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Sequencing-Based Detection of Low-Frequency Human Immunodeciency Virus Type 1 Drug-Resistant Mutants by an J
OURNAL OF
V
IROLOGY
, July 2004, p. 71127123
Vol. 78, No. 13
0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.13.71127123.2004
Copyright � 2004, American Society for Microbiology. All Rights Reserved.
Sequencing-Based Detection of Low-Frequency Human
Immunodeciency Virus Type 1 Drug-Resistant Mutants by an
RNA/DNA Heteroduplex Generator-Tracking Assay
Amit Kapoor,
1,2
Morris Jones,
1,2
R. W. Shafer,
3
Soo-Yon Rhee,
3
Powel Kazanjian,
4
and Eric L. Delwart
1,2
*
Department of Medicine, University of California San Francisco,
1
and Blood Systems Research Institute,
2
San Francisco, and Department of Medicine, Stanford University, Stanford,
3
California, and
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan
4
Received 19 November 2003/Accepted 20 February 2004
Drug-resistant viruses may be present as minority variants during early treatment failures or following
discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population se-
quencing method is its inability to detect human immunodeciency virus type 1 (HIV-1) variants present at
frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing
of minority HIV protease variants is presented here. A multiple-codon-specic heteroduplex generator probe
was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated
with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of
protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease
gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21
HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or
following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the rst episode of
virologic failure identied protease resistance mutations not seen by population-based PCR sequencing. In 8
of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during
the rst episode became detectable by population-based PCR sequencing at the later time point. Distinct sets
of protease mutations could be linked on different genomes in patients with high-frequency protease gene
lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may
improve the efcacy of genotype-assisted salvage therapies.
A frequent cause of treatment failure in human immunode-
ciency virus type 1 (HIV-1)-infected persons is the emergence
of viruses resistant to antiretroviral (ARV) drugs. A number of
studies have shown that viral drug resistance genotyping can
improve virologic outcome (6, 9, 10, 22, 74). Resistance to
ARV drugs can be determined by identifying primary drug
resistance mutations known to confer increased resistance to
specic ARV drugs and secondary drug resistance mutations
that further increase resistance and can improve the replicative
tness of viruses carrying primary drug resistance mutations
(25). Recent studies have also indicated that the presence of
minority drug-resistant variants may also be an independent
predictor of virological failure (37, 40). This may be particu-
larly relevant in persons in whom drug-resistant variants are
only beginning to emerge or who have discontinued treatment
and whose drug-resistant variants become displaced by preex-
isting tter wild-type variants (14, 40).
Sequence-based genotyping can be performed either by di-
rect PCR product sequencing (also called population-based or
bulk sequencing) or by sequencing multiple subclones derived
from a PCR product. Direct PCR sequencing is primarily used
in the clinical setting, but one of its major limitations is its
inability to consistently detect minority variants present at fre-
quencies below 10 to 25% (47, 49, 64, 76). The presence of
mixed bases in clinical samples is also largely responsible for
discordant results when the same samples are analyzed in dif-
ferent laboratories or using different nonsequencing methods
(20, 28, 36, 38, 66). The laborious nature of sequencing mul-
tiple plasmid subclones, where the major variant may be rese-
quenced multiple times (50), largely restricts this approach to
research settings (3, 11, 30, 36, 38, 42, 48, 54, 56, 57, 62).
To increase the sensitivity of current sequencing-based geno-
typing methods, we developed a method for the separation and
sequencing of minority drug-resistant variants. We present
here this method and its application, using clinical samples
from persons in whom HIV-1 developed new drug resistance
mutations while on a failing treatment regimen(s), and we
compare the results to direct PCR population sequencing.
MATERIALS AND METHODS
Synthesis of the HIV-1 protease gene universal heteroduplex generator
(UHG).
The DNA template used for synthesis of the RNA probe was synthesized
by assembling 18 oligonucleotides (each 30 to 48 nucleotides long) into a highly
mutated version of the HIV-1 protease gene. Gene assembly was carried out as
described elsewhere, with minor modications (70). A 250 M concentration of
each oligonucleotide was mixed, and the mixture was subsequently diluted 100-
fold in 50 l of a PCR buffer containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl,
2.5 mM MgCl
2
, 0.1% Triton X-100, a 2.5 mM concentration of each de-
oxynucleoside triphosphate, 3.5 U of Taq polymerase, and 0.05 U of Pfu poly-
merase (Promega, Madison, Wis.). The PCR program consisted of 50 cycles of
94�C for 30 s, 50�C for 30 s, and 72�C for 30 s. The oligonucleotides used were
the following (5 to 3 ): PF1, GAAGCAGGAGCCGATAGACAAGGAAC
TGTATCCTTTAACT; PF2, TCCCTCAGATCACTCTTTGGCAACGACCG
CTCGTCACAAT; PF3, AAAGATAGGGGGGCAACTAAAGGAAGCTCT
* Corresponding author. Mailing address: Department of Medicine,
UCSF, 270 Masonic Ave., San Francisco, CA 94118. Phone: (415)
923-5763. Fax: (415) 791-4220. E-mail: delwarte@medicine.ucsf.edu.
7112 ATTAGATACA; PF4, GGAGCAGATCGATACTGTATTAGAACAAATG
AATTTGCC; PF5, AGGAAGATGGAAACCAAAAAAGATAGGCGGGA
AATGGA; PF6, GGTTTTAATCAAAGTAAGACAGTATGATCAGATAC
TCATA; PF7, GAAATCTGTGGACATAAAGCATTAGGTACAGTATTA
GTAG; PF8, GACCTACACCTGATCAACAATAATTGGAAGTAATCTG
TCTGACTC; PF9, AGATTGGTTGCACTTTAAATTTTCCCATTAGCCC
TATTGAGACTGTACCAG; PR1, CTGGTACAGTCTCAATAGGGCTAA
TGGGA; PR2, AAATTTAAAGTGCAACCAATCTGAGTCAGACAGATTA
CTTCCAA; PR3, TTATTGTTGATCAGGTGTAGGTCCTACTAATACTGT
ACCTAATGCTTTATG; PR4, TCCACAGATTTCTATGAGTATCTGATC
ATACT; PR5, GTCTTACTTTGATTAAAACCTCCATTTCCCGCCTATC
TTTTT; PR6, TGGTTTCCATCTTCCTGGCAAATTCATTTCTTCTAAT
ACA; PR7, GTATCGATCTGCTCCTGTATCTAATAGAGCTTCCTTTAG;
PR8, TTGCCCCCCTATCTTTATTGTGACGAGCGGTCGTTG; and PR9,
CCAAAGAGTGATCTGAGGGAAGTTAAAGGATACAGTTCCTTGTCTA
TCGGCTCCTGCTTC. After the initial gene assembly PCR, the reaction mix-
ture was diluted 40-fold in 100 l of the same PCR buffer, with deoxynucleoside
triphosphates plus 10 pmol of each anking primer: EDPR3, GAAGCAGGAG
CCGATAGACAAGG (HXB2 positions 2211 to 2233); EDPR4, CTGGTACA
GTTTCAATAGGACTAATGG (HXB2 positions 2551 to 2577). The second
PCR program consisted of three cycles of 94�C for 45 s, 57�C for 45 s, and 72�C
for 45 s, followed by 34 cycles of 94�C for 30 s, 57�C for 30 s, and 72�C for 30 s,
and nal extension at 72�C for 5 min. A 100- l aliquot of the PCR product was
run in a 1.5% agarose gel, and the 360-bp band was puried (QIAQuick gel
extraction kit; QIAGEN Inc.). The mutated protease gene DNA fragment was
subcloned into the pGEM-T plasmid vector (Promega), and 10 subclones were
sequenced using M13 reverse primer. One subclone with the expected sequence
was used in all subsequent experiments. The resulting plasmid (pAK1-
pro1UHG), necessary for generating the protease RNA probe, is available upon
request.
RNA probe synthesis.
The mutated protease gene insert of pAK1-pro1UHG
was amplied from 10 ng of plasmid as above but for only 20 PCR cycles with
primers EDPR3 and T7-EDPR4 (T7 RNA polymerase promoter region [TAA
TACGACTCACTATAGGG] added to the 5 end of primer EDPR4). A 100- l
aliquot of the PCR product was run in a 2% agarose gel, and the 370-bp band was
puried from the gel. One microliter of the puried PCR product was added to
100 l of transcription mixture containing 20 l of 5 transcription buffer, 10 l
of 0.1 M dithiothreitol, 20 l of 2.5 mM (each) ribonucleoside triphosphate, 100
U of human placental RNase inhibitor, and 45 U of T7 RNA polymerase. The
mixture was incubated for in vitro transcription at 37�C for 90 min, followed by
70�C for 15 min to inactivate the enzyme.
Virus isolates.
The plasma samples of persons undergoing direct PCR popu-
lation sequencing at Stanford University Hospital Diagnostic Virology Labora-
tory meeting the following criteria were chosen for further evaluation: (i) two
genotypes were performed within the course of 1 year, each following virologic
failure on a protease inhibitor-containing regimen; and (ii) the second genotype
contained at least one major protease inhibitor resistance mutation that was not
observed in the direct PCR-based sequence at the time of t