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Analysis of nevirapine resistance mutations in cloned hiv type 1 variants from hiv-infected ugandan infants using a single-step amplification-sequencing method (ampliseq)AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 24, Number 9, 2008 Mary Ann Liebert, Inc.
DOI: 10.1089/aid.2008.0109 Sequence Note
Analysis of Nevirapine Resistance Mutations in Cloned HIV Type 1 Variants from HIV-Infected Amplification-Sequencing Method (AmpliSeq) William Ian Towler, Jessica D. Church, James R. Eshleman, Mary Glenn Fowler, Laura A. Guay, J. Brooks Jackson, and Susan H. Eshleman Abstract
We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma sampleswere obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by pop-ulation sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplifi-cation-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 vari-ants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealednumerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistancemutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
SINGLE DOSE (SD) NEVIRAPINE (NVP) and other NVP-con- variants. This information can be obtained by sequencing vi-
taining regimens are used in resource-limited settings for ral genomes after cloning6 by single genome sequencing prevention of HIV-1 mother-to-child transmission. These (SGS),7 by ultradeep pyrosequencing,8 or by parallel allele- regimens are effective, but are associated with the emergence specific sequencing (PASS).9 Of these methods, the first three and persistence of NVP-resistant HIV-1 variants in some (traditional cloning/sequencing, SGS, and ultradeep py- women and in some infants who are HIV infected despite rosequencing) provide sequence information. In contrast, prophylaxis.1 Low-level NVP-resistant HIV-1 variants can PASS provides information for individual codons (point mu- persist 2 and may affect treatment outcome.3 tations). For each method, the sensitivity of mutation detec- Several methods can be used to analyze NVP resistance tion depends on the number of HIV-1 templates analyzed.
mutations. However, each method has some limitations.
Pyrosequencing and PASS require specialized equipment, HIV-1 genotyping methods based on population (bulk) se- but can be used to screen large numbers of templates, en- quencing are relatively insensitive for the detection of low- hancing the sensitivity of mutation detection.
level HIV-1 variants with resistance mutations. Point muta- In this report, we used a single-step amplification-se- tion assays, such as allele-specific polymerase chain reaction quencing reaction (AmpliSeq)10 to analyze the genetic link- (PCR), the LigAmp, and the oligonucleotide ligation (OLA), age of NVP resistance mutations and genetic complexity at are more sensitive than routine genotyping assays, but may HIV-1 reverse transcriptase codons associated with NVP re- be labor intensive if multiple mutations are analyzed.4,5 Vari- sistance in six 6-week-old Ugandan infants who were HIV- ations in the HIV-1 nucleotide sequence at oligonucleotide 1 infected despite SD NVP prophylaxis and who had two or or primer binding sites can interfere with hybridization, lig- more NVP resistance mutations detected by population se- ation, and/or priming in these assays.
quencing (Table 1). The samples were collected in three clin- In addition to the limitations mentioned, the assays de- ical trials conducted in Kampala, Uganda: (1) the HIVNET scribed above do not provide information about the genetic 012 trial,11,12 (2) the Repeat Pregnancy Study,13 and (3) the linkage of NVP resistance mutations in individual HIV-1 Breast Feeding Study (“Pathobiology of Breast Milk among Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
TOWLER ET AL.
aThe table shows the source of samples analyzed (Clinical study), the mutations detected by the ViroSeq system, and the HIV-1 subtype (pol region).
bNote that mutations K103R and V179D are associated with NVP resistance only when they HIV-1 Infected Ugandan Women Receiving Intrapartum TABLE 2. NVP RESISTANCE MUTATIONS DETECTED Nevirapine”). HIV-1 genotyping was performed using the ViroSeq HIV Genotyping System (ViroSeq, Celera Diagnos- tics, Alameda, CA) using 0.1 ml of infant plasma. The HIV- 1 subtype of each sample was determined by phylogeneticanalysis of pol region sequences.
DNA cloning was accomplished as follows. Using the ViroSeq system, RNA was extracted from plasma, and the HIV-1 RNA was reverse transcribed to generate comple- K103N ϩ Y188C
mentary DNA. Pol region DNA (encoding the 3Ј portion of HIV-1 gag, HIV-1 protease, and the 5Ј portion of HIV-1 re- verse transcriptase) was amplified using Research Use Only PCR mix provided by Celera Diagnosics, which was identi- cal to the PCR mix included in the ViroSeq system, except that dTTP was substituted for dUTP. Amplification was per- V106A ϩ Y188C
formed without dUTP, because dUTP-containing PCR prod- ucts cannot be cloned using commercially available compe- tent cells. The PCR products were purified using spin columns, and were incubated with Taq polymerase and dNTPs for the addition of terminal adenosine-phosphate groups, and transformed into TOP 10 electrocompetent Esch- erichia coli cells (Invitrogen Corp., Carlsbad, CA). Colonies K103N ϩ Y181C
were isolated on plates containing ampicillin. S-Gal (Sigma Corp., St. Louis, MO) was used to indicate the presence of a DNA insert in the plasmid vector. White colonies were se- K101E ϩ Y181C
K103R ؉ V179Db
AmpliSeq uses commercially available DNA sequencing reagents supplemented with additional dNTPs for the com- bined amplification/sequencing reaction. Two oligonu- cleotides are added to each reaction, with one in molar ex- cess. During the initial reaction cycles, the low-concentration oligonucleotide and the dNTPs are incorporated into ampli- fication products. In later reaction cycles, DNA sequencing K103R ؉ G190Ab
termination products (generated by priming from the high- concentration oligonucleotide and incorporating ddNTPs) predominate. To perform the analysis, a small inoculum (barely visible on a pipette tip) of each bacterial colony wasadded directly to a 20 l reaction containing 4 l Reaction aFifty HIV-1 clones were analyzed by AmpliSeq from each infant Ready Mix (Applied Biosystems, Foster City, CA), 1ϫ Se- sample. The number of clones with 0, 1, or 2 mutations is shown.
quencing Buffer (Applied Biosystems), 0.15 mM dNTPs, 0.05 Mutations shown in bold were detected using the ViroSeq HIV-1 Genotyping System (population sequencing). The following amino M forward primer (5Ј-AGATTTCAGGGAACTCAATAA- acid polymorphisms (not shown in this table) were also detected in the AAGAACTCA-3Ј), and 0.25 M reverse primer (5Ј-GGTTC- samples at codons in HIV-1 reverse transcriptase that are the sites of TTTCTGATGCTTTTTGTCTGGTGT-3Ј). These primers were NVP resistance mutations: 147770: V179A (1 clone), V106S ϩ V108S (1 designed to bind to regions of HIV-1 that are conserved in clone). 147858: V181H (1 clone). 274581: L100V ϩ A98C (1 clone).
288407: K103R (50 clones), V179E (26 clones). 289005: V179I (49 clones), HIV-1 subtypes A, C, and D. The AmpliSeq reaction was per- V179M (1 clone), K103E ϩ A98S (2 clones). 324698: K101R (1 clone).
formed using an Applied Biosystems 9700 thermal cycler as bNote that the mutations K103R and V179D are associated with follows: 95°C for 5 min, followed by 25 three-step cycles of NVP resistance only when they occur together.
ANALYSIS OF NVP RESISTANCE IN HIV-1 CLONES
94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by mutations (Table 2). For each infant, NVP resistance mu- 40 three-step cycles of 95°C for 15 s, 50°C for 15 s, and 60°C tations that were identified in the population sequence 4 min. The reactions were ethanol precipitated, resuspended were also identified in one or more of the 50 clonal se- in 20 l of HiDi formamide (Applied Biosystems), and ana- quences. Clones from five of the six infants also had NVP lyzed on an ABI PRISM 3100 Genetic Analyzer (Applied resistance mutations that were not identified by population Biosystems). Fifty sequences were analyzed for each infant sequencing. Clones with two genetically linked NVP resis- tance mutations were identified in five of the six infants HIV-1 sequence data generated using the AmpliSeq method were analyzed using BioEdit Sequence Alignment We next analyzed the nucleotide sequences of the 50 Editor.14 Sequences included nucleotides encoding HIV-1 re- clones from each infant to determine which codons were verse transcriptase amino acids 95–195. Sequences were ex- present at positions of NVP resistance mutations (Table 3).
amined for the following mutations: A98G, L100I, K101E/P,
Up to six different amino acids were detected at a single po- K103N/S/Q/T, K103RϩV179D, V106A/M, V108I, E138K,
sition (e.g., at codon 179). Furthermore, at 5 of the 10 posi- V179D/E/F, Y181C/I/V, Y188C/H/L, and G190A/E/S/Q. Mu-
tions analyzed, we detected two different codons encoding tations shown in bold are associated with reduced NVP sus- the same amino acid (e.g., GCA and GCG for Ala at codon ceptibility; mutations in bold and italic are associated with 98). In one infant, all 50 clones had K103R, and 24 of those high-level phenotypic NVP resistance and/or reduced viro- clones had V179D; these two mutations are associated with logic response to a clinical regimen.15 DNAStar MegAlign NVP resistance only when they occur together.15,16 Exclud- (DNAStar, Inc., Madison, WI) was used to generate sequence ing K103R and V179D in that infant, the portion of clones alignments. Phylogenetic trees based on 500 bootstraps were that had NVP resistance mutations varied from one posi- generated using DNAStar MegAlign (DNAStar, Inc., Madi- tion to another (from 0 to 140 clones out of 300 clones ex- son, WI). The 50 clonal HIV-1 sequences from each infant grouped together with the corresponding maternal se- The methods described in this report can be performed us- ing DNA remaining from HIV-1 genotyping, which is ideal We first analyzed the amino acid sequences from the 50 for analysis of samples that are limited in volume, such as clones isolated from each infant sample for NVP resistance those from pediatric studies. We were able to obtain read TABLE 3. CODONS DETECTED IN CLONES AT POSITIONS IN HIV-1 REVERSE TRANSCRIPTASE ASSOCIATED WITH NVP RESISTANCEa Arg Glu Glu
Lys Asn Asn Argb Glu Val
GCA GCG TGC AGC CTA TTA GTA AAA AGA GAA GAG AAG AAA AAC AAT AAG GAA GTA GCA AGT
GTA GTG ATA AGT GTT GCT GAT ATT GAA ATG TAT TGT CAT TAT TGT GGA GCA
aFifty HIV-1 clones were analyzed from each of the six infant samples (300 clones total). Sample numbers are indicated on the left. NVP re- sistance mutations occur at codons 98, 100, 101, 103, 106, 108, 179, 181, 188, and 190 in HIV-1 reverse transcriptase. The table shows the nucle-otide sequences and corresponding amino acids detected in clones from each infant sample at each position. The number of clones with eachnucleotide sequence is indicated. Reference sequences/amino acids (those found in reference strain HXB2) are in italics. Sequences/muta-tions associated with NVP resistance are shown in bold.
bNote that mutations K103R and V179D are associated with NVP resistance only when they occur together.
TOWLER ET AL.
lengths of approximately 300 bases with a single AmpliSeq (DHHS). NIH, DHHS (U01-AI-068613), (2) the International reaction, enabling us to analyze the two major regions where Maternal Pediatric Adolescent AIDS Clinical Trials Network NVP resistance mutations occur (i.e., at codons 98–108 and of the NIAID (U01-AI-068632), (3) the HIV Network for Pre- codons 179–190). AmpliSeq has been optimized for read vention Trials (HIVNET) and sponsored by NIAID, NIH, lengths of approximately 500 bases using purified human DHHS, through contract U01-AI-046749 with Family Health and bacterial genomic templates.10 The methods described International, contract U01-AI-046745 with Johns Hopkins in this report also produce cloned plasmids, which can be University, and (4) the Centers for Disease Control and Pre- used for further characterization of specific HIV-1 variants.
vention, Atlanta, GA, including contract number 200-2004- The samples used in this study were selected from six in- M-09279 to Johns Hopkins University. None of the authors fants who had two or more NVP resistance mutations de- has a commercial or other association that might pose a con- tected by population sequencing. Clonal analysis revealed flict of interest with the following exception: Dr. James Esh- that five of the six infants had some HIV-1 variants with two leman (spouse of Susan H. Eshleman) is a co-inventor of the or more genetically linked mutations. HIV-1 variants with AmpliSeq assay and Johns Hopkins University has filed a multiple NVP resistance mutations may have altered fitness, patent application with the US-Patent and Trademark Office.
different patterns of cross-resistance to other nonnucleoside The inventors may receive royalty payments if the applica- reverse transcriptase inhibitors compared to HIV-1 variants with single NVP resistance mutations.15 With the exceptionof one infant who had the K103R polymorphism in all clones References
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