1
Supplementary Methods:
Total, integrated, and episomal 2-LTR circles were quantified by real-time quantitative PCR
(qPCR), using the methods described below. Primers and probes used in the various qPCR are
detailed in supplementary table 1. Calculated HIV DNA copy numbers were normalized for DNA
input by measurement of beta-actin by qPCR, and expressed as HIV DNA copies per 106 cells.
Approximately 500ng DNA was assayed per replicate for each qPCR. This equates to 80,000 cells,
thus the limit of detection (LOD) for each HIV DNA assay, was determine to be 1 copy per 80,000
cells or, 12.5 copies per 106 cells. The limit of quantification (LOQ) for each HIV DNA qPCR was
determined by multiplying the LOD by the lower range of the standard curve used to quantify test
samples (total and 2-LTR 125 copies or integrated 100 copies per 106 cells). The standard curves
for the individual HIV DNA qPCR are detailed below.
Total HIV DNA qPCR
Total HIV DNA was quantified by qPCR using a primer and probe set targeting the HIV gag gene.
All samples were assayed in duplicate and total HIV DNA copies determined using a set of
plasmid standards. All HIV DNA qPCR assays were performed on an iQTM5 real-time PCR
Detection System (Bio-Rad; California, USA).
Reactions contained; 12.5µl iQ Supermix, 800nM SK145 and SKCC1B, 200nM SKLNA2, 5µl
DNA template and dH2O to a final volume of 25µl. Reactions were subject to: 95°C for 3 minutes,
then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
For patient 11001 reactions contained: 12.5µl iQ Supermix, 800nM 11001 Forward and 11001
Reverse, 300nM 11001 TG Probe, 5µl DNA template, and dH2O to a final volume of 25µl.
Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 64.5°C
for 1 minute.
Standard curves were generated using the pNL4-3 plasmid, obtained through the AIDS Research
and Reference Reagent Program (catalogue number 114), Division of AIDS, NIAID, NIH from Dr.
Malcolm Martin [1]. The pNL4-3 plasmid was linearised by EcoRI (New England Biolabs;
2
Massachusetts, USA) restriction enzyme digestion, and linearisation confirmed by agarose gel
electrophoresis. Plasmid DNA concentration was determined by nano-spectrophotometry, and
plasmid copy number calculated based on the nucleotide length of the pNL4-3 plasmid, and the
weight of the individual nucleotides. 10-fold serial dilutions were made 107 to 10 copies per 5µl
and stored as 11µl aliquots at -80°C for use in individual qPCR assays.
For patient 11001: a standard curve was designed by generating a plasmid containing patient
specific PCR product spanning the region of the HIV gag gene targeted by the primer and probe
set. 10-fold serial dilutions were made from 107 to 10 copies per 5µl, and dilutions stored as 11µl
aliquots at -80°C for use in individual qPCR assays.
For use a positive control, genomic DNA was extracted from PBMC isolated from an HIV+
individual, using the QIAGEN DNeasy Blood and Tissue Kit. Extracted DNA was dispensed in
21µl aliquots and stored at -80°C for use in individual qPCR assays.
Integrated HIV DNA qPCR
Integrated HIV DNA levels were quantified by a nested qPCR [2, 3]. This assay specifically
amplifies integrated HIV DNA by utilizing the Alu repeat element of the human genome. The 1 st
round primers target the HIV ltr (L-M667) and human genomic Alu repeat elements (Alu 1 and Alu
2), to specifically amplify integrated HIV DNA. The two Alu-specific primers allow for the
amplification of integrated HIV DNA regardless of its orientation relative to the Alu repeat
elements. The 2nd round primers target the heel of L-M667 (Lambda T) and the HIV ltr (AA55M)
to specifically amplify 1st round products. The 2nd round qPCR includes a probe (LTR-FL) to
quantify HIV DNA by qPCR.
During the 1st round PCR, test samples were assayed in quadruplicate, and standards in a single
replicate. 1st round products were then measured in duplicate by the 2nd round qPCR. A standard
curve generated from the HIV infected T cell clone ACH-2 was used to calculate integrated HIV
DNA copies in test samples. For patients 5004 and 11004, individual primers, probe, and plasmid
standards were designed and optimized to compensate for differences in these patients HIV DNA
3
sequences. 1st round reactions were performed on a Veriti® Thermal Cycler (Life Technologies;
California, USA) or T100TM Thermal Cycler (Bio-Rad; California, USA). 2nd round reactions were
performed on an iQTM5 real-time PCR Detection System (Bio-Rad; California, USA).
For the standard integrated HIV DNA qPCR, 1st round reactions contained: 12.5µl iQ Supermix,
300nM Alu 1 and Alu 2, 100nM L-M667, 1µM MgCl2, 5µl DNA template, and dH2O to a final
volume of 25µl. 1st round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30
seconds, 60°C for 30 seconds then 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round
reactions contained: 12.5µl iQ Supermix, 300nM Lambda T and AA55M, 200nM LTR-FL, 1µM
MgCl2, 5µl 1st round product, and dH2O to a final volume of 25µl. 2nd round reactions were subject
to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
For patient 5004 1st round reactions contained: 12.5µl iQ Supermix, 300nM Alu 1 and Alu 2
primers, 100nM L-M667, 1µM MgCl2, 5µl DNA template, and dH2O to a final volume of 25µl. 1st
round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30 seconds, 60°C for 30
seconds and 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round reactions contained: 12.5µl iQ
Supermix, 300nM Lambda T and AA55M 2, 200nM LTR-FL 2, 1µM MgCl2, 5µl 1st round
product, and dH2O to a final volume of 25µl. 2nd round reactions were subject to: 95°C for 3
minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
For patient 11004 1st round reactions: contained 12.5µl iQ Supermix, 300nM Alu 1 and Alu 2
primers, 100nM L-M667 2, 1µM MgCl2, 5µl DNA template, and dH2O to a final volume of 25µl.
1st round reactions were subject to: 94°C for 7 minutes, 12 cycles of 94°C for 30 seconds, 60°C for
30 seconds and 72°C for 3 minutes, then 72°C for 7 minutes. 2nd round reactions contained: 12.5µl
iQ Supermix, 300nM Lambda T and AA55M 3, 200nM LTR-FL 3, 1µM MgCl2, 5µl 1st round
product, and dH2O to a final volume of 25µl. 2nd round reactions were subject to: 95°C for 3
minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
To generate a standard curve serial dilutions were made using DNA extracted from a latently HIV
infected T cell line (ACH-2). ACH-2 cells were obtained through the AIDS Research and
4
Reference Reagent Program (catalogue number 349), Division of AIDS, NIAID, NIH from Dr.
Thomas Folks [4, 5]. DNA was extracted from ACH-2 cells using the QIAGEN DNeasy Blood and
Tissue kit. DNA quantity and quality were assessed by nano-spectrophotometry. ACH-2 cells
contain 1 copy of integrated HIV DNA per cell, therefore, assuming that 50ng of DNA is equal to
the DNA content of 8,000 cells and hence contains 8,000 copies of integrated HIV DNA, 4-fold
serial dilutions were made from 50ng (8,000 copies) to 0.012ng (2 copies) per 5µl. Serial dilutions
were dispensed in 6µl aliquots and stored at -80°C for use in individual qPCR.
For patients 5004 and 11004 standard curves were designed by generating plasmids containing
patient specific PCR products spanning the HIV 3’LTR region targeted by the primer and probe
sets. 10-fold serial dilutions were made from 107 to 10 copies per 5µl, and stored as 11µl aliquots
at -80°C for use in individual qPCR. The positive control generated for the total HIV DNA qPCR
was also used as the positive control for integrated HIV DNA qPCR.
Episomal 2-LTR HIV DNA qPCR
Episomal 2-LTR HIV DNA circles were quantified using a set of primers and probe specifically
targeting the junction between the 5’ltr and 3’ltr region, unique to 2-LTR HIV DNA circles [6].
For patients 1005, 11003 and 11005, individual primer and probe sets and standards were designed
and optimized to compensate for differences in these patients HIV DNA sequences. All samples
were assayed in duplicate and 2-LTR HIV DNA copies determined using a set of plasmid
standards. Reactions were performed on an iQTM5 real-time PCR Detection System (Bio-Rad;
California, USA).
For the standard 2-LTR HIV DNA qPCR reactions containe: 12.5µl iQ Supermix, 280nM 2-LTR J
F and 2-LTR J R, 200nM 2-LTR Probe 1, 5µl DNA template, and dH2O to a final volume of 25µl.
Reactions were subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 60°C for
1 minute.
5
For patient 1005 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F3 and HIV reverse,
200nM 2-LTR Probe 2, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were
subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 62.8°C for 1 minute.
For patient 11003 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F and HIV reverse,
200nM 2-LTR Probe 1, 5µl DNA template, and dH2O to a final volume of 25µl. Reactions were
subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 65.1°C for 1 minute.
For patient 11005 reactions contained: 12.5µl iQ Supermix, 280nM 2-LTR J F and 2-LTR J R2,
200nM 2-LTR Probe 1, 5µl DNA template and dH2O to a final volume of 25µl. Reactions were
subject to: 95°C for 3 minutes, then 45 cycles of 95°C for 15 seconds and 67.2°C for 1 minute.
A standard curve was generated using a plasmid containing PCR product spanning the 2-LTR
junction. HUT78 cells, obtained through the AIDS Research and Reference Reagent Program
(catalogue number 89), Division of AIDS, NIAID, NIH from Dr. Robert Gallo [7], were infected
using pNL4-3 virus. DNA was extracted from pNL4-3 infected HUT78 cells using the QIAGEN
DNeasy Blood and Tissue Kit. A PCR product spanning the 2-LTR junction specific to 2-LTR
HIV DNA circles was amplified using this DNA. For patients 1005, 11003 and 11005, specific
standard curves were designed by generating patient specific 2-LTR junction PCR products. 10fold serial dilutions of the various plasmid standards were made from 10 7 to 10 copies per 5µl.
Dilutions were dispensed in 11µl aliquots and stored at -80°C for use in individual qPCR.
For use as a positive control: DNA was extracted from HIV (pNL4-3) infected HUT78 cells, using
the QIAGEN DNeasy Blood and Tissue kit. DNA quantity and quality was assessed by nanospectrophotometry. DNA was dispensed in 21µl aliquots and stored at -80°C for use in individual
qPCR.
Beta-Actin qPCR
Beta-actin levels in extracted DNA were quantified using the TaqMan β–actin detection kit (Life
Technologies; California, USA). The primers (Forward CGG AAC CGC TCA TTG CC, and
6
Reverse ACC CAC ACT GTG CCC ATC TA) and probe (FAM/TAMRA labelled) in this kit
target a 289bp conserved region of the β-actin exon 3 [8]. Reactions contained: 12.5µl of iQ
Supermix, 180nM of Forward and Reverse primers, 120nM probe, 5µl DNA template, and dH2O to
a final volume of 25µl. Reactions were performed using an iQTM5 real-time PCR Detection system
(Bio-Rad; California, USA) and were subject to: 1 cycle of 95°C for 3 minutes, then 45 cycles of
95°C for 15 seconds and 60°C for 1 minute. All samples were assayed in duplicate and DNA
concentration determined using a set of DNA standards.
To generate a standard curve serial dilutions were made using DNA extracted from pooled HIV
negative PBMC. PBMC were isolated from HIV negative pooled buffy coats, provided by the
Australian Red Cross Blood Service, by Ficoll-Paque separation. DNA was extracted from the
isolated PBMC using the QIAGEN DNeasy Blood and Tissue Kit, and DNA quality and quantity
were assessed by nano-spectrophotometry. 10-fold serial dilutions were made from 100ng to
0.01ng per µl, dispensed in 11µl aliquots, and stored at -80°C for use in individual qPCR.
A positive control of pooled human DNA at a concentration of 10ng/µl was provided with the
TaqMan Beta-Actin Detection Reagents Kit. The provided DNA solution was dispensed in 11µl
aliquots and stored at -80°C for use in individual qPCR assays.
7
Supplementary Table 1: Primers and probes used for qPCR quantification of HIV DNA species
Primer
Probe
qPCR
/
Sequence (5’ → 3’)
5’ mod.
3’ mod.
bp
Binding
site*
22
1359-1380
17
1402-1418
SK145
AGT GGG GGG ACA TCA AGC AGC C
SKLNA2
AT[C] A[A]T [G]AG GAA [G]CT [G]C
SKCC1B
TAC TAG TAG TTC CTG CTA TGT CAC TTC C
28
1486-1513
11001 Forward
GGT GGG GGG ACA TCA AGC AGC CAT GCA AAT
30
1359-1388
11001 TG Probe
AT[T] A[A]T [G]AG GAG [G]CT [G]C
17
1402-1418
11001 Reverse
TAC TAG TGG TTC CTG CTA TGT CAC TTC C
28
1486-1513
2-LTR J F
GCT AAC TAG GGA ACC CAC TGC TTA AG
26
9582-9607
2-LTR J F3
GCT AGC TAG AGA ACC CAC TGC TTA AG
26
9582-9607
2LTR
2-LTR Probe 1
ACA [C]A[C] A[A]G [G][T]T
6-FAM
BHQ-1
12
58-69
HIV DNA
2-LTR Probe 2
ACA [C]G[C] A[A]G [G][C]T
6-FAM
BHQ-1
12
58-69
2-LTR J R
TGG GTG GTG CCT CAA ACT AGT ACC AGT
27
128-154
HIV reverse
TGG ATG GTG CTA CAA GCT AGT ACC AGT
27
128-154
Alu 1
TCC CAG CTA CTG GGG AGG CTG AGG
24
n/a
Alu 2
GCC TCC CAA AGT GCT GGG ATT ACA G
25
n/a
L-M667
ATG CCA CGT AAG CGA AAC TCT GGC TAA CTA GGG
AAC CCA CTG
42
9579-9607
L-M667 2
ATG CCA CGT AAG CGA AAC TCT GGC TAA CTA GTG
AAC CCA CTG
42
9579-9607
Integrated
AA55M
GCT AGA GAT TTT CCA CAC TGA CTA A
25
9694-9718
HIV DNA
AA55M 2
GCT AGA GAT TTT TCT ACT TCG ATT A
25
9694-9718
AA55M 3
GCT AGA GAT TTT CCA CAC TGA CTA TC
26
9694-9719
LTR-FL
CAC AAC AGA CGG GCA CAC ACT ACT TGA
6-FAM
TAMRA
27
9634-9660
LTR-FL 2
AAC AAC AGA CGG GCA CAC ACT ACT TGA
6-FAM
TAMRA
27
9634-9660
LTR-FL 3
CAC AAC AGA TGG GCA CAC ACT ACT GA
6-FAM
TAMRA
26
9634-9659
Lambda T1
ATG CCA CGT AAG CGA AAC T
19
n/a
Total
DNA
HIV
6-FAM
6-FAM
BHQ-1
BHQ-1
Red letters represent mismatched nucleotides from patient specific qPCR; Brackets indicate locked
nucleic acids; mod = modification; 6-FAM = 6-Carboxyfluorescein; BHQ-1 = Black Hole
Quencher; TAMRA = Tetramethylrhodamine; bp = base pairs. *Relative to HIV B reference
genome (HXB2). 1Binds to the underlined heel of L-M667/LM667 #2.
8
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