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Antimicrobial Agents and Chemotherapy, February 2004, p. 444-452, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.444-452.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Gag Non-Cleavage Site Mutations Contribute to Full Recovery of Viral Fitness in Protease Inhibitor-Resistant Human Immunodeficiency Virus Type 1

Lay Myint,1 Masakazu Matsuda,1 Zene Matsuda,1 Yoshiyuki Yokomaku,1 Tomoko Chiba,1 Aiko Okano,1 Kaneo Yamada,2 and Wataru Sugiura1*

AIDS Research Center, National Institute of Infectious Diseases,1 Japanese Foundation for AIDS Prevention, Tokyo, Japan2

Received 28 July 2003/ Returned for modification 25 September 2003/ Accepted 4 November 2003


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ABSTRACT
 
It is well documented that human immunodeficiency virus type 1 (HIV-1) Gag cleavage site mutations (CSMs) emerge in conjunction with various HIV-1 mutations for protease inhibitor (PI) resistance and improve viral replication capacity, which is reduced by acquisition of the resistance. However, CSMs are not the only mutations that emerge in Gag during treatment; many mutations other than CSMs (non-CSMs) have been found to accumulate in the Gag region. In the present study we demonstrate the important role of Gag non-CSMs with regard to viral fitness recovery. We selected three Gag-protease sequences with different PI resistance-associated mutations and CSMs from patients with antiretroviral treatment failure. To clarify the significance of CSMs and non-CSMs, four types of recombinant viruses with different patterns in each sequence were constructed. These were the GP type (patient-derived Gag and protease), the P type (HXB2 Gag and patient-derived protease), the GP-c type (CSMs removed from the GP type), and the P+c type (CSMs in the HXB2 Gag frame and patient-derived protease). By comparison of these four types of recombinant viruses in each patient-derived Gag-protease sequence, we found that non-CSMs, which had no systematic pattern, make a significant contribution to viral fitness recovery. Our findings demonstrate a delicate interaction between the in vivo evolution of Gag and protease to evade drug selective pressure and the importance of Gag in evaluating drug-resistant viruses.


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INTRODUCTION
 
Human immunodeficiency virus type 1 (HIV-1) protease (PR) is the crucial enzyme for HIV-1 replication (23, 26). The enzyme cleaves the Gag precursor protein p55gag into six functional proteins (30, 33), which form the inner structure of the HIV-1 virion (19, 20). The ordered cleavage of the Gag p55 precursor protein by viral PR, the step which is required for viral maturation, is essential for the acquisition of infectivity of HIV-1 (27, 34, 39, 40, 42). Indeed, viral infectivity was lost by replacing the enzyme catalytic locus consisting of an aspartic acid at position 25 with asparagine (26). Hence, PR has been the major target of antiretroviral treatment (1, 11), and regimens with PR inhibitors (PIs) have been successful in suppressing viral replication to virtually undetectable levels in vivo (21). Still, a considerable number of patients were found to be unresponsive to such treatment because of the emergence of drug-resistant viruses (6, 16, 32, 37). Thus, more attention has been given to drug-resistant virus pathogenesis as the major impediment to successful antiretroviral therapy (ART). Recent studies have shown that, in general, acquisition of drug resistance may reduce virus replication capacity, i.e., viral fitness (13, 29, 43). This phenomenon seems to be evident, especially with regard to PI resistance-associated mutations (3, 7, 9). Many of the major PI resistance-associated mutations are located within the active site of the PR and significantly reduce PR activity (4, 9, 22, 38). This impact on fitness reduction on treatment outcome is noteworthy, as a previous study has suggested that drug-resistant viruses might be less pathogenic than wild-type viruses (12).

However, drug-resistant viruses have the potential to undergo further evolution to recover impaired PR activity. The continuation of drug selective pressure by ART may lead viruses to acquire additional mutations, which allows the viruses to recover their viral fitness (4, 22, 38). These compensatory mutations can appear not only in the PR itself but also in the natural substrate Gag region of the virus (10, 14, 17, 24, 44). Mutations in Gag cleavage sites are the mutations that can improve the replication capacity of PI-resistant viruses (31). Cleavage site mutations (CSMs) were most commonly found in the p2/p7, p7/p1, and p1/p6 cleavage sites (8). Several studies confirmed that p7/p1 (A431V) and p1/p6 (L449F) CSMs were important for viral fitness recovery in PI-resistant HIV-1 (2, 5, 28, 29, 36), and the importance of CSM is now widely accepted. When analyzing clinical samples, however, in addition to CSMs, many other mutations outside the cleavage sites, i.e., non-CSMs may also be found. The importance of non-CSMs in PI-resistant HIV-1 in vitro has been reported previously (18). However, the significance in the viral fitness recovery process in clinical isolates has heretofore not been clarified. Therefore, in this study we attempted to clarify the relevance of non-CSMs in clinically derived gag-pol sequences covering the p24 to p6 region. We found that non-CSMs also play a role as important as that of CSMs in the recovery of fitness of PI-resistant viruses.


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MATERIALS AND METHODS
 
Construction of PI-resistant molecular clones with combinations of p7/p1 and p1/p6 CSMs. To confirm the impact of p7/p1 (A431V) and p1/p6 (L449F) Gag CSMs on viral fitness in PI-resistant viruses, wild-type PR and two PI-resistant mutation patterns, (i) D30N, N88D, and L90M and (ii) L90M, were selected. With regard to the Gag region, wild-type Gag and three different CSM combinations, (i) A431V, (ii) L449F, and (iii) A431V and L449F, were selected. Recombinant viruses with these mutation patterns were constructed on a strain HXB2 backbone, as described previously (41). Preceding mutagenesis and construction of the recombinant clones, 448-bp gag regions (1849 to 2296) with ApaI (position 1856) and NotI (position 2275) restrictions sites were cloned into a pT7 cloning vector and used as the template in the subsequent site-directed mutagenesis to introduce CSMs. Primer pair DR431vF (5' TTG TAC TGA AAG ACA GGT TAA TTT TTT AGG GAA GG) and DR431vR (5' CCT TCC CTA AAA AAT TAA CCT GTC TTT CAG TAC AA) was used to introduce the A431V mutation. Primer pair DR449fF (5' GGA GGC CAG GGA ATT TTT TTC AGA GCA GAC CAG AA) and DR449fR (5' TTC TGG TCT GCT CTG AAA AAA ATT CCC TGG CCT CC) was used to introduce the L449F mutation. Mutagenesis was performed with KOD DNA polymerase (Toyobo Co. Ltd., Osaka, Japan) with PCR conditions of 94°C for 20 s, 52°C for 10 s, and 72°C for 90 s for 25 cycles. The results of the mutagenesis were confirmed by sequencing with an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, Calif.). Finally, a 420-bp gag ApaI-NotI fragment (positions 1856 to 2275) with A431V or L449F, or both, was cloned into the corresponding gag portion of each PI-resistant clone (Fig. 1a).



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  		FIG. 1. Construction of recombinant viruses. (a) Preparation of viruses with artificially induced PI resistance with or without CSMs. Twelve clones with artificially induced PI resistance were constructed in the following manner: four ApaI to NotI gag fragments with different CSM patterns, and three NotI to XmaI protease fragments with three different PI resistance-associated mutation patterns, were prepared; by using these Gag and PR fragments, 12 different Gag-PR combinations were made and inserted into an HXB2 virus expression vector. (b) Construction of recombinant virus with a patient-derived Gag-PR fragment. Four different types of recombinants were constructed according to the pattern of the insert: (i) the GP type, (ii) the P type, (iii) the GP-c type, and (iv) the P+c type. LTR, long terminal repeat.

Patient sample selection and construction of recombinant viruses with patient-derived Gag and PR sequences. To investigate the significance of CSMs and non-CSMs in patient samples, we constructed the recombinant viruses with patient-derived Gag and PR sequences. For comparison with the results of earlier molecular cloning studies, we surveyed patient samples for CSMs in Gag (A431V or L449F, or both) and a D30N or an L90M mutation (or both) in the PR region. As D30N and L90M were targeted for the survey, samples from patients who failed a nelfinavir-containing regimen were focused on when samples sent to the AIDS Research Center of the National Institute of Infectious Diseases of Japan for routine drug resistance testing were analyzed.

Four different types of recombinant viruses were constructed for each patient Gag-PR fragment. These were (i) the GP type, which contained patient-derived Gag and PR sequences; (ii) the P type, which contained a patient-derived PR sequence and the Gag region of wild-type strain HXB2; (iii) the GP-c type, which contained a patient-derived Gag-PR sequence and wild-type CSMs, which were inserted by site-directed mutagenesis (therefore, there are only non-CSMs in the Gag region of this clone); and (iv) the P+c type, which contained a patient-derived PR sequence and HXB2 wild-type Gag with the CSMs found in the corresponding GP clone (Fig. 1b).

The four types of recombinant viruses were constructed as follows. HIV-1 RNA was extracted from 200 µl of patient plasma by using a commercially available extraction kit (Roche Molecular Biochemicals, Mannhein, Germany). The extracted RNA was reverse transcribed with avian myeloblastosis virus reverse transcriptase (RT; TaKaRa, Otsu, Japan), and a 757-bp fragment (from positions 1849 to 2605), including p24, p2, p7, p1, p6, and PR, was amplified by nested PCR with Pyrobest DNA polymerase (TaKaRa). For this amplification, primers DRGAG5 (5' TGT TAA AAG AGA CCA TCA ATG AGG AAG CTG) and DRPRO2 (5' ATT TTC AGG CCC ATT TTT TGA) were used for the outer PCR and primers DRGAG7 (5' ATA AAG CAA GAG TTT TGG CGG AAG CGA TGA GC) and DRPRO4 (5' CTG GCT TTA ATT TTA CTG GTA) were used for the inner PCR. Subsequently, the 757-bp fragment that was amplified was cloned into the pCR-Blunt II-TOPO vector (Invitrogen, Carlsbad, Calif.). The sequences of the cloned fragments were verified by sequencing, and three fragments were selected for the study.

To insert the selected gag-pol fragment into the modified HXB2 vector (HXB2cv), unique ApaI and XmaI restriction sites were introduced at the 5' ends and the 3' ends of the fragments, respectively, by using forward primer DRGagPol-1 (5' GGA GGG CCC GGC CAT AAA GCA AGA TTT TG) and reverse primer DRGagPol- 2 (5' GGG CCA TCC ATC CCG GGC TTT AAT TTT ACT GG). Forward primer DRPolF1 (5' AAC TCT TTG GCA GCG GCC GCT CGT CAC AAT AAA GAT) and reverse primer DRGagPol-2 were used to introduce the restriction sites NotI at the 5' end and XmaI at the 3' end of the 346-bp PR fragment (2260 to 2605). By this process, NotI and XmaI restriction sites were added to the 5' and 3' ends of the PR fragment, respectively. The GP-type and P-type recombinant viruses were constructed by using the introduced restriction sites.

To construct GP-c clones, CSMs were removed and replaced with the wild-type HXB2 amino acid pattern by site-directed mutagenesis. Primer pair DR431aF-1 (5' GAT TGT ACT GAA AGA CAG GCT AAT TTT TTA GGG AAG GTC) and DR431aR-1 (5' GAC CTT CCC TAA AAA ATT AGC CTG TCT TTC AGT ACA ATC) and clone 1-1 were used to revert the A431V nucleotide to the alanine found in the wild type. Since the primer target site of clone 2-2 had a different sequence, a different primer pair, DR431aF-2 (5' GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG GTC) and DR431aR-2 (5' GAC CTT CCC TAA AAA ATT AGC CTG TCT CTC AGT ACA ATC), was used. Primer pair DR449LF (5' GGA GGC CAG GGA ATT TTC CTC AGA GCA GAC CAG AAC C) and DR449LR (5' GGT TCT GGT CTG CTC TGA GGA AAA TTC CCT GGC CTC C) and clones 1-1 and 1-2 were used to revert the L449F nucleotide to the leucine found in the wild type. For the preparation of P+c clones, 316-bp NotI-XmaI PR fragments (positions 2275 to 2590) were inserted into the HXB2 proviral DNA with the corresponding CSMs, which were prepared in the molecular clone experiments that had been carried out earlier as described above.

Evaluation of recombinant virus clone fitness in MT-2 cells. To evaluate the growth kinetics of the recombinant viruses, 10 µg of the constructed recombinant proviral DNA was introduced into 5 x 106 MT-2 cells by electroporation (250 V, 250 µF) with a Gene Pulser II apparatus (Bio-Rad, Hercules, Calif.). After the transfection, the cells were cultured in 10 ml of RPMI 1640 medium (Sigma Chemical, Taufkirchen, Germany) with 10% fetal calf serum (HyClone Laboratories, Inc., Logan, Utah). Half of the culture medium was harvested and replaced with fresh medium every 2 to 3 days, and the culture was maintained for up to 28 days posttransfection. The growth kinetics of the viruses were monitored through observation of the RT activities of the harvested supernatants. The RT activities were measured by a standard RT assay protocol described previously (15). For each recombinant virus, the supernatant with the highest RT activity was selected as the virus stock for the subsequent infectivity studies. The 50% tissue culture infective doses of the virus stock were determined by the limiting dilution method with the supernatant and were calculated by the Reed-Muench method. The growth kinetics of each recombinant virus were reproduced twice by use of independent transfections.

Evaluation of recombinant virus clone fitness by one-to-one competition cultures. To confirm the levels of replication competencies of the reconstructed clones, one-to-one growth competition cultures were performed. Each test virus (50% tissue culture infective dose, 100) was independently used to infect 2 x 106 MT-2 cells by incubation for 2 h at 37°C in individual test tubes. After the incubation, the infected MT-2 cells were washed once with fresh complete medium to remove excess virus inocula, and then the two tubes were combined in a T12.5 culture flask. Each competition culture was maintained for up to 28 days postinfection. Half of the culture medium was replaced with fresh complete medium every 2 to 3 days, and 106 MT-2 cells were added every 5 to 7 days. The proportion of the two test viruses in each competition culture was evaluated by cloning the gag-protease fragments of the viruses in the culture supernatant on days 0, 7, 14, 21, and 28 postinfection. Viral RNA was extracted from 200 µl of the culture supernatant, and a 757-bp gag-PR fragment was amplified by the same protocol described above to amplify the patient viruses. The amplicons were cloned into the pCR-Blunt II-TOPO vector and transformed into TOP10 competent cells by a heat shock procedure. Twenty clones were selected and sequenced at each time point, and the proportions of the two viruses were assessed.


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RESULTS
 
Impact of p7/p1 CSM A431V and p1/p6 CSM L449F on replication of PI-resistant clones. To confirm the significance of the p7/p1 and p1/p6 CSMs, the growth kinetics of 12 clones with artificially induced PI resistance in independent cultures were monitored by measuring the RT activities in the culture supernatants. The results are shown in Fig. 2a to c.



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  		FIG. 2. Replication kinetics of viruses with artificially induced PI resistance. Replication kinetics result in three different PRs: (a) wild-type PR; (b) a PR with an L90M mutation; and (c) a PR with D30N, N88D, and L90M mutations. Ten micrograms of recombinant virus DNA was transfected into 5 x 106 MT-2 cells by electroporation on day 0, and virus cultures were monitored for RT activity every 2 to 3 days. Solid circles, wild-type virus with wild-type Gag and PR; open circles, virus with wild-type Gag; open triangles, virus with the p1/p6 L449F CSM; solid diamonds, virus with p7/p1 A431V CSM; multiplication signs, virus with both the A431V and the L449F CSMs.

For the wild-type PR, the virus with wild-type Gag and the virus with the L449F CSM grew at the same rate, and the peak RT activities of these two clones were observed at 11 days posttransfection. It appears that the L449F mutation had little or no effect on virus replication when the wild-type PR was present. A delay in viral growth was observed in the viruses with the A431V mutation and the A431V and L449F mutations. The time of peak RT activity of the virus with the A431V mutation and the virus with the A431V and L449F mutations was 13 days posttransfection (Fig. 2a). A similar growth kinetics pattern was observed in the PR with the L90M mutation. The PR with the L90M mutation was selected as a representative PI resistance-associated mutation that has little effect on viral fitness. The viruses with the L90M mutation and both the wild-type Gag sequence and the L449F CSM demonstrated peak RT activity on day 10, whereas the viruses with the A431V mutation and that with the A431V and L449F mutations demonstrated peak RT activities on day 13 (Fig. 2b).

The growth pattern of the clone with the D30N, N88D, and L90M mutations was different from that of the viruses with the wild-type sequence or the PR with the L90M mutation. Viruses with PI resistance associated with the D30N, N88D, and L90M mutations were selected as representatives of viruses with severely impaired PR activities. Viruses with the D30N, N88D, and L90M mutations and the wild-type Gag sequence demonstrated a substantial delay in growth compared to the time of growth for the HXB2 wild type. The peak RT activity of the clone with the D30N, N88D, and L90M mutations was observed at day 17 posttransfection, 7 days after the times of peak activity for the wild-type virus and the virus with the L90M mutation. For the virus with impaired PR activity, the L449F CSM slightly improved the growth kinetics in the presence of the D30N, N88D, and L90M mutations; and peak RT activity was shifted to a point 2 days earlier than that for the virus with the wild-type Gag sequence. It appears that the contribution of L449F to viral fitness is essential in viruses with less fit PRs. In contrast, the A431V CSM was not beneficial to viral replication in viruses with the D30N, N88D, and L90M mutations. Viruses with the A431V mutation or the A431V and L449F mutations demonstrated reduced levels of growth (Fig. 2c).

The p1/p6 L449F CSM improved the replication of viruses with the D30N, N88D, and L90M mutations, whereas the p7/p1 A431V CSM was deleterious to virus replication in all three viruses tested.

Sequence profiles of selected patient-derived Gag-PR recombinant viruses with CSMs and non-CSMs. Samples from 43 patients who failed nelfinavir-containing regimens were selected from among the samples sent to the AIDS Research Center, National Institute of Infectious Diseases. Clones were carefully selected according to their drug resistance mutation patterns and Gag CSM patterns so that the population carrying the required CSM and PI resistance-associated mutations was represented. Two clones that had mutation patterns similar to those detected in the molecular clone analyses with viruses with artificially induced PI resistance described above were selected, the three Gag-PR fragments were cloned, and the following recombinant viruses were constructed: (i) a clone with the D30N, N88D, and L90M mutations in the PR sequence and the A431V and L449F mutations in the Gag sequence; (ii) a clone with the D30N, N88D, and L90M mutations in the PR sequence and the L449F mutation in the Gag sequence; and (iii) a clone with the L90M mutation in the PR sequence and the A431V mutation in Gag sequence. The major difference between the patient-derived clones and the clones with artificially induced PI resistance was that the Gag-PR fragments of the patient-derived clones had additional mutations in the Gag and PR regions. The sequences of the three Gag-PR fragments are shown in Fig. 3a and b. Clone 1-1 had both the A431V and the L449F CSMs, and there was one base deletion and 15 substitutions in the non-cleavage site and an APP duplication after position 459. The PR region contained three major mutations for PI resistance (D30N, M46I, and L90M) and seven minor mutations for PI resistance (L10I, L23I, I54V, L63P, A71T, V77I, and N88D). In addition, there were three mutations (K43T, I72E, and I97L) that are not recognized as drug resistance-associated mutations. As we could not determine the baseline sequence before PI treatment, we could not determine the source of the last three mutations, that is, whether they occurred by natural polymorphism or were introduced during PI treatment. However, we were able to obtain the sequence data for virus obtained in the early PI treatment phase, prior to the emergence of mutations for PI resistance. By comparison of the sequence of clone 1-1 with the sequence of the virus obtained at this early time point, we estimated that I72E seemed to be a natural polymorphism and that K43T and I97L probably appeared during PI treatment. One amino acid deletion at position T371 and five substitutions (I401V, E468A, Q474L, R479I, and A487S) appeared in the Gag region after PI resistance acquisition.



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  		FIG. 3. Gag and PR sequences of patient-derived clones. (a) Gag sequences of three patient-derived clones (clones 1-1, 1-2, and 2-2) aligned according to the sequence of reference strain HXB2. To clarify that the mutation appeared at the time of treatment, the baseline sequences are also demonstrated. Sequence data for the virus present prior to treatment were not available for the clones from the first patient (clones 1-1 and 1-2). Therefore, reference is made to the sequence data for virus obtained at an earlier time point of treatment (ETP), a time point at which no PI resistance-associated mutations had emerged. These data are used for comparison to the baseline sequence. Sequence data for the virus present prior to the initiation of PI treatment (PrePI) were obtained for the virus from the second patient (clone 2-2). The shaded areas indicate the cleavage sites, and open triangles indicate the cleavage points. Mutations that appeared after treatment are highlighted in open squares. (b) PR sequences of the three patient-derived clones (clones 1-1, 1-2, and 2-2) aligned according to the sequence of reference strain HXB2. For the clone from the first patient; sequence data for virus obtained at an earlier time point of treatment were used as the baseline sequence, and for the clone from the second patient, sequence data for the virus present prior to the initiation of PI treatment were used as the baseline sequence. The shaded loci indicate major PI resistance-associated mutations, and underlining indicates minor mutations. Mutations that appeared after PI treatment are highlighted in open squares.

Since clone 1-2 originated from the same patient sample as clone 1-1, the Gag sequence of clone 1-2 was nearly identical to that of clone 1-1. The differences between these clones were that clone 1-2 did not have the A431V CSM or the I401V non-CSM. We found more differences in the PR regions between clones 1-1 and 1-2. Fewer PI resistance-associated mutations accumulated in clone 1-2. Compared to the sequence of clone 1-1, clone 1-2 did not have the M46I, L23I, or I54V mutations. In addition, two mutations not related to resistance, K43T and I62V, were missing from clone 1-2.

The Gag sequence pattern of clone 2-2 selected from the sample from the second patient was quite different from those of clones 1-1 and 1-2. In addition to the A431V CSM in the Gag cleavage site, the clone had two additional mutations within the R380K (p2/p7) and P453L (p1/p6) cleavage sites. The clone also had 10 substitutions in the non-cleavage site. We were able to obtain a plasma sample before the initiation of therapy with the PI and determined that the mutations appeared after PI treatment. Four mutations (I389T, P453L, T456S, and P497L) were found to have appeared after PI treatment and resistance acquisition. Two major PI resistance-associated mutations (M46I and L90M) and four minor PI resistance-associated mutations (L10V, L63P, A71V, and G73S) were observed in the PR region. Eight additional mutations not related to resistance were also found in the PR region.

Using the three patient Gag-PR clones as templates, we successfully constructed recombinant viruses with four different combinations of Gag and PR fragments, types GP, P, P+c, and GP-c.

Contributions of CSMs and non-CSMs and highly significant relationship between CSM and non-CSM in the virus fitness recovery process. To evaluate the contributions of the A431V and L449F CSMs and to clarify the relevance of non-CSMs, four different types of recombinant virus were constructed by using the three patient-derived Gag-PR clones as templates. The viruses were cultured independently, and the growth kinetics were monitored. The results of the independent culture studies are shown in Fig. 4. For clone 1-1, the GP type was the most actively growing virus, and the P+c type was the second most actively growing virus. The P type and GP-c types did not grow very well and showed only slight increases in RT activity from the baseline (Fig. 4a). Thus, the level of growth of the four 1-1-derived recombinant viruses was, in order from the most to the least active, GP > P+c > P = GP-c. A competition culture was performed to confirm the replication capacities of the GP and P+c types. As shown in Fig. 5a, the GP and P+c types grew in similar proportions up to day 14, but the GP type dominated after day 21 and comprised 70 to 80% of the virus population in the supernatant. Thus, the GP type had better growth than the P+c type, and the result was consistent with that of the independent culture study. In clone 1-1, two CSMs were important for fitness recovery, as the P+c type grew better than the P type. However, by comparison of the GP and P+c types, the CSMs were not sufficient and 17 additional non-CSMs were required for full recovery of viral fitness. Surprisingly, the combination of 17 non-CSMs observed in clone 1-1 seemed to be deleterious to viral fitness without CSMs, since the GP-c type failed to grow. It appears that this combination of non-CSMs was functional only in the context of the A431V and L449F mutations.



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  		FIG. 4. Replication kinetics of recombinant viruses with patient-derived Gag-PR sequences in independent cultures. The replication kinetics of the recombinant viruses with patient-derived Gag-PR sequences were evaluated by using independent cultures. The results for three different patient-derived Gag-PR sequences are shown: (a) clone 1-1, (b) clone 1-2, and (c) clone 2-2. Ten micrograms of recombinant virus DNA was transfected into 5 x 106 MT-2 cells by electroporation on day 0, and virus cultures were monitored for RT activity every 2 to 3 days. Open circles, HXB2 wild-type control virus; solid circles, GP-type recombinants; open squares, P-type recombinants; open triangles, P+c-type recombinants; solid diamonds, GP-c-type recombinants.



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  		FIG. 5. Replication kinetics of recombinant viruses with patient-derived Gag-PR sequences in competition culture. To confirm the results of the independent cultures, competition assays were performed with the cultures. The results of seven competitions assays are shown. (a) Clone 1-1 of the GP versus clone 1-1 of the P+c type; (b) clone 1-2 of the GP type versus clone 1-2 of the P type; (c) clone 1-2 of the GP type versus clone 1-2 of the P+c type; (d) clone 1-2 of the P+c type versus clone 1-2 of the P type; (e) clone 2-2 of the GP type versus clone 2-2 of the P+c type; (f) clone 2-2 of the GP type versus clone 2-2 of the GP-c type; (g) clone 2-2 of the GP-c type versus clone 2-2 of the P+c type. Solid circles, GP type; open squares, P type; open triangles, P+c type; solid diamonds, GP-c type.

Both the GP and the P+c types of clone 1-2 (another clone from the same patient from which clone 1-1 was derived) grew efficiently. The P type of this clone also grew, but the GP-c type failed to grow (Fig. 4b). Thus, the order of fitness for the clone 1-2-derived recombinants was, in order from the most to the least fit, was GP = P+c > P > GP-c. To confirm this order, three pairs of competition assays with the GP type versus the P type, the GP type versus the P+c type, and the P+c type versus the P type were performed (Fig. 5b to d). In the competition with the GP and P+c types, the growth of the two viruses was similar during the culture period. In the competition assays with the GP and P types, the GP type dominated after 14 days. In the competition with the P+c type and the P type, the P+c type was always dominant and comprised 90 to 100% of the virus population. Thus, the results were consistent with those of the independent cultures. A reduced level of replication of clone 1-2 was observed, and the L449F CSM improved the growth kinetics up to a level similar to that for the GP type. The combination of 16 non-CSMs without the L449F mutation was deleterious to replication in this clone as well.

Clone 2-2, which was derived from a different patient, demonstrated a kinetics pattern different from those of the other two clones. The GP type was the most actively growing virus, and this finding was consistent with those for clones 1-1 and 1-2 (Fig. 4c). Different from the other two clones, the GP-c type was the next most actively growing, followed by the P+c type. The P type failed to grow. Thus, the order of fitness, from the most to the least fit, was GP > GP-c > P+c > P. The A431V CSM was critical to the recovery of fitness for this clone but was not sufficient for full recovery, and additional non-CSMs were required. To confirm the order of fitness, three pairs of competition assays were performed: the GP type versus the P+c type, the GP versus the GP-c type, and the GP-c type versus the P+c type (Fig. 5e to g). In the competition assay with the GP type versus the P+c type, the GP type dominated after 7 days, and 80 to 90% of the virus population was of this type. In the competition assay with the GP type versus the GP-c type, the GP type was always found at a higher percentage throughout the culture period, but the GP-c-type virus was also observed in 20 to 40% of the virus population. It appears that the GP type had a slight competitive advantage compared to that for the GP-c type. In the competition assay with the GP-c type versus the P+c type, the GP-c type grew better than the P+c type and more than 90% of the clones were of the GP-c type at day 28. Thus, the data were consistent with those of the independent culture study. In clone 2-2, Gag mutations were critical for viral replication. CSM A431V and a combination of 10 non-CSMs both had a positive effect on the recovery of viral fitness and appeared to be most effective when CSMs and non-CSMs coexisted.


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DISCUSSION
 
The complementary relationship of Gag mutations within substrate sites, i.e., CSMs, with PI resistance has been reported in many studies (10, 14, 17, 24, 44). However, our analysis of patient-derived Gag sequences showed that CSMs are not the only mutations that appear together with PI resistance-associated mutations, but additional Gag mutations accumulated in non-CSMs, outside the cleavage sites. In this study, we focused on the mutations found outside of the cleavage sites and attempted to clarify their importance in terms of the recovery of viral fitness. Some of the non-CSMs may have existed as baseline Gag mutations prior to ART, and some of them may have emerged after the initiation of ART.

We started the study by confirming the impact of CSMs on viral fitness in PI-resistant clones, and we selected two PI resistance-associated mutation patterns, the D30N, L90M, and N88D mutation pattern, which is representative of a highly impaired PR, and the L90M mutation pattern, which is representative of a fairly impaired PR. We constructed several recombinants with different CSM patterns and compared their replication kinetics. From the results of these experiments, we verified that the p1/p6 L449F CSM has the potential to improve the fitness of the virus with the D30N, L90M, and N88D PI resistance-associated mutations. We did not see a significant positive effect of L449F in the virus with the L90M mutation or the virus with wild-type PR, nor did the mutation hinder replication of the viruses.

We observed different patterns of kinetics among viruses with the p7/p1 mutation. This mutation has also been reported to improve viral fitness, especially with regard to M46IL PI resistance-associated mutations (8). Indeed, patient-derived clones 1-1 and 2-1, which had the A431V mutation, also had the M46I mutation in the PR gene. However, for wild-type virus and viruses with the L90M mutation and the D30N, N88D, and L90M mutations tested in these studies, the p7/p1 mutation demonstrated deleterious effects on virus growth. A similar negative impact of the A431V mutation on viral growth was also reported in two previous studies (14, 29). All six clones with the A431V mutation demonstrated slower viral replication compared to the rate of replication of clones without the A431V mutation. The data also support a significant linkage and the synergistic evolution of the A431V and M46IL mutations.

Interestingly, the growth kinetics of viruses with p7/p1 and p1/p6 double cleavage sites were the worst for wild-type viruses and viruses with the D30N, N88D, and L90M mutations. It seems that there might be interference between these two CSMs, and introduction of additional Gag and PR mutations might be required to overcome this negative effect.

To evaluate the contributions of CSMs and non-CSMs in viral replication, we compared the replication kinetics of four types of recombinant viruses. We performed two different types of experiments. One was an independent culture assay in which viral DNA was transfected into MT-2 cells, and the other was a competition culture assay in which MT-2 cells were infected with recombinant viruses. The results of the two methodologies were precisely in line with one another, and from these results we ascertained the three important features of mutations in the Gag region and the PR region.

First, we found that not only CSMs but also non-CSMs contribute to the recovery of fitness in PI-resistant viruses. Clones of the GP type, which had both CSMs and non-CSMs, demonstrated the highest level of viral growth for all three patient-derived gag-pol sequences. Interestingly, in clones 1-1 and 1-2, GP-c-type recombinants did not grow, suggesting that the non-CSMs accumulated after PI treatment (probably the T371 deletion and the I401V, E368A, S473P, Q474L, and A487S mutations) were deleterious to viral growth, and these mutations were functional only in the context of CSMs. In contrast, clone 2-2 of the GP-c type grew well and grew even better than clone 2-2 of the P+c type. The non-CSMs observed in clone 2-2 were different from those observed in clones 1-1 and 1-2, suggesting that a wide variety of Gag mutations may emerge with PI treatment and that non-CSMs can have different impacts on viral growth, depending on the corresponding Gag and PR mutation patterns.

Second, we ascertained that two PR types are differentiated by Gag sequence independence. One is the Gag-independent PR that can be functional without any mutations in its target Gag region. The PR of clone 1-2 can be classified as this type. In clone 1-2, although the GP and P+c types demonstrated better viral replication abilities, the P-type recombinant could also replicate. It appears that the mutations that accumulated in this PR could allow the viral replication capacity to recover. The second type of PR was Gag dependent, which requires Gag mutations to be functional PR. The PRs of clones 1-1 and 2-2 were of this type. In these clones, P-type recombinants had the lowest level of virus growth, and mutations in Gag, especially CSMs, were required to achieve better replication. We noted with interest that the PR sequences of clones 1-1 and 1-2, which were isolated from samples from the same patient, were quite similar to each other. The PR of clone 1-1 had additional L23I, K43T, M46I, I54V, and I62V mutations compared to the sequence of clone 1-2; and the additional mutations found in clone 1-1 made the activity of the PR dependent on the Gag mutations. This finding is an important issue with regard to drug resistance phenotyping with recombinant virus technology. Our findings indicate that the inclusion or exclusion of the HIV-1 Gag sequence may affect the nature of subsequent virus populations recovered by recombination procedures and may affect drug resistance levels. In this study, we focused on PR activity and not drug resistance phenotypes. Further studies that include phenotypic analysis and other PIs should be performed to obtain a better understanding of the nature of PI resistance and to improve treatment protocols in a practical manner.

Third, we confirmed that the A431V CSM is sufficient as a compensatory Gag mutation in certain mutation patterns. According to the results of previous studies and from our results, it appears that the M46IL mutation in the PR has a key relationship to the A431V CSM (8, 25). The ternary structures of the p7/p1 cleavage site and the PR also support the significant interaction of A431V and M46IL, as A431V is located at the S2 position of the p7/p1 cleavage site and M46I is located within the P2 site of the PR (35).

Thus, all three findings indicate that PR and mutations in its substrate, Gag, are vitally linked. In conclusion, our study demonstrates that non-CSMs are as important as CSMs for the recovery of viral fitness in drug-resistant HIV-1 with impaired PR activities. This essential relationship is the result of the survival competition evolution process of the virus during antiretroviral treatment in vivo.


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ACKNOWLEDGMENTS
 
We thank the patients and physicians who were involved in our study. We also thank Yumi Kanekama and Mary Phillips for help in preparing the manuscript.

This study was supported by a grant from the Organization of Pharmaceutical Safety and Research of Japan and the Ministry of Health, Labour, and Welfare of the Japanese Government.


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FOOTNOTES
 
* Corresponding author. Mailing address: AIDS Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuenn, Musashimurayama, Tokyo 208 0011, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-7746. E-mail: wsugiura{at}nih.go.jp. Back


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Antimicrobial Agents and Chemotherapy, February 2004, p. 444-452, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.444-452.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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