JOURNAL OF VIROLOGY, June 2004, p. 6122–6133
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 12
Resting CD4?T Cells from Human Immunodeficiency Virus Type 1
(HIV-1)-Infected Individuals Carry Integrated HIV-1 Genomes
within Actively Transcribed Host Genes
Yefei Han,1† Kara Lassen,1† Daphne Monie,1Ahmad R. Sedaghat,1Shino Shimoji,1Xiao Liu,1
Theodore C. Pierson,1Joseph B. Margolick,2Robert F. Siliciano,1,3* and Janet D. Siliciano1
Department of Medicine, School of Medicine,1and Department of Molecular Microbiology and Immunology,
School of Public Health,2Johns Hopkins University, and Howard Hughes Medical
Institute,3Baltimore, Maryland 21205
Received 19 January 2004/Accepted 17 February 2004
Resting CD4?T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals
include cells with integrated HIV-1 DNA. In individuals showing suppression of viremia during highly active
antiretroviral therapy (HAART), resting CD4?T-cell populations do not produce virus without cellular
activation. To determine whether the nonproductive nature of the infection in resting CD4?T cells is due to
retroviral integration into chromosomal regions that are repressive for transcription, we used inverse PCR to
characterize the HIV-1 integration sites in vivo in resting CD4?T cells from patients on HAART. Of 74
integration sites from 16 patients, 93% resided within transcription units, usually within introns. Integration
was random with respect to transcriptional orientation relative to the host gene and with respect to position
within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that
were actively expressed in resting CD4?T cells, as directly demonstrated by reverse transcriptase PCR
(RT-PCR). These results predict that HIV-1 sequences may be included in the primary transcripts of host
genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence of HIV-1 sequences
within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results
demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4?T cells in vivo.
Human immunodeficiency virus type 1 (HIV-1) replicates
preferentially in activated CD4?T cells (44). However, at any
given time, most of the CD4?T cells in the body are in a
profoundly quiescent state that is not fully permissive for viral
replication. In resting CD4?T cells, replication is restricted at
several different steps in the virus life cycle. For R5 viruses,
entry is restricted due to the absence of CCR5 on most resting
CD4?T cells, particularly those belonging to the naı ¨ve subset
(48, 52). Reverse transcription is kinetically restricted in rest-
ing CD4?T cells (49, 69) and can take as long as 3 days to
complete (49). There is also a block at the level of nuclear
import of the preintegration complex (PIC) (9). Despite the
karyophilic properties of the PIC that allow nuclear import in
some nondividing cells, such as macrophages (8, 23, 27, 70),
import does not readily occur in resting CD4?T cells, possibly
because of the absence in resting lymphocytes of sufficient
levels of ATP to drive the import of large structures, such as
the PIC (9, 38). Thus, the infection does not proceed to the
point of integration. As a result, in untreated patients, most of
the HIV-1 DNA in resting CD4?T cells is linear, unintegrated
viral DNA localized to the cytoplasm (9, 10, 14). This uninte-
grated HIV-1 DNA represents a labile, inducible reservoir for
the virus; if a T cell is activated before the PIC decays, then
subsequent steps in the life cycle can occur and virus particles
can be produced (10, 38, 49, 61, 69).
Although direct infection of resting CD4?T cells does not
generally proceed to integration, low levels of integrated
HIV-1 DNA can be demonstrated in purified resting CD4?
T-cell populations from infected individuals (14, 16). This ap-
parent paradox can be explained by assuming that resting
CD4?T cells with integrated HIV-1 DNA are actually derived
from activated CD4?T cells that became infected and then
reverted back to a resting memory state. Reversion to a mem-
ory state is a normal physiologic process that generates the
long-lived cells required for immunologic memory (reviewed in
reference 34). Most of the resting CD4?T cells that carry
integrated HIV-1 DNA in vivo have a memory phenotype (6,
14, 48). The presence of integrated HIV-1 DNA in long-lived
memory T cells provides a mechanism for long-term viral per-
sistence even in patients who are on highly active antiretroviral
therapy (HAART) and who show complete suppression of
detectable viremia (13, 20, 21, 47, 57, 67).
Resting CD4?T-cell populations from patients on HAART
generally do not release virus unless the cells are stimulated in
some manner (12, 14, 16, 28). Even when reverse transcriptase
(RT) PCR assays sensitive to 50 copies of HIV-1/ml are used,
the production of HIV-1 virions cannot be detected (12). Some
form of activating stimulus is needed to induce HIV-1 gene
expression from infected resting CD4?T cells. Treatment of
resting CD4?T cells with mitogens (13, 21, 67), cytokines (15,
56, 65), or phorbol esters (37, 40) can induce virus production.
HIV-1 nef can induce macrophage production of factors that
increase the permissivity of resting CD4?T cells to productive
* Corresponding author. Mailing address: Department of Medicine,
Ross 1049, School of Medicine, Johns Hopkins University, 720 Rut-
land Ave., Baltimore, MD 21205. Phone: (410) 955-2958. Fax: (443)
287-6218. E-mail: firstname.lastname@example.org.
† Y.H. and K.L. contributed equally to this work.
infection (63). In vivo, activating stimuli may be supplied in the
context of lymphoid tissue microenvironments (18, 63, 71), par-
ticularly in patients who are viremic. Viremia is associated with
increased levels of T-cell activation, and in viremic patients, in-
fected CD4?T cells with a resting phenotype display abnormal
patterns of gene expression that may allow some degree of virus
production (12, 71). Nevertheless, in patients who are on
HAART and who have shown prolonged suppression of viremia,
purified resting CD4?T cells from the blood produce little if any
virus without stimulation.
The presence of integrated viral genomes in cells that do not
produce virus raises the question of how virus production is
curtailed in these cells. Clearly, some cells may contain viral
genomes that are defective. Among resting CD4?T cells, the
frequencies of cells harboring replication-competent HIV-1
that can be rescued by cellular activation are on the order of
10?6to 10?7, at least 100-fold lower than the frequencies of
cells harboring integrated HIV-1 DNA (13, 14). These findings
suggest that most of the integrated HIV-1 DNA in resting
CD4?T cells exists in an irreversibly nonproductive state,
while a minor fraction exists in a reversibly nonproductive
(latent) state. Although posttranscriptional regulatory mecha-
nisms for limiting virus production have been proposed (43,
50), it is difficult to demonstrate functional HIV-1 mRNAs,
particularly multiply spliced RNAs, in rigorously purified pop-
ulations of resting CD4?T cells from infected individuals on
HAART (28). The bulk of the evidence suggests that the
absence of virus production by resting CD4?T cells with in-
tegrated HIV-1 DNA is due to factors affecting transcriptional
initiation (5, 17, 24, 32, 33, 45, 64) or elongation (1, 25, 29–31,
35). For example, resting cells lack nuclear forms of the acti-
vation-dependent host transcription factors NF-?B and NFAT,
which are important for gene expression from the HIV-1 long
terminal repeat (LTR) (5, 17, 45, 64).
Recently, there has been considerable interest in the idea
that the nonproductive nature of infection in resting CD4?T
cells may reflect proviral integration into chromosomal sites
that are or that become repressive for transcription (26, 32,
33). Resting CD4?T cells are profoundly quiescent cells with
densely heterochromatic nuclei (19). The silencing of impor-
tant genes in T cells involves changes in chromatin structure or
repositioning to heterochromatic regions (58). Elegant studies
by Jordan et al. have suggested that HIV-1 latency may involve
integration into regions of heterochromatin (32). Transformed
cell lines infected in vitro and then selected for reversibly
nonproductive infection showed preferential integration into
centromeric regions known to be repressive for transcription.
In contrast, in vitro infections of T-cell lines conducted without
selection revealed preferential integration into transcribed
genes (54, 68).
The nature of HIV-1 integration sites in resting CD4?T
cells from infected individuals has not been analyzed yet. We
used inverse PCR to characterize integration sites in resting
CD4?T cells from patients on HAART and to explore
whether the nonproductive nature of infection in resting CD4?
T cells is related to the characteristics of the integration sites.
MATERIALS AND METHODS
Purification of resting CD4?T cells. Peripheral blood was obtained from
patients who achieved and maintained suppression of viral replication to below
the limit of detection of ultrasensitive clinical assays (?50 copies of HIV-1
RNA/ml of plasma) while on HAART. Informed consent was obtained from all
patients. This study was approved by an institutional review board.
Resting CD4?T cells were purified by using a previously described two-stage
process (14, 16, 21). Peripheral blood mononuclear cells were negatively selected
to remove CD8?T cells, B cells, monocytes, NK cells, and activated CD4?T
cells by using appropriate monoclonal antibodies and magnetic beads conjugated
with antibodies to mouse immunoglobulin G (14, 21). The depletion of activated
CD4?T cells was accomplished by using antibodies to both early (CD69 and
CD25) and late (HLA-DR) activation markers. Further purification of resting
CD4?T cells was accomplished by sorting for small lymphocytes with high levels
of CD4 and low levels of HLA-DR surface expression (14, 21). The resulting
populations of resting CD4?T cells showed ?1% contamination with activated
cells. In some experiments, the CD45RO?CD45RA?subset of resting CD4?T
cells was isolated by including anti-CD45RA antibody in the initial depletion and
then sorting for CD4?CD45RO?HLA-DR?cells. The purity of these popu-
lations was ?95%.
Analysis of HIV-1 integration sites. A modification of a previously described
inverse PCR strategy was used for analysis of HIV-1 integration sites (14, 16).
Genomic DNA from highly purified resting CD4?T cells was digested with PstI,
which cuts frequently in genomic DNA but only once in most HIV-1 isolates, at
nucleotide (nt) 1419 in gag (HXB2R coordinates) (39). Digested DNA was
ligated under dilute conditions favoring intramolecular ligation. Circularized
DNA was amplified with outwardly directed primers in conserved regions of the
HIV-1 LTR and gag: outer LTR primer, 5?-TAACCAGAGAGACCCAGTAC
AGGC-3?, nt 468 to 445 in the LTR; outer gag primer, 5?-GGTCAGCCAAAA
TTACCCTATAGTG-3?, nt 1170 to 1194 in gag. This amplification captured the
junction between the 5? end of the viral genome and host cell DNA. PCR
conditions were as follows. DNA was denatured at 94°C for 3 min. This step was
followed by 30 cycles at 94°C for 30 s, 65°C for 1 min, and 68°C for 2 min. A final
extension was carried out for 4 min at 68°C. A second PCR was carried out with
the same cycling parameters and a nested set of outwardly directed, HIV-1-
specific primers (inner LTR primer, 5?-TGGTACTAGCTTGAAGCACCATC
CA-3?, nt 152 to 128 in the LTR; inner gag primer, 5?-TGTTAAAAGAGACC
ATCAATGAGGAAG-3?, nt 1388 to 1414 in gag). The nested reaction produced
bands visible on agarose gels. The bands were eluted, cloned, and sequenced. By
using the UCSC Bioinformatics Human Genome database (http://www.genome.
ucsc.edu; July 2003), the human genomic sequence in each inverse PCR product
was identified as a unique best-hit Blat ranking joined directly to the end of the
5? LTR of HIV-1. A similar inverse PCR strategy that involved an initial diges-
tion with NdeI was also used.
Analysis of gene expression in resting CD4?T cells by RT-PCR. RNA was
isolated (RNeasy minikit; Qiagen) from highly purified resting CD4?T cells and
phytohemagglutinin (PHA)-activated CD4?T lymphoblasts from uninfected
donors or from selected cell lines or primary cells from other tissues. RNA was
treated with DNase and then reversed transcribed into cDNA by using random
hexamers and Superscript II RNase H?RT (Invitrogen) at 42°C for 1 h, followed
by incubation at 70°C for 15 min and cooling to 4°C. The resulting cDNA was
used in PCRs with gene-specific primers designed to span an intron so that
amplification of genomic DNA would not occur. The amplification conditions
were as follows: 94°C for 2 min; 30 cycles at 94°C for 30 s, 60°C for 1 min, and
68°C for 2 min; and a final extension at 68°C for 4 min. In each experiment,
control reactions from which RT was omitted were run to ensure that genomic
DNA was not amplified.
Analysis of transcription from upstream cellular genes. RNA was isolated
from highly purified resting CD4?T cells from HIV-1-positive donors who had
shown suppression of viremia to ?50 copies/ml while on HAART as described
above. For each analysis, 106sorted resting CD4?T cells were lysed in 350 ?l of
lysis buffer (RNeasy minikit), and total RNA was isolated according to the
manufacturer’s protocol. RNA was treated with DNase (DNase I, amplification
grade; Invitrogen) to remove genomic DNA. RNA was then reverse transcribed
as described above with a 2 ?M concentration of an HIV-specific primer (HIV-
RT, 5?-AGTCGCCGCCCCTCGCCTCCTGC-3?, nt 720 to 742 in gag), 130 ng of
random hexamers (Invitrogen), 1 ?l of RNase inhibitor (RNAguard; Amersham
Pharmacia Biotech), and Superscript II according to the manufacturer’s proto-
col. One-fourth of the isolated RNA was used for each RT reaction. Control
reactions in which RT was omitted were run to ensure that genomic DNA was
PCR amplification of the resulting cDNA was carried out with Expand high-
fidelity PCR enzyme, a 1 ?M concentration of each gene-specific primer, a 0.2
?M concentration of each deoxynucleoside triphosphate, and 5 ?l of cDNA in a
50-?l reaction volume. Primers used for amplification were as follows: HIV-
START5?, 5?-GGGTCTCTCTGGTTAGACCAGATCTGAGCC-3?, nt 454 to
VOL. 78, 2004HIV-1 INTEGRATION SITES IN RESTING CD4?T CELLS6123
483, repeat region; HIV-UP5?, 5?-GGCGAGCCCTCAGATCCTGC-3?, nt 406
to 425, U3 region; and HIV-3?, 5?-CAGCAAGCCGAGTCCTGCGTCG-3?, nt
687 to 708 in gag.
Single-round PCRs of 35 cycles were carried out with either primers HIV-
UP5? and HIV-3? or primers HIV-START5? and HIV-3?. Heminested PCRs
were also performed. Outer PCRs were carried out with either primers HIV-
UP5? and HIV-RT or primers HIV-START5? and HIV-RT (35 cycles). Inner
PCRs were carried out with either primers HIV-UP5? and HIV-3? or primers
HIV-START5? and HIV-3? (30 cycles). Cycling parameters were as follows: for
single-round and outer PCRs: (i) denaturation for 2 min at 94°C; (ii) 35 cycles at
94°C for 45 s, 60°C for 50 s, and 72°C for 1 min; and (iii) a final extension at 72°C
for 5 min. Nested PCRs were carried out with the same parameters for 30 cycles.
PCR products were cloned by TOPO TA cloning (Invitrogen) and sequenced.
The specificity of the PCR was confirmed by Southern blot hybridization with an
HIV-1-specific probe (5?-CTGCTAGAGATTTTCCACACTGAC-3?, nt 612 to
635, U5 region) and by sequence analysis.
Statistical analysis of integration sites. The P values for the integration en-
vironment and the correlation between host gene transcriptional direction and
HIV-1 orientation were calculated based on the chi-square distribution. The
significance of integration site clustering was inferred from the Poisson distribu-
tion. The correlation between integration sites and host transcription start sites
was calculated by using nonparametric runs test of randomness (MINITAB Inc.).
Cloning of HIV-1 integration sites from resting CD4?T
cells of patients on HAART. To determine whether the non-
productive nature of HIV-1 infection of resting CD4?T cells
is due to transcriptional repression resulting from integration
into regions of heterochromatin, we cloned integration sites
from highly purified resting CD4?T cells from the peripheral
blood of infected patients who showed long-term suppression
of viremia while on HAART. In such patients, other more
labile reservoirs decay, leaving the stable integrated form of
HIV-1 in resting memory CD4?T cells as the principal reser-
voir (3, 14, 21). A two-stage purification procedure gave prep-
arations of resting CD4?T cells that contained ?0.1% con-
tamination with activated (HLA-DR?) cells (Fig. 1). Resting
CD4?T cells purified in this way do not produce virus without
cellular activation (12, 14, 16, 21, 28).
HIV-1 integration sites and the host cell DNA sequences
immediately upstream were amplified by inverse PCR (Fig.
2A). Genomic DNA from purified resting CD4?T cells was
digested with PstI or NdeI. The restricted DNA was diluted
and then ligated under conditions favoring intramolecular li-
gation. The resulting circles were used as templates in PCRs
with outwardly directed primers located in conserved regions
within the terminal fragment of the viral genome. This ampli-
fication captured the junction between the viral genome and
upstream host cell DNA. A second PCR with a nested set of
outwardly directed primers provided sufficient sensitivity to
detect rare single-copy integration events. These were visible
as bands on an agarose gel suitable for cloning (Fig. 2B).
Because integration is random with respect to PstI or NdeI
sites, each integration event gives rise to a band of a distinct
size, reflecting the distance to the nearest upstream restriction
site in the host DNA (Fig. 2A). Distinct sets of bands were
obtained in separate PCRs from the same patient (Fig. 2B).
Controls for the inverse PCR included reactions from which
ligase was omitted (Fig. 2B). These were generally negative
except for occasional spurious reaction products which could
be readily identified and excluded by sequence analysis. The
positive control for the inverse PCR was a previously described
LTR-gag-containing plasmid, p1418-PstI (14).
After cloning and sequencing, integration sites were vali-
dated by sequence analysis. Integration sites were considered
valid only when they contained a direct junction between the 5?
end of the HIV-1 LTR, beginning with the sequence
5?-TGGAA, and a sequence that was uniquely homologous to
a continuous segment of the human genome (Fig. 2A). The AC
dinucleotide found at the 5? end of linear, unintegrated HIV-1
DNA (41, 46, 60) is removed during the integration reaction
and is absent except in about ?1/16 of the integration sites
where host nucleotides of this sequence occur fortuitously.
Based on genome region-specific sequence data (11, 53, 55),
we estimate that short (?4-kb), clonable PstI fragments rep-
FIG. 1. Purification of resting CD4?T cells from patients on HAART. Flow cytometric analysis of unfractionated peripheral blood mononu-
clear cells (left panel) and highly purified resting CD4?T lymphocytes (right panel) stained with phycoerythrin-conjugated anti-CD4 and
fluorescein isothiocyanate-conjugated anti-HLA-DR antibodies. Numbers indicate the percentages of cells in each quadrant.
6124HAN ET AL. J. VIROL.
resent 30 to 40% of the total sequences in euchromatic, cen-
tromeric, and telomeric regions of the human genome. There-
fore, this method does not introduce an obvious bias for or
against integration sites in particular regions of the genome.
Integration sites identified with PstI or NdeI had similar char-
Most integrated HIV-1 genomes in resting CD4?T cells
reside in transcription units. Using the approach described
above, we characterized 74 integration sites from 16 patients
(Table 1). Each site was unique. Gene-rich chromosomes and
gene-dense regions were highly favored as integration sites.
Importantly, almost all (93%) of the viral genomes resided
within transcription units (Table 1). A total of 76% of the
integration sites were in well-characterized genes listed in the
curated RefSeq database of molecularly characterized genes
(51). Another 18% were within transcription units predicted
from sequenced human mRNAs or predicted by the Genscan
algorithm. Transcription units occupy about 33% of the human
genome (42). Therefore, the fraction of integration sites that
lie within genes in vivo in resting CD4?T cells deviates sig-
nificantly from that which would occur by random insertion (P
? 0.001). Among the 69 integration sites residing within genes,
94% were in introns, probably due to the high percentage of
sequence length represented by introns (42).
Among the 74 sites identified, we observed for different
patients several clusters of independent integration events that
fell within 1 Mb of each other (Table 1, bold and italic type).
All of these clusters were in gene-dense regions. Only 5 out of
74 integration events occurred in intergenic regions, and 4 of
these were found within 10 kb of RefSeq transcription units.
There was no apparent tendency for HIV-1 integration into
repetitive elements, despite their frequency within the human
genome (Table 1). Despite previous work suggesting that
HIV-1 latency involves integration into centromeric DNA (32),
no sites were identified in alphoid regions of centromeres.
Although there was a striking preference for integration into
transcriptional units, the orientation of the HIV-1 genome
with respect to the host gene and its location within the host
gene appeared to be random (Table 1 and Fig. 3). Unlike the
findings for endogenous human retroviruses (59), there was no
correlation between the orientation of integrated HIV-1 in
vivo and the direction of cellular gene transcription (P ? 0.5)
(Table 1). In contrast to the findings for murine leukemia virus,
which integrates preferentially in the vicinity of transcription
start sites (68), there was no significant correlation between
HIV-1 integration site and the start site for the transcription of
the targeted host gene (P ? 0.8955) (Fig. 3). For both RefSeq
and non-RefSeq genes, HIV-1 integration sites in resting
CD4?T cells were found along the entire length of the host
genes (Fig. 3).
Integration sites in infected resting CD4?T cells are in host
genes that are actively expressed. To determine whether inte-
gration sites in resting CD4?T cells were in host genes that
were transcriptionally active, we analyzed the expression status
of the targeted host genes in populations of resting CD4?T
cells. Because each integration site is unique and is likely
FIG. 2. Detection of HIV-1 integration sites in resting CD4?T lymphocytes from patients on HAART by inverse PCR. (A) Inverse PCR
strategy for cloning HIV-1 integration sites. Genomic DNA from highly purified resting CD4?T cells was digested with PstI, which cuts frequently
in genomic DNA but only once in most HIV-1 isolates, at nt 1419 in gag. Digested DNA was subjected to intramolecular ligation and nested PCR
amplification as described in the text. Boxed nucleotides represent the 5? end of the LTR. (B) Inverse PCR products from a representative patient.
(Top panel) Results of 12 independent inverse PCRs for the same patient. Sequence analysis demonstrated that each band represented a unique
integration event from a single infected resting CD4?T cell. (Bottom panel) Control reactions carried out either without ligase or without DNA.
VOL. 78, 2004HIV-1 INTEGRATION SITES IN RESTING CD4?T CELLS6125
TABLE 1. Characteristics of HIV-1 integration sites in infected resting CD4?T cells
Host nt at the
nt from host
Arg-Glu dipeptide (RE) repeats
FUS-interacting protein 1
Polycystic kidney disease 1 related
Polycystic kidney disease 1 related
Transcription repressor p66 beta
Adenosine deaminase, RNA isoform
Brain and reproductive organ expressed
Transforming growth factor ? receptor II
Dystroglycan 1 precursor
Synapse-associated protein 97
Aminopeptidase regulator of shedding
Lysyl oxidase preproprotein
Solute carrier family 12
FK506-binding protein 51
v-myb myeloblastosis homolog
Phosphatidylinositol 3-kinase, catalytic, gamma
GTP-binding protein, q protein
Adducin 3 isoform a
Nucleosome assembly protein
Splicing factor 1
Suppressor of K transport defect 3
Chromosome 12 open reading frame 6
Heterochromatin protein 1
6126HAN ET AL.J. VIROL.
Ring finger protein 111
Inhibitor of activated STAT
A-kinase (PRKA) anchor protein 13
Kinesin family member 22
Nuclear factor of activated T cells
Nuclear receptor corepressor 1
A-kinase anchor protein 10
DNA topoisomerase II alpha
Mesenchyme homeobox 1
Growth factor receptor-bound 2
PRPP synthetase associated
Epidermodysplasia verruciformis 1
?-Tubulin cofactor D
DNA methyltransferase 1
Intercellular adhesion molecule 3
Janus kinase 3
Vaccinia virus-related kinase 3
v-crk oncogene homolog
LTR (endogenous retrovirus 1)
Eukaryotic translation initiation factor 3
aJunction between the 5? end of the HIV-1 LTR (first five letters) and the host cell DNA.
bThe host nucleotide number at the junction was determined by using the UCSC Bioinformatics Human Genome Database (July 2003 assembly freeze). Bold type indicates clusters of two integration events within
a 1-Mb window (P, 3.9 ? 10?5). Italic type indicates clusters of three integration events within a 1-Mb window (P, 2.6 ? 10?5).
cNature of the integration site: I, intron; E, exon; Int, intergenic. Overall, 93.2% (69 of 74) of the integration sites were in defined or predicted genes, while 6.8% (5 of 74) were in intergenic regions. Among integrations
in genes, 94.2% (65 of 69) were in introns and 5.8% (4 of 69) were in exons.
dTranscriptional orientation: ?, the host gene and the HIV-1 insert have the same transcriptional orientation; ?, the gene and the insert have the opposite orientation. Of genes in transcription units, 49.3% (34 of
69) were in the ? orientation and 50.7% (35 of 69) were in the ? orientation.
eDistance, in nucleotides, between the start site for transcription of the host gene and the HIV-1 integration site. Numbers in parentheses indicate the relative position of the integration site within the gene, with 0
representing the start of transcription and 1 representing the end of the transcript.
fFor integration sites within known or predicted genes, RT-PCR was carried out with gene- specific primers spanning an intron on total RNA isolated from purified resting or activated (with PHA) CD4?T cells. ?,
presence of a PCR product of the predicted size; ?, no PCR product under conditions that gave a readily detectable band for a ubiquitously expressed gene (GAPDH) and, for characterized genes, a correct product
from cell lines or primary tissues known to express the gene. These included chondrocytes (LOX), kidney cells (ADD3), and mesenchymal stem cells (MEOX1). The expression of TOP2A, MEOX1, and a human mRNAfrom chromosome 12p11.21 was detected in activated but not resting CD4?T cells. For integration events in well-characterized (RefSeq) genes, expression of the targeted gene in resting CD4?T cells was observed for
91.1% (51 of 56) of the genes.
gGene predicted by the Genscan algorithm.
hhmRNA, human mRNA from GenBank.
iThe start site for transcription has not yet been determined.
VOL. 78, 2004HIV-1 INTEGRATION SITES IN RESTING CD4?T CELLS6127
present in only a single cell in any blood sample, it is not
possible to determine simultaneously both the nature of the
integration site in a given cell and the level of expression of the
targeted host gene in that particular cell. However, infected
resting CD4?T cells from patients on HAART express little
HIV-1 RNA (7, 28) and are therefore very similar to unin-
fected resting CD4?T cells. A recent study by Chun et al.
showed that gene expression profiles for resting CD4?T-cell
populations from aviremic patients on HAART are similar to
profiles for resting CD4?T cells from healthy donors (12).
Therefore, the expression of targeted host genes was analyzed
by RT-PCR with RNA from purified resting CD4?T cells
from HIV-1-negative donors (Fig. 4 and Table 1). Expression
patterns in mitogen-activated CD4?T cells and appropriate
control cell lines were also examined.
For each of the integration sites in known or predicted
genes, primers spanning an intron and producing amplicons of
approximately 400 to 500 bases were designed. RT-PCRs were
carried out with a standard set of conditions that gave expected
results for the characterized genes. In all instances when re-
sults were negative with RNA from resting CD4?T cells, the
integrity of the RNA sample was assessed by successful detec-
tion of transcripts from ubiquitously expressed genes, such as
that for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
In addition, we evaluated the ability of the primers to detect
the expression of the relevant gene in activated CD4?T cells
or in positive control cell lines from a tissue known to express
Examples of this analysis are shown in Fig. 4. Under the
conditions used, RNA for the ubiquitously expressed enzyme
(GAPDH) was readily detectable in resting CD4?T cells,
while RNA for the tissue-specific enzyme lysyl oxidase (LOX)
was not detected (Fig. 4A). LOX is an amine oxidase impor-
tant for cross-linking collagen and elastin in the extracellular
matrix. The LOX gene is expressed by fibrogenic cells, and
there is no evidence for expression in lymphocytes (36). Acti-
vated CD4?T cells also failed to express LOX, but expression
was readily detected in a chondrocyte cell line, confirming the
ability of the RT-PCR to detect LOX RNA when present (Fig.
4A). As expected, resting CD4?T cells did not express genes
that are expressed only in cycling cells, such as the gene for
topoisomerase II (TOP2A), an enzyme that is involved in
DNA replication. As shown in Fig. 4B, the expression of the
TOP2A gene was not detected in purified resting CD4?T cells
but was readily detected in activated CD4?T cells. Thus, the
RT-PCR conditions used gave the results expected for the
With this approach, the expression of targeted host genes
was analyzed for all of the integration sites for which there was
sufficient information on intron-exon structure to permit the
design of primers (n ? 68). The results are shown in Table 1.
The most important finding was that a high proportion of the
genes in which integration events were found were actively
transcribed in resting CD4?T cells from healthy donors. For
the 68 genes analyzed, expression in resting CD4?T cells was
clearly detectable for 55 (81%). For five genes for which ex-
pression was not detected, the integration site was located in a
putative gene that was predicted by the Genscan algorithm but
for which no human mRNA had yet been described. It is
therefore possible that the absence of expression reflected
deficiencies in the gene prediction program. When the analysis
is restricted to the 56 integration sites in molecularly charac-
terized genes that are part of the RefSeq database (36), the
results are even more striking. Expression in resting CD4?T
cells was observed for 50 of 56 genes (89%). Some examples
are shown in Fig. 4C. RNAs for both nuclear factor of acti-
vated T cells (NFATc3) and FK506-binding protein 51
(FKBP51) were expressed in resting CD4?T cells as well as
activated CD4?T cells (Fig. 4C). NFATc3 is a member of the
nuclear factors of activated T cells family of transcription fac-
tors, which are present in the cytosol of resting lymphocytes
(22) and which are translocated to the nucleus following de-
FKBP51 is a widely expressed member of the FK506-binding
protein family (2). Two of the genes in which integration sites
were found (those for TOP2A and mesenchyme homeobox 1
[MEOX1]) were expressed in activated but not resting CD4?
T cells; a single gene (that for the activity-dependent neuro-
protector [ADNP]) was expressed in resting but not activated
cells. Overall, 91% (51 of 56) of the targeted genes were
expressed in resting CD4?T cells (Table 1). Patterns of gene
expression were also analyzed by using cDNA arrays (data not
shown). In general, the results were similar, but because RT-
PCR is more sensitive and reliable, results from the RT-PCR
analysis are presented. Taken together, our results suggest that
in infected resting CD4?T cells, HIV-1 genomes reside pre-
dominantly in genes that are actively transcribed in resting
Previous studies suggested that in resting CD4?T cells, the
majority of integrated HIV-1 DNA is present in cells of the
memory subset (6, 14). We therefore confirmed that the ex-
pression of the genes in Table 1 was similar for the memory
subset of CD4?T cells. During the purification of resting cells,
the memory subset was enriched to ?95% by depletion of cells
expressing the RA isoform of CD45 and sorting for cells ex-
pressing the RO isoform. The memory subset was shown to
have a pattern of expression of the relevant genes very similar
to that of the entire resting CD4?T-cell population. For ex-
ample, as shown in Fig. 4D, both populations of cells expressed
FKBP51, NFATc3, and TAP2 but not TOP2A.
FIG. 3. Distribution of HIV-1 integration events along the lengths
of targeted genes. Results are shown both for genes that are part of the
RefSeq database (51) (black bars [bottom portion of bars]) of well-
characterized genes and for genes predicted on the basis of sequenced
human mRNAs or by sequence analysis (gray bars [top portion of
bars]). Each transcript, regardless of its length, was divided into 20
equal parts, and the position of HIV-1 integration within the transcript
was plotted by using these relative length units.
6128 HAN ET AL.J. VIROL.
Incorporation of HIV-1 sequences into transcripts of host
genes. The finding that HIV-1 integration sites in resting
CD4?T cells are located predominantly within introns of
genes that are actively expressed in resting CD4?T cells pre-
dicts that the HIV-1 genome should be included within the
primary transcripts of the targeted host genes and then de-
graded along with the remainder of the relevant introns fol-
lowing splicing. Thus, RNA species derived from host promot-
ers and containing HIV-1 sequences may be present in resting
CD4?T cells, although these cells are not permissive for high-
level expression from the HIV-1 LTR. To test this hypothesis,
RT-PCR experiments were designed to detect low levels of
transcripts containing HIV-1 sequences upstream of the HIV-1
transcription start site and to distinguish them from the low
levels of bona fide HIV-1 transcripts produced in infected
resting CD4?T cells (Fig. 5A). RNA from resting CD4?T
cells was reverse transcribed with the gene-specific primer
HIV-RT, which binds upstream of the first HIV-1 splice site.
The resulting cDNA was amplified with two primer sets that
share a 3? primer. One primer set (HIV-UP5? and HIV-3?)
amplifies only HIV-1 sequences produced as a result of tran-
scription of the targeted host gene reading through the HIV-1
genome that is inserted into the gene. Because the forward
primer is located upstream of the transcription start site (nt
456 of the LTR) and the reverse primer is located downstream
of the LTR, only RNA species initiating upstream of the
HIV-1 transcription start site are amplified. The other primer
set (HIV-START5? and HIV-3?) is able to amplify these tran-
scription products in addition to any HIV-1 transcripts that
have initiated at the HIV-1 LTR. To prevent amplification
from HIV-1 DNA, isolated RNA was treated with DNase
before RT-PCR. In addition, control reactions from which RT
was omitted were included in each experiment and were in-
variably negative. In control experiments, both primer sets
were shown to have comparable sensitivities on a plasmid
template (Fig. 5B).
The presence of both types of transcripts in highly purified
populations of resting CD4?T cells from patients on HAART
and showing stable suppression of viremia to below the limit of
detection was analyzed. Single-round, semiquantitative RT-
PCR demonstrated that RNAs containing HIV-1 sequences
upstream of the transcription start site were present, but at
levels that were even lower than the very low levels of tran-
scripts initiating at the HIV-1 transcription start site (data not
shown). This finding may reflect the rapid turnover of the
HIV-1 sequences contained within the introns of other genes
as well as the fact that only half of the integration sites were in
the same transcriptional orientation as the host gene (Table 1).
For these reasons, a more sensitive heminested PCR approach
was used to detect low levels of short-lived RNA species (Fig.
FIG. 4. Analysis of host gene expression in resting CD4?T cells. All transcriptional units in which an integration site was identified were
analyzed by RT-PCR (Table 1). Representative examples are shown. In all three panels, GAPDH served as a positive control for RT-PCR.
RT-PCRs were carried out with (?) or without (?) RT. (A) Patterns of expression of GAPDH and LOX by resting CD4?T cells, by activated
CD4?T cells, and by chondrocytes. In all instances, RT-PCR signals were dependent on the presence of RT. (B) Expression of TOP2A by
mitogen-activated but not resting CD4?T cells. RT-PCR signals were dependent on the presence of RT. (C) Analysis of NFATc3 and FKBP51
expression in resting and activated CD4?T cells and in the thymus. RT-PCR signals were dependent on the presence of RT. (D) Patterns of
expression of genes targeted for integration in resting CD4?T cells and in the memory subset of resting CD4?T cells.
VOL. 78, 2004 HIV-1 INTEGRATION SITES IN RESTING CD4?T CELLS6129
5C). Reverse primers HIV-RT and HIV-3? were used in the
first and second rounds, respectively. Two independent ali-
quots of RNA from 106purified resting CD4?T cells were
analyzed in quadruplicate with each primer set. Both types of
transcripts could be readily detected as bands of the expected
sizes. The nature of the PCR products was confirmed by South-
ern blot hybridization with an internal probe (Fig. 5A) and by
direct cloning and sequencing. With this approach, transcripts
originating from upstream of the HIV-1 LTR could be de-
tected in purified resting CD4?T cells from each of six pa-
tients studied. Control reactions that lacked RT were invari-
ably negative (Fig. 5C, right panel). Taken together, these
results demonstrate that RNA molecules containing HIV-1
LTR sequences upstream of the HIV-1 transcription start site
can be detected in resting CD4?T cells, consistent with the
active transcription of host genes carrying the integrated
We have analyzed sites of HIV-1 integration in vivo in highly
purified resting CD4?T cells from the peripheral blood of
patients on HAART. This cell population is of particular in-
terest because, despite the presence of cells carrying integrated
HIV-1 DNA (13, 14, 16), there is no detectable virus produc-
tion without some form of stimulation (12, 14, 16, 28). It was
therefore of interest to determine whether the nonproductive
nature of the infection of resting CD4?T cells in vivo is related
to the nature of the integration site. HIV-1 integration sites in
transformed cell lines infected in vitro with HIV-1 were pre-
viously analyzed (32, 54, 68). Transcriptional units were fa-
vored sites of integration in two studies (54, 68), but when cells
were selected for reversibly nonproductive viral gene expres-
sion, integration into regions of heterochromatin was favored
In our in vivo analysis of integration sites in resting CD4?T
cells, we found that integration into transcriptional units was
strongly favored (93%). Typically, HIV-1 integration sites were
within introns of the relevant genes. Moreover, many of the
genes found to contain integration sites were actively ex-
pressed in resting CD4?T cells from healthy donors (91%).
This result was obtained by direct RT-PCR analysis of each
gene and did not rely solely on cDNA array analysis. The
finding that HIV-1 genomes reside within introns of genes
actively expressed in resting CD4?T cells predicts that HIV-1
sequences may be included in the primary transcripts of the
relevant host genes. This hypothesis is supported by the results
FIG. 5. Detection of transcripts containing HIV-1 sequences in resting CD4?T lymphocytes from patients on HAART. (A) RT-PCR strategy
for detecting HIV-1 transcripts initiating at the HIV-1 LTR and transcripts initiating upstream of the transcription start site. The positions of the
two forward PCR primers (HIV-START5? and HIV-UP5?) relative to the transcription start site are indicated. (B) Sensitivity of heminested PCR
analysis with, as a template, 10-fold serial dilutions of a plasmid (pLAI) containing the entire HIV-1 genome. The forward primer was either
HIV-START5? or HIV-UP5?. For the first reaction (rxn), the reverse primer was HIV-RT. For the nested reaction, the reverse primer was HIV-3?.
dH2O, distilled H2O. (C) Analysis of transcripts from two independent aliquots (a and b) of 106sorted, resting CD4?T cells from a representative
patient. (Left panel) Transcripts were amplified in quadruplicate reactions with forward primer HIV-START5?. (Middle panel) Transcripts were
amplified in quadruplicate reactions with forward primer HIV-UP5?. (Right panel) Control reactions lacking RT were performed with the
indicated forward primers. GAPDH served as a positive control for RNA isolation and RT-PCRs for each aliquot.
6130 HAN ET AL.J. VIROL.
of RT-PCR analysis with primers that can amplify only tem-
plates containing HIV-1 sequences upstream of the HIV-1
transcription start site. We conclude that for the majority of
resting CD4?T cells with integrated HIV-1 DNA, the absence
of virus production cannot be attributed to integration into
chromosomal regions that are intrinsically repressive for tran-
scription. Furthermore, our results show that the presence of
integrated HIV-1 DNA within an actively transcribed host
gene is not sufficient for productive infection of resting CD4?
T cells. Mechanisms unrelated to the nature of the integration
site must account for the nonproductive nature of the infection
in resting CD4?T cells.
One potential explanation for the nonproductive nature of
the infection in resting CD4?T cells is that these cells carry
defective HIV-1 genomes. The in vivo error rate for HIV-1 RT
is estimated to be 3.4 ? 10?5substitutions/nucleotide/cycle.
For a genome of 9.6 ? 103nt, there is only a 1 in 3 probability
(0.326) that each newly arising HIV-1 genome will contain a
new mutation, and only a fraction of these mutant genomes
will be defective for replication. However, there may be some
selection for defective genomes in vivo. In some of the acti-
vated CD4?T cells that become infected, new mutations aris-
ing during reverse transcription may adversely affect virus gene
expression. As a result, the cells will not express viral proteins
and will not die from viral cytopathic effects or host cytolytic
effector mechanisms. In principle, such cells will have a greater
chance of surviving long enough to revert back to a resting
state. Thus, resting CD4?T cells carrying defective genomes
Nevertheless, many studies have confirmed that replication-
competent virus can persist in a latent form in resting CD4?T
cells for long periods of time, even in patients who show com-
plete suppression of detectable viremia while on HAART (3,
13, 14, 16, 21, 67). Potential mechanisms for HIV-1 latency
include proviral integration into chromosomal sites that are or
that become repressive for transcription (26, 32, 33), the ab-
sence in resting CD4?T cells of activation-dependent host
transcriptional activators necessary for HIV-1 gene expression
(5, 17, 45, 64), premature termination of HIV-1 transcripts due
to the absence of HIV-1 Tat and Tat-associated host factors (1,
25, 29–31, 35), and the failure to export unspliced HIV-1 RNA
due to insufficient levels of Rev (43, 50). Because only a small
fraction of the resting CD4?T cells with integrated HIV-1
DNA can produce replication-competent virus following cel-
lular activation, our data do not exclude the possibility that
HIV-1 latency involves integration into chromosomal regions
that are or that become repressive for transcription (26, 32,
33). Although we did not observe integration into regions of
heterochromatin such as the alphoid repeat regions of centro-
meres, it remains possible that rare integrations into such sites
occur in vivo and that such integrations are associated with
The nature of the HIV-1 integration sites in vivo can provide
insights into the accessibility of different chromosomal regions
and the targeting mechanisms used in the integration process.
Because direct infection of resting CD4?T cells does not lead
to integration due to blocks at the level of entry (48, 52),
reverse transcription (49, 69), and nuclear import (9, 38), it is
most likely that resting CD4?T cells with integrated HIV-1
DNA arise when activated CD4?T cells become infected and
then revert back to a resting state (3). According to this model,
the characteristics of the integration sites in vivo should reflect
host DNA accessibility and viral targeting characteristics op-
erative in activated CD4?T cells. Our results are consistent
with this model in that most targeted genes were actively ex-
pressed in both activated and resting CD4?T cells. This pat-
tern is strikingly different from that observed for some other
retroviruses and retroelements. Avian leukosis virus integra-
tion is directed away from actively transcribed genes (66).
Murine leukemia virus tends to integrate near transcription
start sites (68) and not throughout the targeted gene, as ob-
served here. The Ty1-4 retrotransposons of Saccharomyces cer-
evisiae integrate upstream of genes transcribed by RNA poly-
merase III (4). In contrast to the random transcriptional
orientation of HIV-1 in resting CD4?T cells, endogenous
human retroviruses tend to have an antisense orientation (59).
Despite widely different patterns of integration adopted by
various retroelements, the pattern of HIV-1 integration ob-
served in resting CD4?T cells is essentially the same as that
observed in extensive studies of HIV-1 integration into prolif-
erating cell lines in vitro (54, 68). This finding reinforces the
notion that the nonproductive nature of the infection in resting
CD4?T cells with integrated HIV-1 DNA is not the result of
the nature of the integration site.
It is not yet possible to determine how the present results
bear on the critical issue of HIV-1 latency. Such a determina-
tion will require the development of methods for simulta-
neously determining the integration site and the replication
competence of individual proviruses. Although difficult, this is
an important goal. The capacity of replication-competent
HIV-1 genomes to persist in a latent state in resting CD4?T
cells for many years, even in patients on HAART, represents a
major barrier to HIV-1 eradication (13, 20, 21, 57, 62, 67). If
the general patterns observed here for integrated proviruses in
resting CD4?T cells also apply to the subset of proviruses that
are replication competent, then the latent reservoir may be
more accessible to therapeutic intervention than previously
We thank members of the Siliciano laboratory for many helpful
discussions and Mike Paradise for help in recruiting patients.
This work was supported by NIH grant AI43222, a grant from the
Johns Hopkins Center for AIDS Research, the Doris Duke Charitable
Foundation, and the Howard Hughes Medical Institute.
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