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Journal of Clinical Microbiology
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Virology

Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing

Deirdre O'Meara, Karin Wilbe, Thomas Leitner, Bo Hejdeman, Jan Albert, Joakim Lundeberg
Deirdre O'Meara
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm,
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Karin Wilbe
Department of Clinical Virology, Swedish Institute for Infectious Disease Control/Karolinska Institute, S-171 82 Stockholm,
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Thomas Leitner
Department of Clinical Virology, Swedish Institute for Infectious Disease Control/Karolinska Institute, S-171 82 Stockholm,
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Bo Hejdeman
Department of Dermatovenereology, Södersjukhuset, S-118 83 Stockholm, and
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Jan Albert
Department of Clinical Virology (IMPI), Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm, Sweden
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Joakim Lundeberg
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm,
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DOI: 10.1128/JCM.39.2.464-473.2001
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  • Fig. 1.
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    Fig. 1.

    Schematic diagram of pyrosequencing. The reaction mixture consists of single-stranded DNA with an annealed primer, DNA polymerase, ATP sulfurylase, luciferase, and apyrase. The four nucleotide bases are added to the mixture in a defined order, e.g., A, C, G, and T. If the added nucleotide forms a base pair (in this case, two Ts base pair to the template), the DNA polymerase incorporates the nucleotide and consequently pyrophosphate (PPi) is released. The released pyrophosphate is converted to ATP by ATP sulfurylase, and luciferase uses this ATP to generate detectable light. This light is proportional to the number of nucleotides incorporated and is detected in real time. The pyrosequencing raw data are displayed simultaneously, and in this example the sequence generated reads ATCTT. The height of the signal is proportional to the number of nucleotides incorporated. Excess quantities of the added nucleotide are degraded by apyrase. If the nucleotide does not form a base pair with the DNA template, it is not incorporated by the polymerase and no light is produced. Apyrase then rapidly degrades the nucleotide.

  • Fig. 2.
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    Fig. 2.

    Pyrosequencing the seven codons in the protease gene that are primarily involved in drug resistance. The sequences of these codons (underlined) and their surrounding nucleotides are displayed above the pyrosequencing data. A negative G frameshift (underlined) is observed after sequencing codon 48. Codons 82 and 84 were sequenced (in the reverse direction) using primer 10, and the complete sequence shows that readability is maintained over 33 nucleotides, i.e., codons 84 to 75 (indicated above the sequence). Ambiguous sequence data are in italics. Note that the peaks between the individual codons cannot be directly compared, as the raw data are not to scale.

  • Fig. 3.
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    Fig. 3.

    Pyrosequencing data on defined mixtures of wild-type (MN) and mutant clones. The wild-type clone has the sequence TTGTC, while the mutant has the sequence TCGTC. Arrows correspond to positions where different ratios of the two templates give rise to different signal levels. These peaks are proportional to the percentage of each clone present in the mixture.

  • Fig. 4.
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    Fig. 4.

    Pyrosequencing polymorphic positions in HIV-1 DNA populations derived from PBMC from treatment-naı̈ve HIV-1-infected patients. (A) Comparison of the expected pattern generated from a wild-type sequence with the observed pattern (using primer 1) reveals the presence of mixed variants in sample B. Detailed analysis of the observed pattern reveals that one A peak (arrow) is approximately 40% larger than the first A peak and that the subsequent G peak is 60% of a normal G peak. Therefore the sequence of codon 13 in sample B is RTA. Direct Sanger sequencing of the PCR product shows a 50% mixture of A and G at codon 13. (B) Comparison of the expected pattern generated from a wild-type sequence with the observed pattern (using primer 1) reveals the presence of mixed variants in sample F. Detailed analysis of the observed pattern reveals that the second T peak (arrow) is 50% larger than the first T peak and that the next C peak is 50% of a normal C. The sequence of codon 11 in sample F is therefore GTY. The next base is also ambiguous in that G and A peaks which are approximately 50% of normal G and A peaks appear. The sequence of codon 12 in sample F is therefore RCA. Direct Sanger sequencing of the PCR product shows approximately 50% mixtures of T and C and A and G at codons 11 and 12, respectively. The mixed bases follow the International Union of Biochemistry (IUB) code (R is A or G, and Y is T or C).

  • Fig. 5.
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    Fig. 5.

    Detection of a minor virus variant by pyrosequencing which is not detected by Sanger sequencing. (A) Pyrosequencing patterns observed with primer 4 on a wild-type sample and sample F. The pattern generated for the wild type was also expected for sample F. (B) Predicted pyrosequencing pattern of sample F if composed of mixed variants (75% wild type [CAT] and 25% mutant [TAT]). (C) Sanger sequencing on sample F. Clone 1, sequencing of a clone with the sequence CAT at codon 36; clone 2, sequencing of a clone with the sequence TAT at codon 36; PCR product, direct sequencing of the PCR product revealing the sequence CAT for codon 36 with no peak representing the minor T variant observed.

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    Fig. 6.

    Graphs showing changes in HIV RNA levels and treatment in four patients. Arrows indicate samples from time points 1 to 4 that were subjected to pyrosequencing; dotted and solid boxes indicate NRTI and PI treatments, respectively. Open boxes illustrate that the patients were receiving these drugs before and/or after this 2 1/2-year period. ZDV, zidovudine; ddI, didanosine; 3TC, lamivudine; d4T, stavudine. (A) HIV RNA levels in patient 2. Pyrosequencing data show the development of drug resistance at codons 10 and 46. The amino acids involved in drug resistance are shown beneath the DNA sequence, with the approximate proportions of mixed variants at time point 2 indicated. The mixed bases follow the IUB code (M is A or C; R is A or G). (B) HIV RNA levels in patients 1, 3 and 4.

Tables

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  • Table 1.

    Pyrosequencing primers for the HIV-1 protease gene and the codons that each primer sequences

    PrimerNucleotide sequence (5′–3′)Genomic locationaSequencing directionCodons sequencedbCodons involved in drug resistancec
    1CCCTCARATCACTCTTTGGC2275–2294Forward 8–188, 10, 16
    2TACTGTATYATCWGCYCCTGT2271–2251Reverse14–2520, 23, 24
    3CTATTAGAYACAGGRGCWGA2342–2361Forward30–3530
    4TTTCCATYTYCCTGGYAAA2404–2386Reverse32–3632, 33, 36
    5AAACCTCCAATTCCCCCTAT2433–2414Reverse37–4645,46
    6dTTRCCAGGAARATGGAIRCCAAA2387–2409Forward46–5746, 47, 48, 50, 52
    7ATAGGGGGAATTGGAGGTTT2414–2433Forward54–6054, 55, 57, 60
    8TTTATCAARGTAARACARTATGA2432–2454Forward61–6863
    9CAATTATGTTGACAGGTGTAGGTCC2531–2507Reverse67–7769, 71, 73, 75, 77
    10TGRGTCAACAIRTTTCTTCCA2550–2530Reverse75–8481,82, 84
    11TACACCTRYCAACRTAATTGG2512–2532Forward87–9488,90, 91
    12TTGGAAGAAAYITGWTGACYCA2529–2550Forward93–9997
    • ↵a Genetic location (in nucleotides) refers to the MN sequence (accession no. AF075719 ).

    • ↵b Codons 1 to 7, 26 to 29, 85, and 86 were not sequenced with this set of primers.

    • ↵c The seven codons primarily involved in resistance are in boldface.

    • ↵d The G variant of primer 6 was designed as pyrosequencing failed in a viral DNA sample because of an A-C mismatch at the underlined A.

  • Table 2.

    Clinical data on patients 1 to 4

    ParameterData for patient:
    1234
    Clinical data at start of HAART
     SexMaleMaleMaleMale
     Age (yrs)42463430
     Time since diagnosis of HIV-1 infection (mos)761083712
     Prior AIDS diagnosisNoNoNoYes
     CD4 cell count100100100170
     Log plasma HIV-1 RNA copies/ml5.75.74.24.6
     Prior nucleoside analogue (NA) treatment (mos)3256339
     New NA (at least one) at start of HAARTYesNoNoNo
     PI at start of HAARTRTVIDVIDVIDV
    Clinical data at end of follow-up
     Length of follow-up after start of HAART (mos)23282730
     Time with no or partial HAART (days)1270280
     New AIDS-defining events or deathNoNoNoNo
     CD4 cell count370330280150
     Log plasma HIV-1 RNA copies/ml4.34.64.54.8
  • Table 3.

    Development of drug resistance mutations in patients 1 to 4 over a 2 1/2-year period

    PatientAmino acida (codon) at time point:PI implicatedb
    1234
    1Asp30 (GAT)Asp30 (GAT)Asn30 (AAT)1°, NFV
    2Leu10 (CTC)Leu/Ile10 (MTC)Ile10(ATC)Ile10 (ATC)2°, IDV-SQV
    Leu24 (TTA)Leu24 (TTA)Ile24 (ATA)Ile24(ATA)2°, IDV
    Met46 (ATG)Met/Ile46(ATR)Ile46 (ATA)Ile46 (ATA)1°, IDV; 2°, RTV
    Gly48 (GGG)Gly48 (GGG)Val48(GTG)Val48 (GTG)1°, SQV
    Ala71 (GCT)Ala71 (GCT)Val71 (GTT)Val71(GTT)2°, IDV-RTV-SQV
    Val77 (GTA)Val/Ile77(RTA)Ile77 (ATA)Ile77 (ATA)2°, IDV-RTV-SQV
    Val82 (GTC)Val82 (GTC)Ala82(GCC)Ala82 (GCC)1°, IDV-RTV; 2°, SQV
    Ile84 (ATA)Ile84 (ATA)Val84(GTA)Val84 (GTA)1°, RTV; 2°, SQV
    3Leu/Ile10 (MTC)Ile10(ATC)Ile10 (ATC)Ile10 (ATC)2°, IDV
    Met46 (ATG)Met46 (ATG)Ile46(ATA)Ile46 (ATA)1°, IDV; 2°, RTV
    Gly73 (GGT)Gly73 (GGT)Ser73 (AGT)Ser73(AGT)2°, IDV-SQV
    Leu90 (TTG)Leu90 (TTG)Met90 (ATG)Met90 (ATG)1°, SQV
    4Leu90 (TTG)Leu90 (TTG)Leu90 (TTG)Met90(ATG)1°, SQV
    • ↵a Resistance-associated amino acids are in boldface. Where two amino acids are listed (with their abbreviations connected by a shill), both amino acids were found at the position.

    • ↵b 1°, primary; 2°, secondary.

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Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing
Deirdre O'Meara, Karin Wilbe, Thomas Leitner, Bo Hejdeman, Jan Albert, Joakim Lundeberg
Journal of Clinical Microbiology Feb 2001, 39 (2) 464-473; DOI: 10.1128/JCM.39.2.464-473.2001

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Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing
Deirdre O'Meara, Karin Wilbe, Thomas Leitner, Bo Hejdeman, Jan Albert, Joakim Lundeberg
Journal of Clinical Microbiology Feb 2001, 39 (2) 464-473; DOI: 10.1128/JCM.39.2.464-473.2001
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KEYWORDS

Acquired Immunodeficiency Syndrome
Drug Resistance, Microbial
HIV Infections
HIV Protease
HIV Protease Inhibitors
HIV-1
Polymorphism, Genetic
Virus Replication

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