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Previous Article | Next Article 
Journal of Virology, November 2001, p. 11253-11260, Vol. 75, No. 22
National Centre for HIV Virology Research, Infectious
Diseases Laboratories, Institute of Medical and Veterinary
Science,1 and Department of
Molecular Biosciences, University of Adelaide,2
Adelaide, Australia
Received 11 December 2000/Accepted 17 August 2001
We have developed a novel linker-primer PCR assay for the detection
and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies
of integrated HIV DNA, in a background of 2 × 105
cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a
near-synchronous one-step T-cell infection model to investigate the
kinetics of viral DNA accumulation following HIV infection. We report
here that integrated HIV DNA started accumulating 1 h after the
first appearance of extrachromosomal viral DNA and accounted for
~10% of the total HIV DNA synthesized in the first round of viral
replication. These results highlight the efficient nature of
integrase-mediated HIV integration in infected T cells.
Integration of newly synthesized
viral DNA into the host cell chromosome is common to all retroviruses
and is essential for a productive human immunodeficiency virus (HIV)
infection (12, 22, 28, 30). Upon reverse transcription of
the viral genomic RNA, the resulting linear DNA molecule is actively
transported to the nucleus within a complex of host and viral proteins
known as the preintegration complex, which is thought to be the
immediate precursor to the integration reaction (2, 3, 5, 13, 19,
24, 26). Analyses of the extrachromosomal and total HIV DNA
forms using both Southern hybridization and PCR-based techniques have
indicated that full-length linear DNA is first observed at
approximately 3 to 4 h postinfection (p.i.) (1, 20, 21, 23,
25). In reports on the kinetics of HIV DNA synthesis following
cell-to-cell infection, the circular forms of viral DNA were shown to
first appear at 8 h p.i., with the two long-terminal-repeat
(2-LTR) species constituting a minor population compared to the 1-LTR
and linear forms over the course of infection (1, 25).
In contrast to investigations on both free and total viral DNA forms,
little work has been performed on the accumulation of integrated DNA
within infected cells following HIV infection. This has been primarily
due to the lack of an appropriate assay which can selectively detect
and quantify integrated viral DNA, as chromosomal DNA preparations
isolated from cells infected with HIV invariably contain significant
amounts of contaminating extrachromosomal HIV DNA (1, 27, 30,
34). However, two assays able to distinguish between the
extrachromosomal and integrated HIV DNA have recently been described
and used to quantify the amounts of integrated proviral HIV DNA in
infected patients (6-10). Here we present an alternative
linker-primer PCR assay (LP-PCR) developed to specifically detect and
quantify integrated HIV DNA species. This assay utilizes the presence
of frequently occurring NlaIII restriction enzyme
recognition sites in chromosomal DNA adjacent to the integrated
provirus and at known positions within the proviral sequence. Linkers
are ligated to the DNA termini generated by NlaIII digestion
of chromosomal DNA and serve as templates from which priming can occur
in a subsequent PCR amplifying the 5'-U3 HIV region and upstream
cellular DNA sequence. In conjunction with other PCR-based assays, we
have used LP-PCR to study the kinetics of total, integrated, and 2-LTR
HIV DNA accumulation over time following a high-multiplicity infection
of HuT-78 T cells with HIVHXB2. In addition, we
also present results comparing LP-PCR to a nested Alu PCR method for
the quantification of integrated HIV DNA.
Establishment of LP-PCR for the detection and quantification of
integrated HIV DNA.
To specifically detect integrated HIV DNA in
the presence of contaminating extrachromosomal viral DNA forms, we
modified a previously described linker ligation PCR protocol used for
sequence analysis of the human T-cell leukemia virus type 1 integration junctions (32). Briefly, chromosomal DNA was digested with
the restriction enzyme NlaIII. NlaIII has a 4-bp
recognition sequence and generates a 4-bp 3' overhang to which the
specifically designed oligonucleotide linker LPNV is annealed and
ligated (Fig. 1A). This linker generates
a region from which priming can occur in a subsequent PCR using the
same linker oligonucleotide (LPNV) in conjunction with a primer (U3NV)
designed to anneal within the U3 region of the HIV LTR. Since
retroviral integration is random with respect to cellular sequences,
LP-PCR generates a population of cellular-5' HIV junction DNA sequences
of various lengths. A nested PCR was performed to generate a product of
a defined length, which was then quantified against a known set of
standards (see below).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.11253-11260.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Kinetics of Human Immunodeficiency Virus Type 1 (HIV) DNA
Integration in Acutely Infected Cells as Determined Using a Novel
Assay for Detection of Integrated HIV DNA
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FIG. 1.
LP-PCR method for detection of integrated HIV DNA. (A)
LP-PCR-mediated amplification of the integrated HIV DNA forms. The
nested PCR product was detected using the U3-106 probe fragment
(hatched box) (Table 1). (B) BglII-mediated selection
against amplification of the three main extrachromosomal HIV DNA
forms.
]) polymerase before the ligation
reaction was done. Due to the relative sizes of the restriction enzyme
recognition sites, the chances of a BglII site occurring
prior to an NlaIII site in the chromosomal sequence upstream
of the 5'LTR is once every 16 integration events. Therefore,
theoretically 94% of all integrated forms should be detectable by this technique.
The LP-PCR procedure was performed as follows: chromosomal DNA
(isolated by the method of Hirt [17, 31]) was first
digested to completion with 20 U of BglII (New England
Biolabs) in 2× OPA Plus buffer (Pharmacia) for 3 h at 37°C in a
final volume of 20 µl. Following this digestion, buffering conditions
were then altered to final concentrations of 1× OPA Plus, 20 mM
Tris-acetate (pH 7.9), 0.1 mg of bovine serum albumin (New England
Biolabs) per ml, and 1 mM dithiothreitol (Boehringer Mannheim) prior to
the addition of 10 U of NlaIII (New England Biolabs) and
incubation at 37°C for 3 h in a final volume of 40 µl. All
digestion reactions were confirmed to have proceeded to completion by
both gel electrophoresis and PCR-based assays (data not shown). Two
nucleotides (G and A) of the BglII overhang generated by
digestion were filled in with 5 U of Klenow (3'-5'
exo) (New England Biolabs) after modification
of the buffering conditions to final concentrations of 7.5 mM
dithiothreitol, 0.25 mM dGTP (Promega), and 0.25 mM dATP (Promega) and
incubation at 37°C for 30 min in a final volume of 50 µl. Samples
were then extracted with phenol-chloroform-isoamyl alcohol (25:24:1),
ethanol precipitated in the presence of glycogen (Boehringer Mannheim),
and washed in 70% ethanol prior to resuspension of the pellet in
water. Linker ligation reactions in 1× ligation buffer (New England
Biolabs) using 50 pmol (vast excess) of LPNV (Table
1) were heated to 60°C for 10 min and
snap-cooled to minimize inter- and intramolecular ligation of
NlaIII fragments, followed by the addition of 400 U of T4
DNA ligase (New England Biolabs) and incubation overnight at 16°C.
First-round PCR was performed in 1× PCR Buffer II (Perkin-Elmer), 2 mM
MgCl2, and 0.2 mM deoxynucleoside triphosphates
(dNTPs) (Promega) using 150 pmol of LPNV, 100 pmol of U3NV (Table 1),
and 5 U of AmpliTaq Gold DNA polymerase in a final volume of 100 µl.
PCRs were as follows: 95°C for 12 min; 22 cycles of 94°C for
30 s, 58°C for 30 s, and 72°C for 1 min; and 72°C for
10 min. Nested PCRs were performed on 1/100 of the first-round PCR
product in 1× PCR Buffer II (Perkin-Elmer), 1.5 mM
MgCl2, and 0.2 mM dNTPs (Promega) using 25 pmol
each of primers U3.1(+) and U3-106(
) (Table 1) and 2.5 U of AmpliTaq
DNA polymerase (Perkin-Elmer) in a final volume of 25 µl. PCRs were
cycled as follows: 94°C for 3 min; 22 cycles of 94°C for 45 s,
58°C for 30 s, and 72°C for 45 s; and 72°C for 10 min.
Amplified DNA was analyzed by subjecting 10 µl of each reaction
mixture to electrophoresis through 8% polyacrylamide gels and then
Southern transfer (electroblot apparatus) onto Hybond N+ nylon filters
(Amersham). Following denaturation and fixation using 0.4 M NaOH, the
filters were hybridized using the U3-106 probe (Table 1) in Ultrahyb
solution (Ambion). Following Southern hybridization, bands were
quantified using PhosphorImager ImageQuant analysis and a standard
curve was generated from the simultaneous PCR of known copy numbers of
standards.
|
-globin gene
(31) on six chromosomal DNA extractions from cells counted
independently. The HA8 standards were used as copy number controls for
quantifying total HIV DNA, integrated HIV DNA, and the -globin
content of samples. HuT-78 (15) chromosomal DNA was used
as background DNA. To confirm that all four integration sites within
the HA8 cell mix could be amplified by LP-PCR, the chromosomal sequence
immediately upstream of the 5' HIV LTR region of the integrated
provirus(es) present in H3B, ACH-2, and 8E5 cells was obtained. In all
cases, an NlaIII site preceded the BglII site in
the flanking sequence (data not shown).
By comparison with the HA8 composite integrated HIV DNA standard,
LP-PCR was shown to routinely detect 20 copies of integrated HIV-1 DNA
in a background of 500 cell equivalents of HuT-78 chromosomal DNA (Fig.
2A). In addition, we were able to
reproducibly detect 10 copies of the HA8 integrated standard in the
presence of 2 × 105 cell equivalents of
HuT-78 chromosomal DNA (1.2 µg) when elevated nested-PCR cycle
numbers were used (data not shown). Furthermore, amplification of a
construct precisely mimicking the linear viral DNA form spiked onto 1.2 µg of HuT-78 DNA routinely resulted in a signal intensity equivalent
to ~7.5% of that generated by an equivalent HIV DNA copy number of
the HA8 integrated standard. This result indicated that LP-PCR was
approximately 15-fold more specific for the integrated than
extrachromosomal HIV DNA forms (data not shown). Sample heating and
snap-cooling in the presence of a vast excess of the linker prior to
the ligation reaction (to minimize intermolecular NlaIII
fragment ligation), as well as the use of a hot-start PCR (to fully
dissociate NlaIII fragments and inhibit linker-mediated
amplification of all NlaIII fragments), were found to be
critical to the success of this assay (data not shown). Furthermore,
the efficiency of linker ligation to NlaIII termini was
demonstrated to be approximately 100% (data not shown).
|
) primer. Consequently, amplification between Alu elements upstream of the integration site in 8E5 cells, instead of amplification between the Alu164 and the
PBS-659() primers, would have occurred. In contrast, the analogous
Alu element in ACH-2 chromosomal DNA was present in the correct
orientation for successful amplification with the PBS-659() primer
(data not shown). We therefore propose that the nested Alu PCR
technique allows amplification of only those integrants inserted at
chromosomal sites immediately adjacent to an Alu element present in an
orientation opposite to that of the PBS-659() primer. Statistically,
then, the nested Alu PCR approach would be expected to successfully amplify at best half of all integrated proviral forms. Consequently, we
believe that the LP-PCR assay is a potentially more appropriate protocol for detecting integrated HIV DNA. A comprehensive comparison between LP-PCR, nested Alu PCR, and an alternative assay currently being developed in our laboratory to detect integrated proviral forms
based on the use of degenerate primers will be published elsewhere.
Kinetics of HIV-1 DNA integration following a one-step viral infection of HuT-78 cells. To investigate the kinetics of viral DNA accumulation following infection, a highly synchronous one-step infection of HuT-78 cells with cell-free HIV at a multiplicity of infection of 1 TCID50 per cell was performed as previously described (31). Extensive washing of cells to remove residual noninternalized virus prior to plating minimized the chance of any additional infection events occurring after the initial infection period. Viral release into the culture supernatant following infection (as measured by P24 release using a commercial kit [NEN]) was evident by 26 h p.i., indicating that one round of replication was complete by this stage (see Fig. 4A). Consequently, the proportions of each viral DNA form assessed following infection were calculated at 26 h p.i. to ensure that the results obtained were not skewed by secondary cell-free (or cell-to-cell) infection events.
DNA was extracted by the method of Hirt (17, 31) from infected cells harvested at various time points following infection to ensure that the bulk (>80%) of extrachromosomal forms were separated from the chromosomal DNA forms. Chromosomal DNA preparations were subjected to PCR amplification of the-globin gene to determine the
cell equivalent DNA content of each sample. Samples were volume adjusted based on the results of initial -globin PCR quantification, and upon reanalyses, little variation between samples was observed (Fig. 3D). Extrachromosomal DNA
preparations were equalized between samples based on a semiquantitative
PCR assay (31) measuring the mitochondrial complement of
this fraction. The mitochondrial DNA PCR results showed only minor
variation between all samples, and therefore adjustment was not
necessary (Fig. 3E).
|
|
) (Table 1) in
appropriate amounts of background DNA (data not shown). Quantification
of 2-LTR circular DNA following infection was achieved by performing
PCR on 500 cell equivalents of total DNA in 1× PCR Buffer II
(Perkin-Elmer), 1.5 mM MgCl2, and 0.2 mM dNTPs
using 25 pmol each of primers R7 and U3PNV and 1.5 U of AmpliTaq Gold DNA polymerase. Reactions were cycled as follows: 94°C for 12 min; 26 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for
45 s; and 72°C for 10 min. The initial results (Fig. 3, panel
C.i) clearly showed continuing 2-LTR DNA accumulation from
7 h p.i. onwards, which is in close agreement with previous
studies (21). An unexpectedly low value was obtained for
the 26-h-p.i. sample. Since this time point was to be used for our
endpoint analysis, we reanalyzed this sample and the adjacent time
points using 1,000 cell equivalents of DNA (Fig. 3, panel C.ii). Taken
together, these results showed that the 2-LTR viral DNA form is a minor species compared to the integrated DNA form, with levels of ~0.4 copy/cell (representing ~1% of the total viral DNA species) at 26 h p.i. (Fig. 4B).
In our infection model, nearly full-length DNA species were first
detected at 3 h p.i., with the appearance of integrated forms at
4 h p.i. Integrated HIV DNA as measured by LP-PCR was found to
comprise approximately 10% of the total viral DNA synthesized following one round of infection (Fig. 4B). While we believe such levels to represent an efficient process, care should be taken when the
integration efficiencies observed in cell-free T-cell infection systems
are used for predicting the integration efficiencies in vivo. Our
infection model involves the use of actively growing T cells and a high
multiplicity of infection. Consequently, this model gave rise to a
large number of viral DNA molecules, which might be expected to compete
for cellular factors involved in a variety of early events in HIV
replication, including integration. It is possible that in HIV-infected
patients, where these factors might not be as limiting, the efficiency
of HIV integration would be higher as measured by the amounts of
integrated viral DNA expressed as a percentage of the total viral DNA.
Furthermore, major differences exist in vivo with respect to not only
the various activation states of T cells but also the cell type(s)
infected. Cells of the macrophage/monocyte lineage are generally
considered to be nondividing cells, and the early events in infection
of these cells differ markedly from those observed in proliferating T
cells (29, 33). Thus, the kinetics of integration within
the monocyte/macrophage cell lineage should be considered in a separate study.
In conclusion, we have established a system in which the amounts of
integration can be measured over the course of an infection with HIV-1.
We have also demonstrated that HIV-1 integration is a rapid and
relatively efficient process under one-step infection conditions and
defined the levels of total, integrated, and 2-LTR HIV DNA forms during
the course of infection. While this article was being revised, a short
report was published that supports the conclusions of this work by
demonstrating similar frequencies of proviral integration into host
chromosomal DNA following a one-step, cell-free infection model
(4). Studies are now under way to investigate the
mechanisms of integration and the efficiency with which potential
integrase inhibitors affect the accumulation of integrated proviral DNA
in infected cells under similar infection conditions.
| |
ACKNOWLEDGMENTS |
|---|
We thank Linda Mundy and Helen Hocking for preparing the viral stocks and Melissa Egberton and Steven Young (Merck and Co.) for the sample of L-731,988 used in this study.
This work was supported by the Australian Commonwealth AIDS Research Grant Programme.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: National Centre for HIV Virology Research, Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Rd., Adelaide 5000, Australia. Phone: 61 8 82223544. Fax: 61 8 82223543. E-mail: peng.li{at}imvs.sa.gov.au.
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), integrated (
), and 2-LTR (×) HIV DNA forms. Data were
determined by PhosphorImager quantification of bands in Fig.