Nuclear architecture dictates HIV-1 integration
site selection
Bruna Marini
1
, Attila Kertesz-Farkas
2
*, Hashim Ali
1
*, Bojana Lucic
1
{, Kamil Lisek
1
{, Lara Manganaro
1
{, Sandor Pongor
2
{,
Roberto Luzzati
3,4
, Alessandra Recchia
5
, Fulvio Mavilio
5,6
, Mauro Giacca
1,4
1 & Marina Lusic
1
1{
Long-standing evidence indicates that human immunodeficiency
virus type 1 (HIV-1) preferentially integrates into a subset of
transcriptionally active genes of the host cell genome
1–4
. However,
the reason why the virus selects only certain genes among all
transcriptionally active regions in a target cell remains largely
unknown. Here we show that HIV-1 integration occurs in the outer
shell of the nucleus in close correspondence with the nuclear pore.
This regioncontainsa series of cellular genes, which arepreferentially
targeted by the virus, and characterized by the presence of active
transcription chromatin marks before viral infection. In contrast,
the virus strongly disfavours the heterochromatic regions in the
nuclear lamin-associated domains
5
andother transcriptionally active
regions located centrally in the nucleus. Functional viral integrase
and the presence of the cellular Nup153 and LEDGF/p75 integration
cofactors are indispensable for the peripheral integration of the
virus. Once integrated at the nuclear pore, the HIV-1 DNA makes
contact with various nucleoporins; this association takes part in the
transcriptional regulation ofthe viralgenome. These results indicate
that nuclear topography is an essential determinant of the HIV-1
life cycle.
One important aspectof the interaction between HIV-1 and its target
cells is the encounter between the viral complementary DNA (cDNA)
with the complex architecture of the mammalian nucleus, in which
chromosomes and genes are spatially arranged to occupypreferred posi-
tions within the three-dimensional space
6
.
We analysed the lists of human genes targeted by HIV-1 from six
different studies (Extended Data Table 1), containing altogether 1,136
unique gene integration sites in activated T cells carrying the CD4 anti-
gen (CD4
1
); 126 of these genes recurred in two lists, 24 in three, and six
in at least four lists, for a total of 156 genes, which we named HIV recur-
rent integration genes (RIGs). The probability of detecting this number
of specific genes by chance was extremelylow (P , 1 3 10
29
; Extended
Data Fig. 1a). RIGs were also highly represented in another list of approx-
imately 12,000 integration sites
4
, 5,221 of which were unique genes, as
well as in two integration lists generated from patients’ CD4
1
T cells
7,8
(P , 0.001 of detecting these genes by chance). Thus, RIGs are bona
fine the hottest spots of HIV-1 integration.
We then ranked RIGs according to their frequency and plotted them
onto the human chromosome map
9
. Unexpectedly, they appeared to
cluster into specific chromosomal regions (Extended Data Fig. 1b). In
five out of eight cases, RIGs were also in proximity to the ‘hotter zones’,
previously defined as regions with remarkably high HIV-1 integration
density
1
(Supplementary Table 1). In these areas, observations hinted
at the possibility that the topological distribution of these chromosomal
regions inside the nucleus could determine HIV-1 integration.
By applying three-dimensional immuno-DNA fluorescence in situ
hybridization (FISH), we assessedthe position of RIGsand hotterzones
in primary CD4
1
T cells from healthy donors. Selected FISH probes,
listed in the Supplementary Information, provided topological infor-
mation for a total of 169 RIGs and other integration sites located within
10 megabases (Mb) from the centre of the probe (Extended Data Fig. 2).
When the radial positions of the RIG FISH signals were binned into
three zones of equal area
10,11
(Fig. 1a), a clear gradient in signal localiza-
tion was observed, which decreased from the nuclear envelope towards
the interior(images of 14 RIGs in Fig. 1b, c; four hotter zones in Fig. 1d).
The global distribution of RIGs (n 5 1,420 analysed alleles) was remark-
ably different from that of control genes, all of which were expressed in
CD4
1
T cells
12,13
(n 5 522): 44% of RIGs mapped in zone 1, 41.5% in
zone 2 and only 14.5% in zone 3 versus 25.6%, 47.6% and 26.8%for con-
trol genes, respectively (Fig. 1f; representative images of control genes
are shown in Fig. 1e). Considering an average of about 7 mm for the
nuclear diameter in CD4
1
T cells, 63% of RIGs and hotter-zone alleles
were concentrated within about 1 mm below the nuclear membrane.
We wanted, therefore, to visualize the position of the HIV-1 DNA itself
in infected, primary CD4
1
T-cell nuclei. At 4 days after infection with
theVSV-G-pseudotypedHIV-1
NL4-3/E-R-
14
, the vast majority ofthepro-
viral immuno-FISH signals were in zone 1 (75.2% within 1 mm under
the nuclear envelope) (Fig. 2c). The visualized viral DNA was integrated
15
,
as also detected by real-time Alu PCR (Fig. 2a), and transcriptionally
active (Fig. 2b). A similar distribution was observed in primary macro-
phages and the monocytic cell line U937 (Extended Data Fig. 3a, b, respec-
tively). Peripheral localization was also observed for a fully competent
virus carrying the HIV-1
BRU
envelope
16
(Fig. 2d) and, notably, for the
wild-type viruses found in CD4
1
T-cells from two HIV-infected patients
(Fig.2e, f). Peripheral localizationwas also a feature of lentiviral vectors,
irrespective of their transcriptional activity (Extended Data Fig. 3c, d),
but not of the MoMLV gammaretrovirus, which localized preferen-
tially inside the nuclear interior (Extended Data Fig. 3e).
In contrast, when integration was impaired, the viral cDNA roamed
around the nucleus. This was the case for two HIV-1 clones harbouring
single-point mutations in the integrase catalytic domain (class I IN
mutations: IN(D64E) and IN(D116N))
17,18
or for HIV-1
NL4-3/E-R-
in the
presence of the integrase inhibitor raltegravir; under these conditions,
only 10–20% of viruses were found in zone 1 (Fig. 2g). In these cases, the
detected viral genomes did not correspond to integrated DNA (Fig. 2h)
but were highly enriched in circular forms of viral DNA containing two
long terminal repeats (2-LTR circles) (Fig. 2i). We also downregulated
the chromatin tethering factor LE DGF/p75 (ref. 19) and the inner nuclear
basket protein Nup153 (ref. 20), which are involved in viral DNA inte-
gration (Fig. 2j). FISH was performed 48 h after infection when there
*These authors contributed equally to this work.
1These authors jointly supervised this work.
1
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy.
2
Protein Structure and Bioinformatics Group, International Centre for Genetic
Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy.
3
Struttura Complessa Malattie Infettive, Azienda Ospedaliero-Universitaria, 34134 Trieste, Italy.
4
Department of Medical, Surgical and Health
Sciences, University of Trieste, 34129 Trieste, Italy.
5
Department of Life Sciences, University of Modena and Reggio Emilia, 41121 Modena, Italy.
6
Genethon, 91002 Evry, France. {Present addresses:
Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg and German Center for Infection Research, 69120 Heidelberg, Germany (B.L.; M.L.); Laboratorio Nazionale Consorzio
Interuniversitario per le Biotecnologie (LNCIB), 34149 Trieste, Italy (K.L.); Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA (L.M.); Pazmany University,
Budapest 1083, Hungary (S.P.).
1
was a marked reduction in HIV-1integration (Fig. 2k), and the majority
of the signals labelled unintegrated viral DNA
16
: 72% and 77% of the
FISH signals were in zones 2 and 3 for the LEDGF/p75 and Nup153
knockdowns, respectively (Fig. 2l). The effect of the Nup153 knock-
down was rescued by transfecting an expression plasmid coding for an
RNA interference (RNAi)-resistant Nup153 (Extended Data Fig. 4).
Most of the HIV-1 targets are common in different cell types; how-
ever, subtle differences exist. For example, HIV-1 almost never targets
the IKZF3 locus in CD34
1
haematopoietic stem cells
21
(P , 1 3 10
212
),
whereas the TAP2 gene fromthemajor histocompatibility complex class
II locus is never targeted in CD4
1
T cells (P , 1 3 10
213
). Strikingly,
we observed that IKZF3 localized in zones 1 and 2 in peripheral blood
CD4
1
T cells (.80% of alleles), while it was almost absent from zone 1
in cord blood CD34
1
cells (,6% alleles; P , 0.001). Conversely, the
TAP2 locus was absent from zone 1 in CD4
1
T cells (,8% of alleles),
while it was distributed between zones 1 and 2 in CD34
1
cells (.90%
of alleles; P , 0.001; Fig. 3a).
To understand the chromatin features of RIGs, we compared the avail-
abledata from chromatin immunoprecipitationsequencing (ChIP-seq)
obtained in CD4
1
T cells for RIGs
22
, cold genes (defined as transcrip-
tionally inactive genes never targeted by HIV-1; F.M. and A.R., unpub-
lished observations) and a list of genes corresponding to the 1,000 most
expressed (active) and 1,000 least expressed (silent)genes fromthe GNF
SymAtlas
13
. Association of RNA Pol2 with RIGs had a pattern super-
imposable to that of active genes, peaking at the transcription start sites
(TSSs) (Fig. 3b). In a similar manner, distribution of markers of active
transcription (H3K9ac, H3K36me3, H3K4me3, H4K16ac and H4K20me)
was identical for RIGs and active genes (Fig. 3c–e and Extended Data
Fig. 5). In contrast, markers of facultative (H3K9me2) and constitutive
(H3K9me3 and H3K27me3) chromatin were found enriched both on
cold genes (where HIV-1 never integrates) and on silent genes, but not
on RIGs (Extended Data Fig. 5). Of interest, active genes and RIGs had
a superimposable distribution of H3K4me2, which is enriched at the
lamin-associated domain (LAD) borders
5
(Fig. 3f).
Heterochromatic LADs contain approximately 4,000 transcriptionally
inactive genes
5
. We found that more than 90% of HIV RIGs lay outside
LADs, while almost 80% of cold genes were inside LADs (P , 0.001
compared with a random gene distribution; Fig. 3g). Immuno-FISH
images for three of these cold genes confirmed their localization close
to the nuclear envelope in primary CD4
1
T cells (Fig. 3h). Finally, when
all the 1,344 known LADs were aligned by their left or right borders,
87.2% of RIGs were found outside the LADs, in contrast to a random
distribution of genes (68.2%; P , 0.001, also taking into account the lower
gene density within LADs; Fig. 3i).
Transcriptionally activegenes at the nuclear periphery are often asso-
ciated with the nuclear pore complex (NPC)
23–27
. Wethereforeassessed
interaction of the HIV-1 provirus with the NPC by ChIP assays in primary
CD4
1
T cells (primer scheme and controls in Extended Data Fig.6a, b).
At 4 days after infection, when RNA Pol2 and the USF1 and p65/RelA
transcription factors were associated with the viral DNA as expected
14
,
both the mAb414 antibody, which recognizes phenylalanine–glycine
(FG)-repeats in nucleoporins, and specific antibodies against Nup153,
b
e f
Percentage of nuclear radius
Nuclear
envelope
Nuclea
r
centre
Zone 1 Zone 2 Zone 3
3.0
2.0
1.0
0
0 19 28 43 100
RIGs and HZs
(n = 1,420)
Control genes
(n = 522)
Loci frequency (×10
–2
)
NFATC3
RP11-67A1 16q22.1
n = 68
123123123
1 2 3
123 123 123 123 123 123 123
1 2 312 312 312 312 312 312 3
Alleles (%)
Alleles (%)Alleles (%)
Alleles (%)
HEATR7A
RP11-714N16 8q24.3
n = 78
123 123 123
n = 94
RPTOR
RP11-28G8 17q25.3
n = 72
SPTAN
RP11-216B9 9q34.11
SMG1
RP11-1035H13 16p12.3
n = 86
100
0
50
75
25
100
0
50
75
25
Zone: Zone:
n = 132
GRB2
RP11-16C1 17q25.1
n = 102
KDM2A
RP11-157K17 11q13.2
n = 136
DNMT1
CTD-2240E14 - 19p13.2
n = 100
HZ 2
CTD-2517M22 8q24.3
n = 62
HZ 3
CTD-2587H24 19q13.42
n = 154
HZ 4
RP11-1057N3 8q24.21
n = 118
HZ 1
RP11-349I23 4q.25
100
0
50
75
25
n = 90
Zone:
NPLOC4
RP11-765O14 17qter
FKBP5
RP3-340B19 6p21.31
n = 128
100
0
50
75
25
n = 126n = 52
Zone:
n = 100 n = 52
GAPDH
RP11-711C24
12p13.31
ACTN1
RP11-226F19
14q24.1
CD28
RP5-940J5
2q33.1
CD4
RP4-761J14
12p13.31
n = 106
n = 42
n = 44
PACS2
RP11-521B24
14q32.33
KDM2B
RP11-44F24
12q24.31
HEATR6
RP11-178C3
17q23
r
CD4
+
T-cell
nucleus
Zone: 3
100019 43
21
a
cd
mAb414 BAC
mAb414 BACmAb414 BAC
mAb414 BAC
Figure 1
|
Localization of HIV RIGs at the nuclear periphery. a, Subdivision
of nucleus into three concentric zones of equal area. b–e, Three-dimensional
immuno-DNA FISH of ten HIV RIGs (b, c), four hotter zones (d) and
seven control genes (e) in activated CD4
1
T cells (green: bacterial artificial
chromosome (BAC) probe labelled with DIG (dUTP-digoxygenin) and FITC
(fluorescein isothiocyanate); red: NPC staining by mAb414). Below each
representative image, the distribution of the analysed alleles into the three
nuclear zones is shown, normalized over nuclear radius. Evenly distributed
random genes would be enriched equally in the three zones (red dashed line).
The number of alleles analysed is shown at the bottom of each panel. HZ, hotter
zone. f, Distribution of the relative distances of all measured alleles from the
nuclear envelope (HIV RIGs and hotter zones: n 5 1,420; control genes:
n 5 522). The three zones are shown by grey shading. The dashed line indicates
approximately 1 mm from the nuclear edge of the T-cell nucleus.
2
Nup98, Nup62 and Tprall immunoprecipitated the HIV-1 DNA; bind-
ing was also observed for the NPLOC4 RIG gene, but not for the LAD
gene PTPRD (Extended DataFig. 6c). WhenChIP was performed on the
IN-defective D64E virus, no viral DNA was detected using the mAb414
and anti-Nup153 antibodies (Extended Data Fig. 6d).
Next, we aimed to verify whether HIV-1 localization changed when
the virus reverted from a transcriptionally inactive to an active state.
In the latent T-cell J-Lat clone 15.4 (ref. 28), the HIV-1 DNA retained
its gross peripheral localization both in inactive and in TPA (12-O-
tetradecanoylphorbol-13-acetate) phorbol ester-reactivated conditions
(Extended Data Fig. 7a, b). Similar results were obtained in a primary
model of HIV-1 latency
14
(Extended Data Fig. 7c–e). However, when
localization was analysed at molecular resolution by ChIP using the
mAb414,anti-Tpr and anti-Nup153antibodies, bindingof the proviral
region located downstream of the TSS to the nucleoporins was observed
upon transcriptional activation but not in latent conditions (Extended
Data Fig. 7f). We also observed that nucleoporins directly participated
in HIV-1 transcriptional regulation. When Tpr and Nup153 were silenced
by RNAi in latent J-Lat cells, proviral transcription was significantly
reduced (Extended Data Fig. 7g, h). Similarly, downregulation of Tpr
also blunted LTR-driven gene expression in HIV-1-infected HeLa cells
(Extended Data Fig. 8a–e).
Our findings show that the cellular genes that are highly targeted by
HIV-1 are distributed in a topologically non-random manner, being
positioned within 1 mm from the nuclear edge; these genes are enriched
in open chromatin marks, excluded from the LADs and associatedwith
the NPC. Thus, the HIV-1 pre-integration complex preferentially targets
those areas of open chromatin that are proximal to the nuclear pore,
while excluding the internal regions in the nucleus as well as the peri-
pheral regions associated with the nuclear lamina (model in Extended
Data Fig.9). The localization of HIV-1 proviral DNAinclose association
with the nuclear pore is consistent with several observations showing
that different NPC components play a role in HIV-1 infection
20,29,30
.
Why does the viral DNA integrate into the NPC compartment? A
possibility that we favour is that the virus simply integrates into the first
open chromatin regions it meets along its route into the nucleus. This is
mAb414
-
HIV-1
c
CD4
+
T cells/HIV-1
NL4.3
Ex vivo infection
Proviruses (%)
Proviruses (%)
Proviruses (%)
Proviruses (%)
100
0
50
75
25
Zone:
mAb414
-
HIV-1
HIV-1 patient 1
e
HIV-1 patient 1
Natural infection
100
0
50
75
25
Zone:
mAb414
-
HIV-1
HIV-1 patient 2
HIV-1 patient 2
Natural infection
100
0
50
75
25
Zone:
f
mAb414
-
HIV-1
d
CD4
+
T cells/HIV-1
BRU
Ex vivo infection
100
0
50
75
25
Zone:
CD4
+
T-cells + HIV-1
BRU
CD4
+
T-cells + HIV-1
NL4.3
g
h
75
100
Raltegravir:
HIV-1
NL4.3
Integrated HIV DNA
(% of control)
0
25
50
–––+
Raltegravir:
0
5
10
15
20
ND
D116ND64E
HIV-1
NL4.3
D116ND64E
2-LTR circles
(fold over control)
j
siRNA: NT2 LEDGF
WB: tubulin
WB: LEDGF
NT2 Nup153
WB: Nup153
WB: tubulin
HIV-1
NL4.3
150
100
–NT
siRNA:
Integration (%)
0
50
LEDGF Nup153
HIV-1
mAb414
Control siNT2/NT5
n = 163
123
siLEDGF
n = 164
123
n = 116
100
0
50
75
25
123
123
123
123
123
123 123 123
Zone:
siNup153
n = 129
123
HIV-1
m414bA
Lamin
IN(D116N) WT + raltegravirIN(D64E)
n = 160
n = 42
n = 28
n = 27
Viruses (%)
100
0
50
75
25
Zone:
n = 66
n = 159
i
k
l
ba
Integrated HIV DNA
(% of infected cells)
0
75
100
50
25
HIV-1
NL4.3
–+
HIV-1 mRNA
(fold over 18S × 10
–2
)
0
30
10
20
HIV-1
NL4.3
–+
–+––
Figure 2
|
Integrated, transcriptionally active HIV-1 is found at the nuclear
periphery. a, b, Quantification of integrated HIV-1
NL4-3/E-R-
DNA (a) and
HIV RNA (b) by real-time Alu PCR in infected CD4
1
T cells. c–f, Three-
dimensional immuno-DNA FISH of HIV-1 DNA (green) in primary CD4
1
T cells infected ex vivo with HIV-1
NL4-3/E-R-
(c) and HIV-1
BRU
(d), or directly
obtained from two patients infected with HIV-1 (e, f). g, Three-dimensional
immuno-DNA FISH of HIV-1 DNA in activated CD4
1
T cells infected with
the mutant viruses IN(D64E) or IN(D116N) or with HIV-1
NL4-3/E-R-
upon
raltegravir treatment. h, i, Real-time Alu PCR (h) and 2-LTR quantification (i)
in the cells treated as in g. ND, not determined. j, Western blot (WB) showing
protein levels for LEDGF/p75 and Nup153 at the moment of HIV-1 infection,
36 h after short interfering RNA (siRNA) transfection. NT2, non-targeting
siRNA. k, Real-time Alu PCR in Jurkat cells infected with HIV-1
NL4.3
and
previously transfected with a non-targeting siRNA (NT) or an siRNA targeting
LEDGF/p75. Samples were normalized over control-infected cells. l, Three-
dimensional immuno-DNA FISH for HIV-1 DNA visualization upon Jurkat
cell treatment with the indicated siRNAs. NT2 and NT5 are two non-targeting
siRNAs. All graphs, except those relative to patients’ cells, show mean and
s.e.m. of at least three independent experiments.
3
likely to be related to the short life of viral integrase
16
and thus the need,
for the pre-integration complex, to achieve rapid integrationinto geno-
mic DNA upon its entry into the nucleus. This interpretation is consis-
tent with our observation of more dispersed, unintegrated viral cDNA
in all conditions in which integrase function is impaired.
Finally, while adding a three-dimensional view to the process of HIV-1
integration, our results also indicate that the localization of the HIV-1
DNA in closecorrespondence with the nuclearporehas functional rele-
vance, since itappears important for productive HIV-1 gene expression.
Online Content Methods, along with any additional Extended Data display items
and Source Data, are availablein the online version of the paper; references unique
to these sections appear only in the online paper.
Received 5 December 2013; accepted 9 January 2015.
Published online 2 March 2015.
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***
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***
Ref. 3
Ref. 2
Wang et al.
Han et al.
Liu et al.
Ikeda et al.
RIGs
Cold genes
Mavilio et al.
Genes inside LADs (%)
HIV-1 inte
g
ration sites
g
Normalized counts (×10
–6
)
Relative positions (kb)
–400 –200
0
+200 +400
0
1.0
2.0
3.0
HIV RIGs
Random genes
Inside LADsOutside LADs
0
20
40
60
80
n = 30
n = 30
n = 56
100
0
50
Zone:
RP11-63O1 3p26.3
CNTN4
RP11-487A2 13q32
GPC5
RP11-338L20 9p23
PTPRD
mAb414
DNA
b
Counts
RNA Pol2
0
0
8
16
HIV RIGs
Cold genes
Silent genes
Active genes
HIV RIGs
Cold genes
Silent genes
Active genes
Counts
Position relative to TSS
c
H3K9ac
–2,000 –1,000 0 +1,000 +2,000
0
4
8
12
f
H3K4met2
Position relative to TSS
−4,000 −2,000 0 +2,000 +4,000
0
2
4
Position relative to TSS
H3K36met3
–20,000 –10,000 0 +10,000 +20,000
0
2
4
6
d
H3K4met3
Position relative to TSS
−4,000 −2,000 0 +2,000 +4,000
0
40
80
e
a
n = 54
n = 52
RP11-94L15 17q12
IKZF3
Alleles (%)
Alleles (%)
100
0
50
75
25
Zone:
mAb414
BAC
123123
1 2 312 3
123 123 123
CD4
+
CD34
+
TAP2
RP11-10A19 6p21.3
n = 60n = 54
CD4
+
CD34
+
***
Position relative to TSS
+1,000 +2,000–1,000–2,000
Figure 3
|
HIV RIGs are transcriptionally active genes that are excluded
from the LADs. a, Localization of the IKZF3 (a) and TAP2 (b) genes (green) in
CD4
1
T cells and CD34
1
haematopoietic stem cells. b–f, Distributions of Pol2,
acetylated H3K9, H3K36me3 and H3K4me2/3 around the TSSs of HIV
RIGs (red) and cold genes (green), compared with highly active (black) and
silent (blue) genes in activated CD4
1
T cells. g, Cross-comparison of different
lists of integration loci, including HIV RIGs, with the lists of genes present
inside LADs: HIV integration loci are significantly depleted in LADs compared
with a null distribution (indicated by a red dotted line). ***P , 0.001,
**P , 0.01, *P , 0.05. References with authors’ names can be found in the
reference list at the end of the Methods section. h, Three-dimensional
immuno-DNA FISH in activated CD4
1
T cells of three cold genes predicted to
be inside LADs by bioinformatics analysis. i, Distribution of HIV RIGs (red)
and of a random set of genes (black) around aligned LAD border regions.
The light grey area with positive genomic coordinates indicates the regions
inside LADs; the white area with negative coordinates is outside LADs.
4
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Acknowledgements This work was supported by grants from the Italian National
Research Programme on AIDS of the Istituto Superiore di Sanita
`
, Italy, to M.G. and M.L.
and from the Young Investigator Grant RF2007-16 of the Italian Ministry of Health to
M.L. The authors are grateful to S. Kerbavcic for editorial assistance.
Author Contributions B.M., B.L., K.L. and M.L. performed the immuno-DNA FISH and
ChIP experiments; A.K.-F., B.M., S.P., M.L. and M.G. analysed the data; A.K.-F., B.M. and
S.P. performed the bioinformatics analysis; H.A. and M.L. performed the experiments
using infectious virus; L.M. generated and analysed integrase-defective HIV-1
molecular clones; R.L. contributed to studies in primary cells from patients with HIV;
A.R. and F.M. generated lentiviral vectors and analysed integration into CD4
1
T cells
and CD34
1
bone marrow cells; M.L. and M.G. conceived and supervised the
experiments and wrote the paper with help from the other authors.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper. Correspondence
and requests for materials should be addressed to M.G. (giacca@icgeb.org) or
M.L. (marina.lusic@med.uni-heidelberg.de).
5