-1_NL4-3 diminished CypA binding. It should be noted that H219Q represents a polymorphic amino acid substitution and is often seen particularly in clades F and G isolates (27). In the present study, we found that His-219 in HIV-1_NL4-3 obtained from freshly transfected COS-7 cells spontaneously changed to glutamine over 10 passages in one of three independent experiments. In the two other experiments, A224V and V218M emerged when the H219Q substitution did not emerge, whereas no mutations occurred in common among clones in other regions of the entire gag gene (Table II). These data strongly suggest that the H219Q substitution brings about a conformational change in the loop formed by the stretch of the amino acids positioned 217-225 (corresponding to CypA binding loop).

Based on these data, we postulated that the exposed loop of p24 might exert a negative effect(s) on the replication of HIV-1 either at the stages of assembly or disassembly and that the binding of CypA to the loop might induce conformational changes so that the negative effect(s) are canceled, thus restoring replicative fitness. Therefore, we compared the amounts of viral particle-associated CypA in H219Q-carrying and H219Q-lacking HIV-1 (HIV-1_AR^{L75R/H219Q/V390} and HIV-1_AR^{L75/H219/V390}, respectively) using the Western blot assay, and we found that HIV-1_AR^{L75R/H219Q/V390} contained less CypA (Fig. 6) but replicated faster than HIV-1_AR^{L75/H219/V390} (Fig. 2, A and F). These data suggest that H219Q substitution changed the loop conformation so that the rate of replication became greater and presumably relatively independent of the binding of CypA. Indeed, in our preliminary structural characterization of HIV-1 matrix-capsid antigens, the N terminus of capsid antigen appears to take multiple conformations.⁴ It is possible that the cleavage of matrix and capsid antigens shifts the equilibrium toward a certain conformation (28), and CypA binding is required for the stabilization of that conformation. In this respect, the H219Q substitution per se might stabilize that conformation, and the requirement of CypA binding is reduced. It is worth noting that Western blot analysis has a limited utility in quantification and that enzyme-linked immunosorbent assay or its related method should enable us to quantitate more precisely the levels of virion-associated cyclophilin A than Western blot analysis. However, to date, no cyclophilin-specific monoclonal antibody has been generated or reported, and the establishment of a more quantitative method for cyclophilin A is awaited for further detailed investigation into the role of cyclophilin A in the replication of HIV-1.

Following the emergence of protease gene mutations in HIV-1_NL4-3 propagated in the presence of APV, two substitutions R409K and E468K emerged in the Gag protein. Both codons at 409 and 468 are known to be highly conserved (27). A newly generated HIV-1 clone carrying these two mutations in addition to the cleavage site mutation L449F (HIV-1_AR^{R409K/L449F/E468K}) predominated two HIV-1 clones lacking either of these two mutations in the presence of APV in the replication kinetics assay and the CHRA (Fig. 7B and Fig. 8, C and D). In contrast, HIV-1 carrying these two mutations and wild type protease (HIV-1_NL^{R409K/L449/E468K}) was outgrown by HIV-1 lacking the two mutations (HIV-1_NL^{R409/L449/E468}) (Fig. 8E). Based on these data, we thought that these two mutations altered the conformation of the tertiary structure of the Gag precursor p7-p1-p6 and made the cleavage site more accessible to the mutated protease, the rate-limiting step for virus maturation (29-31). However, Western blot analysis failed to reveal the difference in Gag cleavage profiles. There is a possibility that the functions of Gag proteins are altered by L75R, H219Q, R409K, or E468K substitutions, resulting in enhanced viral replication. One of known Gag functions is the packaging of genomic RNA into HIV-1 particles (19). Therefore, we semi-quantified the amount of viral particle-associated genomic RNA in five different clonal HIV-1 preparations (HIV-1_AR^{L75/H219/V390/R409K/L449F/E468K}, HIV-1_AR^{L75R/H219Q/V390/R409K/L449F/E468K}, HIV-1_JR^{L75/H219/ V390/R409K/L449/E468}, HIV-1_JR^{L75/H219Q/V390/R409K/L449/E468}, and HIV-1_NL^{L75/H219/V390/R409/L449/E468}) using Northern blot analysis (Fig. 5) and RT-PCR (Table III), but the amounts of RNA were all comparable.

Most of the Gag mutations identified in this study have not been reported to be associated with the acquisition of HIV-1 resistance against PIs. For example, R409K occurred when HIV-1_NL4-3 was passaged in the presence of all four PIs, APV, JE-2147, KNI-272, and UIC-94003 (Table I). However, we failed to identify R409K in any of ~30 clones generated from each of four heavily PI-treated HIV-1-infected patients whose HIV-1 was proved to harbor a number of mutations in the protease-encoding gene (2). It appears that the R409K substitution occurs specifically with the HIV-1_NL4-3 genetic background. In all the selection experiments in this study, we used a newly generated HIV-1_NL4-3, a highly pure clonal HIV-1 preparation, which enabled us to identify such a mutation after drug selection procedure. In this context, it is likely that HIV-1 in patients represents the quasi-species carrying a myriad of genetic backgrounds and, in response to PI(s) including APV, HIV-1 develops a number of genetic background-specific Gag mutations, so that sequencing analyses of relatively few clones (i.e. ~30 clones in our study) failed to identify common mutations.

The present data, taken together, suggest that HIV-1 resistance to PIs is associated with primary and secondary mutations in the viral protease and is also associated with the cleavage site amino acid substitutions in Gag together with substitutions at non-cleavage sites. Such non-cleavage site Gag mutations should render the polyprotein cleavage sites more accessible to the protease, make polymerization of viral proteins more efficacious, and/or make assembly and disassembly more efficient. Alteration(s) of other unknown functions of Gag proteins may also contribute to the HIV-1 acquisition of resistance to PIs, but it appears that HIV-1 resistance to PIs is acquired with multiple mechanisms.

	ACKNOWLEDGEMENT

We thank Louis E. Henderson for helpful discussions.

	FOOTNOTES

^* This work was supported in part by a Grant from Research for Future Program JSPS-RFTF 97L00705 of the Japan Society for the Promotion of Science, a grant-in-aid for Scientific Research (Priority Areas) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Monbu-Kagakusho), a grant for Promotion of AIDS Research from the Ministry of Health, Welfare and Labor of Japan (Kosei-Rohdosho), and by Science Applications International Corp. NCI-Frederick Grants N01-CO 56000 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported in part by the grant from the Japanese Foundation for AIDS Prevention.

^§§ To whom correspondence should be addressed: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, NCI, Bldg. 10, Rm. 5A11, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-9238; Fax: 301-402-0709; E-mail: hmitsuya@helix.nih.gov.

Published, JBC Papers in Press, December 10, 2001, DOI 10.1074.jbc.M108005200

² K. Nagashima, unpublished data.

³ H. Gatanaga, unpublished data.

⁴ C. Tang and M. F. Summers, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PIs, protease inhibitors; HIV-1, human immunodeficiency virus type 1; APV, amprenavir; CPE, cytopathic effect; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; CHRA, competitive HIV-1 replication assay; RT, reverse transcriptase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CypA, cyclophilin A; MA, matrix protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES