Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 Loop Library
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m414360200
ISSN1083-351X
AutoresKeisuke Yusa, Yosuke Maeda, Aki Fujioka, Kazuaki Monde, Shinji Harada,
Tópico(s)Immune Cell Function and Interaction
ResumoThe human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 × 106 independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 μm in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure. The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 × 106 independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 μm in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure. Entry of R5 human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; env, envelope; GP, glycoprotein. into target cells requires sequential interaction of the envelope glycoprotein GP120 with CD4 and the coreceptor CCR5 (1Berger E. Murphy P. Farber J. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1890) Google Scholar). The binding of GP120 to CD4 leads to exposure of the coreceptor-binding site, designated the third hypervariable loop (V3 loop), which is composed of ∼35 amino acid residues (2Platt E.J. Kuhmann S.E. 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Roca G. Wyatt R. Sodroski J. Zhao L. Olson W. Kwong P.D. Sattentau Q.J. J. Virol. 2000; 74: 1948-1960Crossref PubMed Scopus (290) Google Scholar). The presence of at least one basic substitution at V3 position 11 or 25 can change coreceptor usage (26De Jong J.J. De Ronde A. Keulen W. Tersmette M. Goudsmit J. J. Virol. 1992; 66: 6777-6780Crossref PubMed Google Scholar, 27Fouchier R.A. Brouwer M. Broersen S.M. Schuitemaker H. J. Clin. Microbiol. 1995; 33: 906-911Crossref PubMed Google Scholar), and sequence changes in the GPG motif in the V3 loop can modulate coreceptor usage (17Hu Q. Trent J. Tomaras G. Wang Z. Murray J. Conolly S. Navenot J. Barry A. Greenberg M. Peiper S. J. Mol. Biol. 2000; 302: 359-375Crossref PubMed Scopus (49) Google Scholar, 18Shimizu N. Haraguchi Y. Takeuchi Y. Soda Y. Kanbe K. Hoshino H. Virology. 1999; 259: 324-333Crossref PubMed Scopus (44) Google Scholar). Variation in the residues flanking the GPG motif can alter the stability of the β-sheet and/or alter the surface accessibility of this element, thereby influencing coreceptor usage (28Catasti P. Fontenot J.D. Bradbury E.M. Gupta G. J. Biol. Chem. 1995; 270: 2224-2232Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Chesebro B. Wehrly K. Nishio J. Perryman S. J. Virol. 1992; 66: 6547-6554Crossref PubMed Google Scholar, 30Tugarinov V. Zvi A. Levy R. Hayek Y. Matsushita S. Anglister J. Structure Fold. Des. 2000; 8: 385-395Abstract Full Text Full Text PDF Scopus (60) Google Scholar). If we can manipulate a mixed population of HIV-1 with diversity in the V3 loop in vitro, it would be a useful system for screening and analyzing the biological features of HIV-1 with regard to V3 variation. There has been a report regarding the construction of a heterogeneous retrovirus population in vitro (31Berkhout B. Klaver B. Nucleic Acids Res. 1993; 21: 5020-5024Crossref PubMed Scopus (36) Google Scholar), and we recently constructed an in vitro system that created an HIV-1 library containing mutations associated with protease inhibitor resistance (32Yusa K. Song W. Bartelmann M. Harada S. J. Virol. 2002; 76: 3031-3037Crossref PubMed Scopus (7) Google Scholar). In the current report, we constructed an R5 HIV-1 V3 loop library containing diverse structures of the V3 loop. We chose 10 amino acid positions in the V3 loop (residues 302, 303, 304, 305, 306, 312, 314, 317, 318, and 321; V3 loop starting from Cys293 to Cys327 within V3 of HIV-1JR-FL Env) (Fig. 1A), and we incorporated the amino acid substitutions in 31 R5 HIV-1 strains obtained from the Los Alamos HIV data base (//hiv-web.lanl.gov). Theoretically, the number of substitution combinations in a library carrying a set of random combinations of 0–10 substitutions is 27,648 possibilities. A new class of antiretroviral drugs that prevent virus entry are being developed. TAK-779 inhibits HIV-1 replication by blocking the interaction of GP120 with CCR5 (34Baba M. Nishimura O. Kanzaki N. Okamoto M. Sawada H. Iizawa Y. Shiraishi M. Aramaki Y. Okonogi K. Ogawa Y. Meguro K. Fujino M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5698-5703Crossref PubMed Scopus (748) Google Scholar). This compound has been shown previously to inhibit cell-to-cell fusion and cell-free virus infectivity in vitro (34Baba M. Nishimura O. Kanzaki N. Okamoto M. Sawada H. Iizawa Y. Shiraishi M. Aramaki Y. Okonogi K. Ogawa Y. Meguro K. Fujino M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5698-5703Crossref PubMed Scopus (748) Google Scholar). The binding site for TAK-779 is located near the CCR5 extracellular surface, within a cavity between transmembrane helices (35Dragic T. Trkola A. Thompson D.A. Cormier E.G. Kajumo F.A. Maxwell E. Lin S.W. Ying W. Smith S.O. Sakmar T.P. Moore J.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5639-5644Crossref PubMed Scopus (423) Google Scholar). Recently, the viruses resistant to an entry inhibitor enfuvirtide (T20) were sensitive to TAK-779 (36Reeves J.D. Lee F.H. Miamidian J.L. Jabara C.B. Juntilla M.M. Doms R.W. J. Virol. 2005; 79: 4991-4999Crossref PubMed Scopus (134) Google Scholar). However, development of TAK-779 resistance remains to be elucidated. To address the contribution of the V3 loop to susceptibility to an entry inhibitor, we selected TAK-779-resistant variants from the HIV-1 library, and we isolated R5 entry inhibitor-resistant HIV-1 with mutations restricted to the V3 loop. Cell Culture and Molecular Clones—PM1 (37Schwenk H.U. Schneider U. Blut. 1975; 31: 299-306Crossref PubMed Scopus (66) Google Scholar) and HeLa-CD4-LTR-β-gal (MAGI) cells (38Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Crossref PubMed Google Scholar) were provided by the National Institutes of Health AIDS Research and Preference Reagent Program. PM1-CCR5 cells were generated by standard retrovirus-mediated transduction of PM1 cells with pBABE-CCR5 provided by the National Institutes of Health AIDS Research and Preference Reagent Program. PM1 and PM1-CCR5 cells were grown in RPMI 1640-based complete medium supplemented with 10% fetal calf serum (Vitromex, Bayern, Germany), 200 units/ml penicillin, and 200 units/ml streptomycin. The transduced cells were selected with complete medium plus 1 μg/ml of puromycin (Sigma), and a single clone was selected based upon the expression level of CCR5 assessed by flow cytometry analysis. Similarly, MAGI-CCR5 was isolated by standard retrovirus-mediated transduction of MAGI cells with pBABE-CCR5 (39Chen Z. Zhou P. Ho D.D. Landau N.R. Marx P.A. J. Virol. 1997; 71: 2705-2714Crossref PubMed Google Scholar). MAGI and MAGI-CCR5 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 200 units/ml penicillin, and 200 units/ml streptomycin. The HIV-1 proviral expression vector pJR-FL was kindly provided by Dr. Y. Koyanagi (Kyoto University). pJR-FLan was created from pJR-FL by incorporation of AflII and NheI sites into env at nucleotides 6395 and 6564, respectively. There were no amino acid substitutions caused by incorporation of the AflII site (CTG AAA (Leu Lys) → CTTAAG (Leu Lys)). Incorporation of the NheI site led to two amino acid substitutions (GTT ATA (Val Ile) → GCT AGC (Ala Ser)). HIV-1JR-FLan was used as the parental virus. Construction of an R5 HIV-1 V3 Loop Library—The strategy used to construct the HIV-1 library carrying a set of random amino acid substitutions is shown in Fig. 2A. The 2077-bp StuI-XhoI fragment from pJR-FLan was subcloned into pCRΔZ digested with StuI and XhoI, resulting in pCR-SANX. pCRΔZ was created from pCR-blunt (Invitrogen) by deleting a PmlI fragment containing a Zeocin cassette. Six oligonucleotides, V31, V32, V33, V34, V35, and V36, were commercially synthesized to generate three short DNA fragments with sticky ends. The respective pairs of complementary oligonucleotides at 0.35 μg/μl were denatured for 2 min at 90 °C in a heat block and then left in the switched-off heat block for 30 min in 10 mm MgCl2 and 10 mm Tris-HCl (pH 8.0) at room temperature for annealing. The resultant short DNA fragments were designated V312 (48 bp), V334 (65 bp), and V356 (64 bp), respectively. V334 contained 10 degenerated codons (ARA for Lys or Arg; RGT for Ser or Gly; RTA for Ile or Val; HMY for His, Pro, Asn, Ser, Thr, or Tyr; MTR for Ile, Met, or Leu; TKK for Phe, Leu, Trp, or Cys; RCW for Thr or Ala; SMM for Glu, Asp, Gln, Ala, His, or Pro; RTA for Ile or Val; and RAT for Asp or Asn) (Fig. 2A). The single letter code used here is as follows: R represents A or G; H represents A, C, or T; M represents A or C; Y represents C or T; K represents G or T; W represents A or T; and S represents C or G. The three DNA fragments (0.25 μg each) were ligated by T4 DNA ligase (New England Biolabs, Beverly, MA), and the resultant 185-bp DNA fragment was purified by 1.5% agarose electrophoresis. One hundred nanograms of the purified DNA fragment was used for PCR as the DNA template with Pfx DNA polymerase (Invitrogen). The upstream primer was VV-Af (5′-ACAGCTTAAGGAATCTGTAGAAATTAATTG-3′), and the downstream primer was VV-Nh (5′-ATTTGCTAGCTATCTGTTTTAAAGTGTCAT-3′). The amplification conditions for the first-round PCR were 94 °C for 1 min, 16 cycles of 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 60 s and a final extension at 72 °C for 10 min. PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems Inc., Foster City, CA). The PCR product (5.0 μg) was purified using the Concert Rapid PCR Clean-up System (Invitrogen), digested with AflII and NheI and then ligated into the AflII and NheI sites of pCR-SXΔAN (10.0 μg). pCR-SXΔAN was created from pCR-SANX by replacing the AflII-NheI fragment with a linker. The ligation mixture was purified by Microcon-30 (Millipore Corp., Bedford, MA) and used to transform Escherichia coli strain DH5α by electroporation with an ECM 600 (BTX, San Diego) at 2.5 kV, generating pCR-SX-V3Lib containing 7.1 × 106 independent clones with an AflII-NheI fragment. After purification of the pCR-SX-V3Lib DNA, the StuI-XhoI fragment from 15 μg of the plasmid was cloned into the StuI and XhoI sites of pJR-FLΔSX. pJR-FLΔSX was created from pJR-FL by replacing the StuI-XhoI fragment of pJR-FL with a linker. The ligation products (0.2 μg) were transformed into E. coli strain JM109 by electroporation as described above. Finally, the HIV-1 library, designated pJR-FL-V3Lib, contained 6.6 × 106 independent clones containing a 2077-bp (StuI-XhoI) env DNA fragment (ligation efficiency, 77%) (Table I).Table IHIV-1 library (pJR-FL-V3Lib) containing combinations of 0-10 amino acid substitutions in the V3 loopNo. combinations of amino acid substitutionsNo. independent clonesaAfter transformation, 1/1000 volume of the cell suspension was inoculated on an LB agar plate containing 100 μg/ml ampicillin and incubated overnight. The number of independent clones was then counted.No. clones containing a mutated V3 loopLigation efficiencybThe ligation efficiency was obtained from >70 independent clones.27,648%pCR-SX-V3Lib8.5 × 1067.1 × 10684pJR-FL-V3Lib9.3 × 1066.6 × 10677a After transformation, 1/1000 volume of the cell suspension was inoculated on an LB agar plate containing 100 μg/ml ampicillin and incubated overnight. The number of independent clones was then counted.b The ligation efficiency was obtained from >70 independent clones. Open table in a new tab Analysis of Replication—All viral stocks, including the virus library, were prepared by transfecting 293T cells as described previously (32Yusa K. Song W. Bartelmann M. Harada S. J. Virol. 2002; 76: 3031-3037Crossref PubMed Scopus (7) Google Scholar). Viral stocks for analysis of the replication of the virus library HIV-1V3Lib were generated by transient transfection of 293T cells with pJR-FL-V3Lib. Then 5 × 105 of PM1-CCR5 or MT-2 cells were infected with HIV-1JR-FLan or HIV-1V3Lib stocks containing 600 ng of p24 Gag antigen/ml. Viral replication was monitored by measuring the p24 Gag antigen concentration in the supernatants of the challenged cells. p24 Gag antigen was determined by p24 Gag antigen enzyme-linked immunosorbent assay system using RETRO-TEK (ZeptoMetrix, Co., Buffalo, NY). Selection of TAK-779-resistant HIV-1V3Lib—PM1-CCR5 cells (1 × 106) were infected with HIV-1V3Lib (600 ng of p24 Gag antigen) at passage 1 and incubated for 3 days in the presence of 0.1 μm TAK-779. Virus passages were then performed at 3–4-day intervals using PM1-CCR5 cells (2 × 105) in the absence or presence of 0.10 μm TAK-779. The concentration of TAK-779 was increased from 0.10 to 0.30 μm at passage 3. The infectivity of the supernatant at each passage was evaluated using MAGI-CCR5 cells in the presence or absence of 0.1 μm TAK-779. Determination of Susceptibilities—The susceptibilities of the viruses to entry inhibitors were determined by the MAGI assay using previously titrated virus preparations (80 blue foci/well). MAGI-CCR5 cells (1 × 104 cells/well) were plated into 48-well tissue culture plates 1 day prior to infection. After absorption of the virus for 2 h at 37 °C in the presence or absence of 0.001–1.0 μm TAK-779 or AMD3100, the cells were washed twice with phosphate-buffered saline and then further incubated for 48 h in the presence or absence of 0.0010 nm to 3.0 μm TAK-779 or AMD3100 in fresh medium. The cells were stained, and the number of blue foci in each well was counted (37Schwenk H.U. Schneider U. Blut. 1975; 31: 299-306Crossref PubMed Scopus (66) Google Scholar). All experiments were performed in triplicate. Sequencing—The nucleotide sequences of the V3 loops in the virus library were determined as follows. The virus mixture was precipitated and subjected to reverse transcription-PCR using the ImProm-II transcription system (Promega, Madison, WT). A 380-bp fragment containing a V3 loop sequence was amplified by PCR in a 50-μl reaction volume comprising 50 mm KCl, 10 mm Tris-HCl (pH 8.3), 2 mm MgCl2, 0.01% gelatin, and 2 units of AmpliTaq (Applied Biosystems Inc.) with primers VV1 (5′-AATGGCAGTCTAGCAGAAGAAG-3′) and VV2 (5′-TTTCTGGGTCCCCTCCTGAGGA-3′). The PCR products were purified by 1% agarose electrophoresis and cloned into the pCR-TOPO vector (Invitrogen). The cloned sequences were sequenced using an ABI Prism 310 (Applied Biosystems Inc.). Construction of the Proviral DNA for the HIV-1 V3 Loop Library—A variety of amino acid substitutions was observed in the V3 loop of 31 R5 HIV-1 derived from the Los Alamos HIV data base (Fig. 1). To construct a proviral DNA mixture containing a set of random substitution combinations derived from the envelope V3 loops of the R5 isolates, we chose 10 amino acid positions for substitutions (residues 302, 303, 304, 305, 306, 312, 314, 317, 318 and 321; V3 loop starting from Cys293 to Cys327 in HIV-1JR-FL) (Fig. 2A). AflII-NheI DNA fragments containing a set of random combinations of these mutations were prepared with overlapping oligodeoxynucleotides comprising both DNA strands and were subsequently annealed and ligated to form the complete gene. In the AflII-NheI DNA fragments, the residues at positions 302, 303, 304, 314, 318, and 321 contained two possible amino acid residues, whereas the residues at positions 305, 306, 312, and 317 contained 6, 3, 4, and 6 possibilities, respectively (Fig. 2A). Amino acid substitutions (Phe to Cys at 312, Gln to His or Pro at 3317; Fig. 2A, underlined) that were not detected in the R5 isolates (Fig. 1) were inevitably incorporated into the library because of unintended combinations of nucleotide substitutions. Theoretically, the number of possible combinations of 0–10 amino acid substitutions was calculated to be 27,648 (Table I), and the lowest frequency combination of the substitutions was 0.00204%. We finally obtained a proviral DNA, designated pJR-FL-V3Lib, for the HIV-1 library that contained 6.6 × 106 independent clones. This size of the library contained all possible combinations of the recombinants. Incorporation of random combinations of the amino acid substitutions into the V3 loops was confirmed by the sequencing of randomly selected clones from the library DNA (Fig. 3A). There was no marked difference in the composition of the amino acid residues at the 10 positions in V3 between the library DNA and the virus library prepared for the following experiments (Fig. 3B). Coreceptor Usage of the Virus Library—We tested the coreceptor usage of the virus library using a single-round assay (Fig. 4A). HIV-1JR-FLan, which was used as a background for the construction of the V3 loop library, showed similar infectivity to HIV-1JR-FL. The infectivity of the virus library HIV-1V3Lib into MAGI-CCR5 cells was 26% lower than that of parental HIV-1JR-FLan. This result suggested that most of the viral clones in the library were replication-competent. In fact, 16 of 21 (76%) viral clones randomly selected from the library were competent in PM1-CCR5 cells (data not shown). No blue-stained foci were detected in MAGI cells infected with HIV-1JR-FLan and HIV-1JR-FL, whereas the X4 virus HIV-1NL4–3 did generate blue foci. To examine whether the library contained viral clones that acquired the ability to use CXCR4 as a coreceptor by a combination of mutations restricted to the V3 loop, a large amount of p24 was used for infection of MAGI cells. No blue-stained cells were obtained with HIV-1V3Lib (Fig. 4A). To further confirm this result, MT-2 cells were infected with HIV-1V3Lib (Fig. 4B). p24 production from PM1-CCR5 cells infected with HIV-1JR-FLan was saturated on day 3 because of the intense cytopathic effect caused by the high efficiency of infection at time 0. p24 generation from HIV-1V3Lib-infected PM1-CCR5 cells reached a higher level than that of HIV-1JR-FLan-infected cells because of a slower cell death rate, although no increase in p24 was observed in the supernatant of MT-2 cells infected with the same amount of p24 of HIV-1V3Lib, even at day 7 post-infection. Similarly, there was no increase in the amount of p24 released from MT-2 cells infected with HIV-1JR-FLan. We repeated the same experiments, but we did not obtain any viral clones from the library that could use CXCR4 for entry. These results indicated that none of the combinations of the 0–10 substitutions (27,648 possibilities) in the V3 loop derived from R5 viruses could use CXCR4 as a coreceptor in an HIV-1JR-FLan background. The V3 loop of clinical isolates has a global positive charge that can vary from +2 to +10, and an increase in the positive charge of the V3 loop enabled the use of CXCR4 (7Hoffman T.L. Doms R.W. Mol. Membr. Biol. 1999; 16: 57-65Crossref PubMed Scopus (97) Google Scholar, 25Moulard M. Lortat-Jacob H. Mondor I. Roca G. Wyatt R. Sodroski J. Zhao L. Olson W. Kwong P.D. Sattentau Q.J. J. Virol. 2000; 74: 1948-1960Crossref PubMed Scopus (290) Google Scholar). The virus library had V3 loops with a positive charge from +3to +7 and did not contain the X4 type. Selection of TAK-779-resistant HIV-1 from the Library—In the presence of 0.1 μm TAK-779, infectivity of HIV-1JR-FLan or unselected HIV-1V3Lib was suppressed by 86–92%, as evaluated by a single-round assay using MAGI-CCR5 cells (Fig. 5, passage 0). After three passages of HIV-1V3Lib in the presence of 0.1 μm TAK-779, we increased the concentration of TAK-779 to 0.30 μm at passage 3. HIV-1JR-FLan could not be passaged in PM1-CCR5 cells in the presence of 0.1 μm TAK-779 because of poor replication, whereas HIV-1V3Lib started to replicate after a delay of 3 or 4 days compared with HIV-1V3Lib in the absence of 0.30 μm TAK-779 at passage 3 (data not shown). The inhibitor-sensitive population of the virus library seemed to be replaced with the inhibitor-resistant variants from passages 3 to 8 (Fig. 5). The infectivities of the variants at passages 8 and 10 were not even inhibited by the presence of 0.10 μm TAK-779. In contrast, HIV-1V3Lib at passage 8 in the same passage schedule without the inhibitor did not acquire resistance to TAK-779. We determined the IC50 of the selected HIV-1V3Lib using a single-round assay (Table II). The selected variants showed 15-fold resistance to TAK-779 compared with HIV-1JR-FLan, whereas unselected HIV-1V3Lib at passage 8 did not. To elucidate the alteration of the V3 loop responsible for the resistance in the selected HIV-1V3Lib, we subcloned and sequenced the V3 loops of the variants. The V3 loops contained five incorporated substitutions, namely I304V, H305N, I306M, F312L, and E317D (Fig. 6A). In addition to these substitutions, 25% of the clones (3/12) contained A332D in the GP120 C3 region. No other mutations were detected in other regions of GP120 in the selected viruses by direct sequencing (data not shown). In the absence of the entry inhibitor, the viruses at passage 8 only contained three types of V3 loop in 13 clones (Fig. 6A), although the virus at passage 0 contained complexity in all 10 amino acid positions (Fig. 3A). These V3 loops, especially V3 loop number 01, containing I304V, H305Y, I306L, E317H, I318V, and D324N (85%; 11/13), may confer a substantial replicative advantage among the 27,648 possible substitution combinations in PM1-CCR5 cells without selective pressure.Table IISusceptibilities of selected HIV-1Lib-P8 to entry inhibitorsVirusIC50aIC50 indicates the concentration required to inhibit 50% of the blue foci formation in MAGI-CCR5 cells at 2 days post-infection.TAK-779AMD3100μmHIV-1JR-FLan0.032 ± 0.07bDegree of resistance (x-fold) as compared with HIV-1JR-FLan. (1.0)>3HIV-1JR-FL0.034 ± 0.05 (1.1)>3HIV-1NL4-3>10.0023 ± 0.0003HIV-1V3Lib-P8- - TAK-779cVirus library passaged eight times without TAK-779.0.041 ± 0.12 (1.4)>3HIV-1V3Lib-P8+ + TAK-779dVirus library selected with 0.1 μm TAK-779 from passages 1 to 3 and 0.3 μm TAK-779 from passages 4 to 8.0.48 ± 0.15 (15)>3a IC50 indicates the concentration required to inhibit 50% of the blue foci formation in MAGI-CCR5 cells at 2 days post-infection.b Degree of resistance (x-fold) as compared with HIV-1JR-FLan.c Virus library passaged eight times without TAK-779.d Virus library selected with 0.1 μm TAK-779 from passages 1 to 3 and 0.3 μm TAK-779 from passages 4 to 8. Open table in a new tab Fig. 6A, sequences of the V3 loop in TAK-779 escape viruses (passage 8) in the absence (HIV-1V3Lib-P8-) or presence (HIV-1V3Lib-P8+) of TAK-779. The numbers above the sequences indicate the positions of amino acid residues in JR-FLan GP120. HIV-1JR-FLan has been also passaged for same the duration without drug pressure; however, no mutations were detected in the V3 loop (data not shown). B, susceptibilities of recombinant viruses containing TAK-779 resistance associated mutation(s). IC50 indicates the concentration required to inhibit 50% of the blue foci formation in MAGI-CCR5 cells at 2 days post-infection. Degree of resistance (x-fold) is compared with HIV-1JR-FLan.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that resistance was conferred by changes in the V3 loop, we cloned the AflII-NheI DNA fragment into env genes containing the V3 loop of numbers 31 and 32. These were then used to replace the V3 loop of the parental virus HIV-1 JR-FLan, and their dr
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