REVIEWS

Nuclear landscape of HIV1 infection

and integration
Marina Lusic1 and Robert F.Siliciano2
Abstract | To complete its life cycle, HIV1 enters the nucleus of the host cell as reverse-transcribed
viral DNA. The nucleus is a complex environment, in which chromatin is organized to support
different structural and functional aspects of cell physiology. As such, it represents a challenge for
an incoming viral genome, which needs to be integrated into cellular DNA to ensure productive
infection. Integration of the viral genome into host DNA depends on the enzymatic activity of
HIV1 integrase and involves different cellular factors that influence the selection of integration
sites. The selection of integration site has functional consequences for viral transcription, which
usually follows the integration event. However, in resting CD4+ Tcells, the viral genome can be
silenced for long periods of time, which leads to the generation of a latent reservoir of quiescent
integrated HIV1 DNA. Integration represents the only nuclear event in the viral life cycle that can
be pharmacologically targeted with current therapies, and the aspects that connect HIV1
nuclear entry to HIV1 integration and viral transcription are only beginning to be elucidated.

HIV1 infection can be controlled, but not cured, by
antiretroviral therapy (ART). ART suppresses HIV1
replication to undetectable levels, but, if therapy
is interrupted, viral loads rapidly rebound to pre-
treatment levels. This is due to the formation of a reser
voir of latently infected resting CD4+ Tcells that harbour
integrated HIV1 and persist in all infected individuals,
regardless of the duration of ART15. Most of the provi
ruses in resting CD4+ Tcells are defective and cannot
replicate, but a minority remains transcriptionally
competent and can cause viral load to rebound when
treatment is stopped6,7.
The life cycle of HIV1 begins with binding of the
Department of Infectious viral envelope glycoproteins to receptors on the target
1
transcriptional activity (see BOX1 for the organization
of the human genome and chromatin). Several factors
influence the selection of the target site, including the
route of nuclear entry and cell cycle phase, chromatin
structure and underlying sequence specificities and
interaction of the viral integrase with chromatin tether
ing factors such as lens-epithelium-derived growth fac
tor (LEDGF; also known as PSIP1). The latest findings
confirm that the nuclear architecture also has a central
role in directing the PIC towards a permissive nuclear
environment that can support transcription and repli
cation of the viral genome. In contrast to the actively
transcribed viral genome that is seen in productively
infected cells, the integrated viral genome can undergo

Diseases, Integrative Virology,

University Hospital Heidelberg
and German Center for
Infection Research (DZIF),
ImNeuenheimer Feld 324,
69120 Heidelberg, Germany.
2
Howard Hughes Medical
Institute; Department of
Medicine, Johns Hopkins
University School of Medicine,
Room 879, Edward D.Miller
Research Building, 733North
Broadway, Baltimore,
Maryland 21205, USA.
Correspondence to M.L.
marina.lusic@med.uni-
heidelberg.de
doi:10.1038/nrmicro.2016.162
Published online 12 Dec 2016
cell, followed by fusion of the viral and cellular mem
brane and release of the viral core into the cytoplasm8.
The viral RNA genome is then reverse transcribed
into DNA by the viral reverse transcriptase; the newly
generated viral DNA forms a pre-integration complex
(PIC), which comprises viral DNA, the viral integrase
and capsid proteins, and some cellular proteins. The PIC
enters the nucleus and mediates the integration of viral
DNA into the cellular genome (FIG.1). Nuclear entry is an
active process that requires passage through the nuclear
pore complex (NPC) in non-dividing cells, in which
the chromosomes are separated from the cytoplasm
by the nuclear envelope (see BOX1for the anatomy of the
nuclear periphery)9. Although viral integration can take
place at various locations in the host cell genome, it pref
erentially targets regions that have high gene density and
transcriptional silencing in resting CD4+ Tcells, leading
to the generation of a latent viral reservoir, which is a
major obstacle for the eradication of HIV1.
In this Review, we discuss the integration of the HIV1
genome into host cell DNA, including the influence of
the nuclear architecture on this step in the viral life cycle,
and its role in the outcome of infection. In addition, we
will provide an overview of current and possible future
approaches for the treatment of HIV1 that act on the
integration of, or on integrated, viral genomes.
Towards a preferred nuclear neighbourhood
HIV1 can infect non-dividing cells and, hence, it enters
the nucleus through the NPC channel. As the PIC by far
exceeds the size of molecules that can pass through the
NPC channel, it is generally accepted that the nuclear

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 1

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Viral envelope glycoproteins

Surface proteoglycan proteins
that are products of the env
gene. The env gene encodes
glycoprotein 160 (gp160),
which forms a homotrimer and
is cleaved into gp120 and gp41
by host cell proteases. The
outer glycoprotein gp120 and
the transmembrane protein
gp41 are embedded in the viral
envelope, which forms the
outermost layer of the virion.
Viral core
A viral component that
contains the viral capsule
protein p24, which surrounds
two single strands of HIV1
RNA, bound to the
nucleocapsid protein p7
andlate assembly protein p6.
Italso contains enzymes that
are needed for the replication
of HIV1, such as reverse
transcriptase, protease,
ribonuclease and integrase,
and numerous cellular proteins.
Central polypurine tract
A specific structural element
that is involved in viral DNA
structure and nuclear entry.
Itis formed during the
second-strand DNA synthesis
of reverse transcription as a
central 99nucleotide-long
plus-strand overlap in the
linear DNA molecule; it is
located centrally in the
genome, in the integrase
openreading frame, and acts
as a cis determinant of
lentiviral DNA nuclear import.
Twolong terminal repeat
circles
(2LTR circles). Unintegrated,
circular molecules of viral
DNAthat contain two adjacent
LTR promoter regions. They
are considered byproducts
of integration, and their
quantification by PCR-based
methods is used as an
indicator of the efficiency of
nuclear import.
Nuclear-localization signal
(NLS). A motif that is present
inproteins that are imported
into the nucleus by importins
and adaptor proteins, which
interact with the nuclear
porecomplex.
Phe-Gly repeats
(FG repeats).
Phenylalanine-rich and
glycine-rich domains found in
nucleoporins that facilitate the
transport of cargo through the
channel of the nuclear pore
complex (NPC).
Maturation
Assembly
Receptor binding Reverse
and fusion transcription Synthesis
of viral
proteins
Genomic
RNAs
Splicing and
nuclear export
of RNAs
Integration Transcription
Latency
Figure 1 | An overview of the life cycle of HIV1. After fusion of the HIV1 virion with receptors on the plasma
membrane, the viral genomic RNA is reverse transcribed into DNA, followed by uncoating (disassembly of the viral capsid),
Nature Reviews | Microbiology
nuclear entry and integration of the viral DNA into cellular chromatin. During productive infection, viral transcription is
followed by splicing and nuclear export of viral RNA, and the production and assembly of new virus particles, which bud
from the plasma membrane and become infectious after maturation. Alternatively, integrated viral DNA can be silenced
through several mechanisms, thus enabling the creation of silent viral reservoirs (proviral latency).
import of the HIV1 PIC is an energy-dependent The matrix protein was the first component of the
active process that is governed by different viral and PIC proposed to have a role in nuclear import in non-
cellularfactors. dividing cells as it has a nuclear-localization signal (NLS)17.
However, the role of matrix in nuclear import seems to
Viral factors. Several viral components, including the be redundant, because even without a functional matrix
capsid, matrix, Vpr, integrase and the central polypurine HIV1 can still infect macrophages18.
tract, have been reported to have a role in nuclear import Vpr, which is a small viral protein that is best known
of the HIV1 PIC9,10 (FIG.2). for its capacity to arrest the cell cycle in the G2M phase,
The capsid protein is a major determinant for also contains an NLS, although a non-canonical one, and
nuclear entry and for the infection of non-dividing it enters the nucleus in a non-classical way through its
cells. This was first determined when the replication interaction with importin19. Vpr was among the first
of a chimeric HIV1 virus that carried the capsid pro viral proteins that were shown to be involved in the dock
tein of murine leukaemia virus was shown to depend ing of the PIC to the NPC and binding to Nups20,21. As
on the cell cycle. Arrest of the cell cycle caused a defect Vpr can move between the cytoplasm and the nucleus, it
in nuclear import of this chimeric virus, as measured was suggested that Vpr has a role in the transport of viral
by the decreased formation of two-long terminal repeat DNA19. Furthermore, GFP-tagged Vpr was used to track
circles (2LTR circles)11. Furthermore, certain HIV1 the trafficking of PICs along cytoplasmic microtubule
capsid mutants are defective only in resting cells and filaments towards the nucleus22. However, the role of Vpr
not in actively dividing cells, which highlights the role in nuclear import remainscontroversial.
of the capsid in nuclear entry 12. The capsid protein
interacts with RANBP2 (also known as NUP358) to Cellular factors. Various host proteins have been shown
dock the virion to the cytoplasmic side of the NPC13,14, to participate in HIV1 nuclear import, many of which
which supports the hypothesis that viral uncoating were revealed in genome-wide short interfering RNA
and the completion of reverse transcription occur at (siRNA) screens; those identified in more than one
the nuclear pore (reviewed in REF.10). A recent study screen include RANBP2, transportin 3 (TNPO3: also
in primary macrophages showed that, intriguingly, known as TRN-SR) and NUP153 (REFS2325). Knock
the capsid remains associated with nuclear PICs 15, down of these factors blocks infection after reverse tran
which indicates that it has a role in the nuclear scription, but before integration, and involves the capsid,
portion of the viral life cycle as well. In addition to although the exact mechanisms remain to be elucidated.
nucleoporins (Nups), the capsid of HIV1 interacts RANBP2 and NUP153 localize to the cytoplasmic
with other cellular factors; disrupting these inter and the nuclear ends of the NPC, respectively, and
actions through mutation can substantially alter the both are attachment sites for molecules that traverse
integration profile14,16. the NPC. Both proteins are enriched in Phe-Gly repeats

2 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Box 1 | The mammalian nucleus

Nuclear architecture and genome organization
Different levels of organization of chromatin structure the nucleus, with chromosome territories being the major
organizing units. The packaging of DNA into the nucleus results in physical interactions between loci, even between
those located on remote parts of chromosomes. These interactions are non-random. High-resolution chromatin mapping,
which is based on chromosome conformation capture (3C), and related methods, showed that recurrent and functionally
significant interactions organize the genome.
Long-range interactions on the same chromosome (intrachromosomal) can occur between promoter and terminator
regions of a gene, as well as between enhancers and promoter regions. These interactions, together with those that are
reported to be driven by insulators, contribute to the organization of each chromosome into distinct structural (and
possibly functional) domains of different length. Topologically associated domains (TADs), which are separated by
genetically defined boundary elements, vary in length from several hundred bases to several million bases. Two regions
in one TAD interact with each other more frequently than with regions outside of the TAD, which means that TADs
represent the physical compartmentalization of the genome. TADs have two basic features: self-association of regions
in TADs and insulation towards neighbouring TADs. The nature of TADs is hierarchical, with sub-TADs, loops and
insulating neighbourhoods representing the local organization of each TAD. Whereas TADs seem to be conserved
between different cell types, sub-TADs, loops and insulating neighbourhoods can differ. Thus, TADs could represent a
larger, invariant feature of genome organization, whereas the underlying hierarchical structures may have roles in
lineage-specific genome regulation. Other chromatin domains that are defined on the basis of their interaction with
thenuclear lamina (lamin-associated domains (LADs)) correlate with low gene expression and late replication timing.
LADs that are present in all cell types analysed thus far are termed constitutive LADs, whereas those that differ between
cell types or change in their association with the nuclear lamina, which is dependent on the cell cycle, are known as
facultative LADs. Changes in the association with the nuclear lamina are a further indication of how dynamic
nuclearorganization really is, not only in terms of differentiation, but also in response to different exogenous stimuli,
such as viral infection (see REFS164166 for comprehensive and detailed reviews on nuclear structure and organization).
The nuclear periphery and implications on genome organization and function
Chromatin domains that have different transcriptional and replicative activities define the nuclear architecture.
Organization occurs at various levels, ranging from chromosome territories and sub-chromosomal organizational
domains to functional nuclear compartments, such as transcription or replication factories, or nuclear bodies
(Polycombbodies, promyelocytic leukaemia (PML) bodies or Cajal bodies)164,165,167.
The nuclear periphery contains the nuclear envelope, the underlying nuclear lamina and nuclear pore complexes (NPCs),
which span from the cytosol into the nucleoplasm. The nuclear envelope consists of two membrane systems, theouter
nuclear membrane (ONM), which faces the cytosol, and the inner nuclear membrane (INM), which surrounds the
nucleoplasm. These two layers are separated by the perinuclear space and intersect at the level of NPCs. The inner
side of the nuclear envelope is covered with lamins, which form a thin protein layer (2030nm) that is tightly associated
with the INM on one side and with the underlying chromatin on the other side. Type A and type B lamins, together with
lamin-associated proteins, constitute lamin filaments and provide structural support to the nucleus. The key function
of nuclear lamin proteins is to maintain the shape and mechanical properties of the nucleus; they also tether
heterochromatic chromosomal domains to the nuclear periphery. In the nuclear lamina, genes are mostly transcriptionally
inactive and this region is enriched in heterochromatin111,168,169, whereas there is no condensed chromatin beneath the NPC170.
The mammalian NPC is composed of 30 different nucleoporins (Nups). Nups are present in multiple copies and
arranged in a ~125nm diameter core that can be structurally divided into a core scaffold (central pore), which is formed
by NUP93 and NUP205, and two rings (the cytoplasmic ring and the nuclear ring) that are composed of NUP107 and
NUP160. The inner side of this structure contains the socalled phenylalanine-glycine Nups (FGNups), such as NUP62,
which regulate receptor-mediated transport through the nuclear pore. Embedded in the nuclear envelope are three
known transmembrane Nups (POM121, NDC1 and GP210 (also known as NUP210)), that link the NPC to the nuclear
envelope. On the cytoplasmic side, eight filaments (formed of NUP214, RANBP2 and NUP88) protrude towards the
protein synthesis machinery and the cytoskeleton. Nuclear basket filaments that are composed of NUP153, TPR and
NUP50 (REFS112,113) have a more direct role in genome-related functions, whereas all other components of the nuclear
pore participate in selective nuclear import and export.

Zinc-finger domains
Protein structural domains that
can coordinate one or more
zinc atoms to act as binding
partners for a wide variety of
substrates, including DNA.
(FG repeats) and establish contacts with nuclear recep
tors. They have homologous zinc-finger domains that
could confer their binding to DNA (or RNA) and could
also explain their capacity to influence target site pref
erences for integration14,16. In fact, NUP153 has been
shown to affect integration site distribution16 and nuclear
import of HIV1 through its interaction with the capsid
protein26,27.
Cyclophilin A (CYPA) is a peptidyl-prolyl isomer
ase that binds to an exposed loop on the surface of
capsid28. It was suggested that CYPA directs HIV1 into
a nuclear entry pathway that involves RANBP2, which
could dock HIV1 to the cytoplasmic side of the NPC.
Nuclear import most likely occurs in synergy with
other Nups, such as NUP153 (REF.14), as the depletion
of RANBP2 or NUP153 decreases the nuclear import of
HIV1 (REFS14,24,26,29). CYPA binding and cis-to-trans
isomerization lead to conformational changes in the
capsid; however, the effect of these processes on
the uncoating and infectivity of HIV1 remain poorly
understood30.
TNPO3 was also identified in siRNA screens23 and
an independent yeast two-hybrid screen showed its
interaction with integrase31. TNPO3 imports splicing

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 3

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

capsid protein (N74D) has been shown to be resistant to

Reverse
transcription
Capsid
CYPA
Vpr
Matrix Integrase
vDNA
RANBB2
TNPO3
Nuclear basket
CPSF6 (including NUP153
and TPR)
Capsid
Integrase LEDGF
5 3
3 5
Target DNA
Figure 2 | Nuclear entry and the selection of integration site. The capsid of
HIV1 seems to be a crucial viral factor for both uncoating Nature
and theReviews | Microbiology
entry of the
pre-integration complex (PIC) into the nucleus. Other than capsid, the viral factors Vpr
and matrix, as well as different cellular partners are involved in the process of nuclear
entry on the cytosolic side of the nuclear pore complex (NPC). Cyclophilin A (CYPA;
also known as PPIA) and RANBP2 have roles in the nuclear entry of the PIC on the
cytosolic side of the NPC, transportin 3 (TNPO3) is involved in shuttling through the
NPC, and nucleoporin 153 (NUP153) and cleavage and polyadenylation specificity
factor 6 (CPSF6) are involved in the import of HIV1 into the nucleus. Both NUP153 and
CPSF6 have a role in target site selection, together with lens-epithelium-derived
growth factor (LEDGF), which is the most prominent chromatin-tethering factor that is
involved in HIV1 integration. vDNA, viral DNA.
factors into the nucleus32 and was suggested to pro
mote the nuclear entry of the PIC13,14,31. The same role
was attributed to another cellular factor, cleavage and
polyadenylation specificity factor 6 (CPSF6), that was
shown to interact with the capsid protein before trans
location into the nucleus3336. CPSF6 is a 68kDa member
of a large protein complex of pre-mRNA splicing factors
that shuttle between nucleus and cytoplasm37. Atrun
cated form of CPSF6, lacking the carboxy-terminal
domain (CPSF61358), that localized mainly in the cyto
sol and blocked nuclear entry of the PIC, was identi
fied in a mouse cDNA screen13. A mutant form of the
CPSF61358. This mutant is insensitive to TNPO3 knock
down, whereas the wild-type CPSF6 changes its locali
zation from the nucleus to the cytosol when TNPO3 is
depleted38. It was proposed that CPSF6 enables HIV1 to
use RANBP2, NUP153 and TNPO3 for nuclear entry 39
and the mutation in the CPSF6 binding site of capsid
(N74D) was shown to activate alternative mechanisms for
the PIC to enter the nucleus16,29. This ultimately results in
integration into gene-poor regions14,16. A recent study has
provided compelling evidence that CPSF6 gene silencing
results in HIV1 integration in sites distinct from gene
bodies and gene-dense chromatin regions40 (see below).
Nuclear docking and uncoating of the virion are
tightly connected with the import of the PIC into the
nucleus and are dependent on the capsid, which enters
the nucleus together with the viral DNA14,15,36,41. Thus
nuclear pore proteins from both the cytosolic and nuclear
sides of the NPC, as well as other transport proteins,
determine viral entry from the cytosol into the nucleus
and towards the susceptible regions of the genome in
which it will integrate.
HIV1 integration
After passing through the NPC, the PIC reaches the
periphery of the nucleus, which is a complex and hetero
geneous environment that provides anchoring sites for
chromosomes.
Integrase. Insertion of the viral DNA into chromosomes
is catalysed by integrase42,43 (FIG.3a). Integrase is encoded
by the pol gene, and the mature enzyme, which is com
posed of three structurally and functionally distinct
domains4446, is derived from the GagPol polyprotein
precursor following cleavage by the viral protease42.
The aminoterminal zinc-binding domain (NTD)
of integrase consists of three -helices that contrib
ute to oligomerization and to the catalytic activity 47,48.
Integrase also contains a conserved HHCC motif,
which comprises two histidine and two cysteine resi
dues. This domain binds to a Zn2+ ion and resembles the
zinc-coordinating residues of zinc fingers47.
The central catalytic core domain (CCD) is highly con
served in all retroviral integrase proteins, and, in addition
to its catalytic activity, has been proposed to be involved
in DNA binding 44. From a structural perspective, the
catalytic core domain belongs to the RNaseH superfamily
of polynucleotidyl transferases, and its key active site46 is
comprised of three acidic amino acid residues (Asp64,
Asp116 and Glu152) with a conserved spacing of 35 amino
acids between Asp116 and Glu152 (D,D(35)E motif).
These residues form coordination bonds with two Mg 2+
ions during nucleophilic substitution and DNA strand
transfer 4446. The CCD interacts with both the donor
(viral) and the target (cellular) DNA, and mutations in the
D,D(35)E motif inhibit integrase activity 49,50. The CCD
also contains a serine residue that can be phosphorylated
by cellular JUN Nterminal kinases and subsequently
isomerized by peptidyl-prolyl cis-trans isomerase
NIMA-interacting 1 (PIN1); this post-translational
modification stabilizes integrase in Tcells51.

4 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

a Pol Figure 3 | Integrase and the integration mechanism.

a | Schematic representation of HIV1 integrase, which is
Gag PRO RT IN
cleaved from the GagPol protein: integrase domains are
shown in the lower part of the panel with numbers
representing amino-acid position boundaries and the
Primary lengths of interdomains and the carboxyterminal
destabilizing Protease tail. Amino acid residues that are conserved between
residue cleavage site
different integrase species are marked by red stars; point
mutations in these residues abolish the activity of the
1 47 59 202 223 270 enzyme. Prototype foamy virus (PFV) integrase, which was
HIV-1 Phe NTD CCD CTD used for the crystal structure of the intasome, is also shown.
D64 D116 E152 288 b | HIV1 integration mechanisms: integrase multimerizes
1 4851 102 123 271 321 374 392 on the ends of the viral long-terminal repeat (LTR) and then
PFV
cleaves two nucleotides from each 3 end of the viral DNA
during 3 processing. The completion of 3 processing
leaves a recessed and chemically reactive hydroxyl group.
b Following nuclear import of the pre-integration complex
IN (PIC), integrase binds to the target DNA and strand transfer
IN occurs. After disassembly of the strand transfer complex,
LTR LTR the DNA recombination intermediate is repaired, probably
HIV DNA
Intasome formation by host cell enzymes, and the provirus, flanked by host
IN DNA, becomes an integrated part of the cellular chromatin.
IN CCD, catalytic core domain; CTD, carboxy-terminal
domain; IN, integrase; NTD, amino-terminal domain;
PRO, viral protease; RT, reverse transcriptase.
3 processing
CAGT-3 5-ACTG
GTCA-5 3-TGAC
The partial structures of these domains, resolved
LTR LTR
either by crystallization46 or by NMR spectroscopy53,
were useful for proposing different models of integrase
in complex with the ends of viral DNA55. However, full
Dissassembly of coverage of invivo events became available only after
strand transfer complex the crystal structure of prototype foamy virus (PFV)
CA-OH-3 5-ACTG
LTR GTCA-5 3-HO-AC LTR integrase in complex with viral DNA was resolved56,57.

Molecular mechanisms of integration. Integrase carries

5 Gap repair and
integration into
3 cellular genome
5 functional integraseviral DNA complex known as
5 3
3 5
Nature Reviews | Microbiology
The Cterminal domain (CTD) has the least
sequence conservation among retroviral integrase pro
teins52, with residues 220270 being responsible for
DNA binding and the formation of dimers of parallel
monomers53. The CTD contains four lysine residues
that are acetylated by cellular histone acetyltrans
ferases. These post-translational modifications regulate
integrase strand transfer activity, viral integration and
ultimately result in decreased viral replication54.
out two essential enzymatic reactions: 3-end processing
of the viral DNA and strand transfer reactions (FIG.3b).
Both 3-end processing and the integration of oligo
nucleotides that match the ends of HIV1 DNA can be
reproduced invitro in the presence of integrase alone58,59.
Moreover, experiments that used hybrid viruses with
altered integration preferences supported the principal
role of integrase in the selection of the integrationsite60.
Integrase activity initiates soon after the synthesis
of a double-stranded viral DNA molecule. In the PIC,
3 integrase multimerizes on the nascent DNA to form the
the intasome56,57. As revealed by the Xray crystal struc
ture of integrase from PFV, the intasome consists of a
tetramer (or dimerofdimers) of integrase subunits56,57,
and integrase activity depends on tetramerisation61. One
subunit of each integrase dimer binds to viral DNA for
3-end processing: integrase removes two nucleotides
from 3 ends of the blunt-ended linear viral DNA and
generates reactive CAOH3-hydroxyl ends.
After import into the cell nucleus, the intasome docks
onto the target DNA to form a target capture complex
(TCC)56 and strand transfer ensues: integrase uses the
3hydroxyl groups on the ends of the viral DNA as
nucleophiles to attack the target DNA across the major
groove. Complementary strands of viral DNA are then
joined with the 5phosphates at the ends of the target
DNA. The resulting DNA recombination intermediate

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 5

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Nucleosomes
The building blocks of
chromatin, which are
composed of an octameric
histone core around which
147bp of DNA are wrapped
(in1.65 turns of a left-handed
superhelix).
contains single-stranded gaps at the joints that can be
completed by the cellular repair machinery. This yields
a 5bp target site duplication that flanks the stably inte
grated provirus62. Modelling that was based on the crys
tal structure of the PFV intasome suggested that HIV1
integrase accommodates target DNA with a severely bent
conformation; integrasetarget DNA interactions prefer
specific, flexible dinucleotides at the centre of integra
tion sites63,64. Several amino acids of PFV integrase were
shown to distort the target DNA57. Homology modelling
between the TTC of HIV1 and the crystal structure of
the PFV intasome showed that the amino acids Ser119
and Arg231 of the HIV1 integrase are in direct contact
with the target DNA65,66. Careful invitro analysis of 13
integrase mutations yielded a total of 1610 unique inte
gration sites, from which a preferred target DNA signa
ture emerged. HIV1 targets (0)RYXRY(4) sequences
(in which R refers to a purine and Y refers to a pyrim
idine), which favours overlapping flexible dinucleo
tides at the centre of the integration site; whether this
sequence is the one that is responsible for the preferred
targeting of gene-dense regions still remains to be deter
mined65. Although the preference for integration into this
sequence is weak, it still predicts integration targeting well
when compared with randomly chosen sequences65,67,68.
Interestingly, polymorphisms at position 119 and posi
tion 231, which are frequently observed, can re-target the
intasome away from transcriptionally active regions66. In
particular, two variants (S119G and R231G) retain cata
lytic activity while having altered requirements for target
DNA binding and decreased electrostatic compatibility
with certain nucleosomes. Consequently, integration was
distorted away from gene-dense chromatin regions; these
polymorphisms were also proposed to accelerate disease
progression in longitudinal follow-updata66.
A single-particle cryo-electron microscopy study
demonstrated how the PFV intasome captures chromo
somal DNA that is wrapped around a mononucleosome.
During this interaction, the histone octamer remains
intact while DNA is lifted from the H2AH2B surface to
enable integration at a strongly preferred location. In the
case of HIV1, this occurs preferentially at 3.5 turns of
the DNA superhelix. On the basis of mutagenesis studies
that changed the intasome preferences for chromatin, the
authors proposed that contacts between the intasome and
the Nterminal tails and Cterminal domains of histones
enable the intasome to decode epigenetic marks69,70.
Finding the right target
Integration can hypothetically occur throughout the
genome; nevertheless, it is not a random process71. HIV1
integrase prefers a specific sequence at the end of the viral
DNA, whereas at the chromatin level the selection of inte
gration site is influenced by three major determinants:
sequence specificity, chromatin structure and cellular
tethering factors.
Sequence specificity and chromatin determinants.
HIV1 preferentially integrates into active genes. The
first integration site mapping showed that HIV1 favoured
integration into transcriptional units in a Tcell line72.
This preference was then confirmed invivo in resting
CD4+ Tcells from infected individuals70. Subsequently,
a detailed map of preferred target sites was generated,
which confirmed that in most cell lines HIV1 integrates
into transcriptionally active regions60,7376. HIV1 integra
tion occurs in active transcriptional units72,77,78. A recent
study of approximately 1 million integration sites in
infected HEK293 cells showed that 75% of integrations
occurred in active, RNA pol II-dependent transcriptional
units that had numerous introns; when corrected for
their relative length, no selection of intronic over exonic
sequences was observed40. Analysis of 1,000 transcrip
tional units that had the largest number of total integra
tions revealed that integration density correlates strongly
with the levels of splicing, and that the cellular protein
LEDGF is required for targeting highly spliced transcrip
tional units, through its direct interaction with numerous
splicing factors79. Large sets of data on the integration
sites of primary Tcells from individuals infected with
HIV1 confirmed that HIV1 preferentially integrates
into highly transcribed genes7,80,81. Interestingly, these
recent studies have provided strong evidence that inte
gration into particular genes can cause clonal expansion
and persistence of the infected cell7,8082. Analysis of patient-
derived samples demonstrated that HIV1 shows a strong
preference towards expressed genes6,74,83 with several
independent integrations identified in certain genes. One
study 81 identified 15 independent integrations in intron4
and intron 5 of the BACH2 gene, all of which were in the
same transcriptional orientation as the host gene. By con
trast, invitro infection of HeLa cells or haematopoietic
stem cells showed integrations throughout the gene in
either orientation. The MKL/myocardin-like2 (MKL2)
gene also showed this interesting pattern of multiple
invivo integrations in a specific region of the gene in the
same transcriptional orientation76. Another study iden
tified BACH2 integrations in two out of three patients80.
BACH2, signal transducer and activator of transcrip
tion5B (STAT5B) and MKL2 are recurrent integration
sites for HIV1, with each site identified multiple times in
different patients7,80,81,83. Interestingly, these genes encode
cellular transcription factors that are involved in cell
growth, with BACH2 and MKL2 being reported to have
proto-oncogenic functions84,85. Frequent identification of
these integration sites are a result of the clonal expan
sion of Tcells after HIV1 integration, which means that
HIV1 integration into these genes increases the long-
term survival of the host cells and possibly also alters the
function of thesegenes.
Many genes that were identified in patient stud
ies7,80,81,83 are also found in invitro HIV1 infections of
primary CD4+ cells, peripheral blood mononuclear
cells (PBMCs) or Tcell lines68,72,86,87. These genes, which
reappear in different data sets, termed recurrent integra
tion genes (RIGs), have common features: they are local
ized in specific regions of chromosomes, as previously also
suggested for the so-called hotspots of integration72, and
are positioned in the outer shell of the Tcell nucleus87.
Considering that integration into some of these genes
may lead to clonal expansion of the target cells, further
analysis of their genetic architecture and transcriptional

6 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Euchromatin
A loosely packed form of
chromatin (DNA, RNA and
histones) that is usually
enriched in genes that are
often being actively
transcribed. Most of the
genome is euchromatic
(predicted at approximately
90%), whereas the rest is
heterochromatic, that is,
tightlypacked and not
activelytranscribing.
Euchromatin is often referred
to as open chromatin.
SWI/SNF chromatin
remodelling complex
A complex that remodels
nucleosomes using the
hydrolysis of ATP to regulate
the accessibility of DNA to the
transcription machinery.
capacity is required. This is of particular importance
because the clonal expansion of infected cells that carry
replication-competent HIV1 could greatly complicate
efforts to eradicate the infection.
Integration into transcriptional units correlates
with several features, such as high GC content, high
CpG island density and specific histone marks; indeed,
euchromatin can easily be accessed by the viral PICs40,88.
Invivo, HIV1 integration favours the major groove
of DNA, facing outwards from the nucleosome core77.
Analysis of approximately 40,000 integration sites in a
Tcell line revealed associations between integration
frequency and transcription-associated histone mod
ifications77. For example, H3K9 acetylation, which is
characteristic of transcriptionally active genes, and
trimethylated H3Lys36 (H3K36me3), which is associ
ated with elongating RNA pol II and productive tran
scription, correlated with HIV1 integration sites87.
Importantly, LEDGF, the main chromatin-tethering
factor of HIV1, binds to H3K36me3.
Other retroviruses also show high preferences for
euchromatin, but usually target promoter regions89,
whereas HIV1 generally integrates into transcriptional
units and introns, which indicates that factors that are
unrelated to chromatin also contribute to the selection
of integrationsite.
Chromatin tethering. LEDGF is probably the most
important host factor for HIV1 integration, determining
both integration efficiency and integration site selection.
LEDGF is a chromatin-tethering factor that has been
implicated in cellular differentiation and response to
stress90,91. It was initially identified as an integrase cofac
tor 92 by coimmunoprecipitation; numerous studies that
followed have confirmed its pivotal role in the replication
of HIV1 (REFS9397) (reviewed in REF.98). The full-length
isoform of 75kDa contains a Cterminal integrase-
binding domain (IBD), which is absent in the trun
cated isoform of 52kDa; both isoforms contain the
conserved Nterminal PWWP domain (Pro-Trp-Trp-Pro),
and both were shown to associate with several splic
ing factors79,99. The IBD tethers integrase to chromatin
through the conserved Nterminal PWWP domain,
which binds specifically to H3K36me3 (REFS99,100).
The association of LEDGF with active regions of chro
matin, which was first probed by DNA adenine methyl
transferase identification (DamID)101,102, most likely
occurs through a direct interaction with splicing fac
tors79,99. LEDGF thus directs the integration of HIV1
into highly spliced transcriptional units and intron-rich
genes79. Depletion of LEDGF shifts integration away
from active genes93,97. Chimeric LEDGF molecules, in
which the chromatin-binding module was replaced by
the chromatin-reader domains of other proteins, also
showed a shift in integration patterns78,103. Hepatoma-
derived growth factor-related protein 2 (HDGFRP2), a
host protein that also has PWWP and IBD domains, is
often associated with the residual integration capacity of
HIV1 in LEDGF knockdown cells; however, studies with
cells that lack both LEDGF and HDGFRP2 have shown
residual, non-random integration in active genes96.
Other factors besides LEDGF are also involved in
chromatin tethering and integration site selection and
some of them are included in the PIC before nuclear
import. High mobility group protein A1 (HMGA1) was
among the first proteins to be identified as an integration-
relevant factor 104 . This non-histone chromatin-
associated protein can interact with viral DNA, but
not with integrase, and might help in bringing the
viral DNA ends together in the intasome104. Barrier-to
autointegration factor (BAF), a protein that is involved in
the assembly and organization of the nuclear envelope,
is included in PICs, in which it interacts with viral DNA
probably to compact it and prevent premature self-inte
gration9. Other cellular factors can interact with integrase
directly, such as integrase interactor 1 (INI1). Although
the importance of INI1 in the stimulation of HIV1 inte
gration is debated, INI1 could, as part of the SWI/SNF chro
matin remodelling complex, facilitate targeted integration
into SWI/SNF-remodelled regions of the genome105.
Several cellular partners that are able to post-
translationally modify HIV1 integrase have also been
reported to affect its enzymatic activity. In particular,
acetylation by histone acetyl transferase (HAT) p300
increases the affinity of integrase for DNA54. Acetylated
integrase can be bound by transcription intermediary
factor 1 (TIF1, also known as TRIM28 and KAP1), a
member of the tripartite motif (TRIM) family of antiviral
proteins that, in a complex with histone deacetylase1
(HDAC1), deacetylates integrase and restricts integra
tion106. It is intriguing to hypothesize that the binding of
TIF1 to integrase could serve to re-target the viral genome
towards the more repressed regions of cellular chromatin
in which the virus could remain silent for certain periods
of time; however, this requires further exploration.
Determinants of HIV1 positioning in the nuclear space.
Recent evidence that CPSF6 has a role in integration site
targeting has focused attention on the role of capsid.
CPSF6, which binds to capsid rather than HIV1 inte
grase, was shown to markedly influence integration into
transcriptionally active spliced genes and regions of open
chromatin40. A tandem knockout of CPSF6 and LEDGF
decreased integration into genes and revealed that CPSF6
had a more dominant role than LEDGF in targeting
HIV1 integration to euchromatin. A two-phase mecha
nism, in which capsidCPSF6 binding directs HIV1 to
actively transcribed chromatin and integraseLEDGF
binding directs integration into gene bodies, seems a
very probable explanation; this reflects the complex
interplay between the incoming PICs and the host cell
chromatin. This hypothesis is supported by data from
a recent independent study in which the spatial nuclear
distribution of HIV1 PICs was assessed in the presence
or absence of LEDGF; it was shown that HIV1 maintains
its spatial distribution in the euchromatic regions of the
genome independent of LEDGF107, which suggests that it
is indeed the capsidCPSF6 interaction that directs the
PIC towards the preferred regions in the nucleus.
The nucleus is a highly dynamic and compart
mentalized environment, in which chromosomes and
genes occupy preferred positions108; this high degree

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 7

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Nuclear basket of the HIV1 genome. Especially interesting is the role of

Nuclear (including NUP153, Nuclear pore complex
lamina NUP98 and TPR)
Nuclear envelope
LAD
CPSF6
HIV-1
LEDGF
Open
chromatin
Higher rate of HIV-1
1 m integration
Lower rate of HIV-1
integration
Figure 4 | HIV1 integration at the nuclear periphery. HIV1 integration
Nature Reviews into
| Microbiology
transcriptionally active genes and regions of open chromatin is enabled by the cellular
factors lens-epithelium-derived growth factor (LEDGF), which is a chromatin-tethering
factor that binds to HIV1 integrase, and cleavage and polyadenylation specificity factor6
(CPSF6), which binds to the capsid protein. HIV1 integration, which occurs in the outer
shell of the nucleus in proximity to the nuclear pore complex (NPC), is also dependent
onnucleoporin 153 (NUP153), NUP98 and TPR. Consistently, highly targeted HIV1
integration sites (or genes) are preferentially positioned in the outer shell of the nucleus,
1m beneath the nuclear envelope. LAD, lamin-associated domain.
of spatial organization determines nuclear functions109.
Visualization of infected primary human CD4+ Tcells
by 3D immuno-DNA fluorescence insitu hybridiza
tion revealed that HIV1 preferentially integrates in the
peripheral nuclear compartment, within 1m from
the edge of the nucleus87. This is consistent with previ
ous findings that showed preferential peripheral nuclear
localization of viral PICs. PICs, visualized either by
EGFP (enhanced green fluorescent protein) fused to
integrase88,107 or by YFP (yellow fluorescent protein)
fused to the host protein APOBEC3F110, were distrib
uted in close proximity to the nuclear envelope and not
throughout the nucleus.
The nuclear architecture could thus determine the
selection of integration site (FIG.4); in fact, highly targeted
RIGs were observed to be prevalently distributed at the
nuclear periphery 87. RIGs are characterized by the pres
ence of RNA polymerase II and association with open
chromatin marks; by contrast, HIV1 strongly disfa
vours the heterochromatic condensed regions in lamin-
associated domains (LADs)111 as well as other centrally
located transcriptionally active regions87. The nuclear
periphery contains open chromatin regions that under
lie the NPC (comprehensively reviewedin REF.112), and
HIV1 DNA is bound by several proteins of the NPC87.
Nups have important roles in cellular functions such as
nuclear trafficking, chromosome segregation during
mitosis and genome integrity 112,113. Tethering to the NPC
could be required for the optimal transcriptional activity
TPR, a protein of the inner basket of the NPC. TPR
knockdown has almost no effect on the efficiency of viral
integration (as measured by Alu-PCR), but markedly
impairs viral transcription87,88,114, which is consistent with
the decreased deposition of open chromatin marks, in
particular H3K36me3 (which associates with the elongat
ing RNA pol II), on HIV1 target genes114. TPR regulates
the expansion of heterochromatic regions in proximity
to the NPC115. Furthermore, TPR stabilizes LEDGF in the
nuclear periphery 114, which implies that the two proteins
might work together to direct HIV1 DNA towards its
preferred sites in the nuclearperiphery 116.
Functional implications of integration
Some aspects of the positioning of integration sites seem
to be a consequence of nuclear entry through the nuclear
pore. An example is the involvement of NUP153 in the
distribution of integration sites16,26,27. Moreover, the pro
moter activity of integrated LTR promoters depends
on an open chromatin conformation and the binding
of different transcription factors117,118. Thus, it is highly
probable that if regions of open chromatin are in close
proximity to nuclear pores, they will be preferred sites
for integration and viral gene expression.
The functional implications of integration site posi
tioning in the human genome are not fully understood,
despite several attempts to address this question experi
mentally. It still remains unclear how strong the relation
ship is between the selection of integration site and the
necessity for the provirus to be efficiently and rapidly
transcribed (and continue with the productive life cycle).
The spatial positioning of the viral genome in proximity
of the nuclear pore is depicted in FIG.5a. Similarly, it is still
unclear whether and how the integration sites relate to
the capacity of the provirus to establish a latent infection.
Following integration into the cellular genome, the fate of
the provirus is determined by several factors. Active tran
scription of the provirus leads to the production of new
viral progeny, whereas silencing, which is caused by sev
eral factors, leads to transcriptional repression and pro
viral latency 119. Among different factors that contribute
to latency, HIV1 chromatin structure as well as provirus
location in the host chromatin depend on the integra
tion site. Taking into account preferential integration into
open chromatin regions, it remains to be determined how
the heterochromatic status of the provirus is established
during latency (FIG.5b,c shows some options).
One possibility is that the virus is initially integrated
into permissive regions of the genome, but then becomes
repressed during the transition of the target cell from an
activated state towards a quiescent state through direct
effects such as repressive chromatin marks deposited
on the sites of integration120122, CpG promoter methyla
tion123,124, decreased binding of transcription factors some
of which are excluded from the nucleus in resting CD4+
Tcells125127, and transcriptional interference from neigh
bouring host genes128130 (reviewed in REFS117,119,131).
Silencing of the HIV1 genome through an association
with alphoid pericentromeric regions from a different
chromosome132 suggests that trans-acting mechanisms

8 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

a Figure 5 | Connecting integration and transcription.

Nuclear pore Nuclear basket
complex (including NUP153 a|HIV1 integration, which is mediated by the cellular
Nuclear lamina and TPR) factors lens-epithelium-derived growth factor (LEDGF)
and cleavage and polyadenylation specificity factor 6
Nuclear envelope (CPSF6), occurs in proximity to the nuclear pore complex
(NPC), where the association of the viral genome with
nucleoporin 153 (NUP153) and TPR facilitates viral gene
expression. The surrounding chromatin environment is
defined as euchromatic (green). Heterochromatic regions
are in orange. b | Changes in cellular activation state could
trigger the modification of chromatin organization,
CPSF6 resulting in a heterochromatic environment surrounding
the integrated HIV1 genome. This can occur following
LEDGF integration of the viral genome in the nuclear periphery.
Association with the nuclear lamina, in which histone
and DNA-methylation patterns contribute to the
heterochromatic environment, could make the viral genome
less accessible to transcription factors. c|Alternatively,
HIV1 can integrate in the inner regions of the nucleus where
it becomes silent upon encountering different nuclear
subcompartments (such as promyelocytic leukaemia (PML)
nuclear bodies or other heterochromatic regions).
b

could also be involved in latency. In fact, the association of

transcriptionally inert HIV1 with promyelocytic leukaemia
bodies (PML bodies) that was recently described as an
example of silencing in trans118,133, adds to the importance
of the spatial organization of the nucleus in determining
the fate of HIV1 (FIG.5c).
Silencing of the viral genome could also occur
through direct integration into heterochromatic
regions, which would not be permissive for transcrip
tion. Genomic location of integration was first pro
posed to contribute to latency when it was shown in a
cell line model that HIV1 transcriptional control is, in
part, dependent on the state of the surrounding chro
matin134. Regulation of HIV1 transcription was shown
to be dependent on the orientation of the integrated
viral genome and on host gene transcriptional read-
c through128,129. However, a comprehensive meta-analysis76
of integration sites in five well-characterized models of
HIV1 latency in combination with known transcrip
tional regulatory genomic features (such as histone
acetylation, histone methylation, CpG islands, alphoid
repeats, gene orientation and gene deserts), failed to find
a statistical correlation between latency and genomic
features that were relevant for all five models of latency.
The features that affect latency seem to be largely local
and model specific76.
The problem of identifying replication-competent
viruses in patient-derived samples has hindered our
understanding of the relationship between HIV1 inte
gration sites and latency. Recent studies have shown
that almost all proviruses (98%) in resting CD4 +
Tcells from patients who are undergoing treatment
have major defects that preclude replication, such as
APOBEC3Ginduced hypermutation and large inter
nal deletions6,135. Latent proviruses that are replication-
PML competent are extremely rare and difficult to identify;
nuclear
body hence a detailed analysis of the factors that were pro
posed to contribute to latency in invitro models, such
as chromatin structure and histone modifications, could

Nature Reviews | Microbiology

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 9

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Promyelocytic leukaemia
bodies
(PML bodies). Nuclear
structures the main component
of which is the promyelocytic
leukaemia protein (or TRIM19).
Several different protein
components as well as
various functions, such as
transcription, apoptosis,
senescence DNA damage
response and antiviral defence,
are associated with these
bodies.
CpG islands
Regions that have a high
frequency of CpG sites, usually
connected with the promoter
regions of genes.
not really be studied invivo. However, recent studies of
integration sites in patients who are undergoing pro
longed ART in whom clonally expanded HIV1infected
CD4 + T cells were identified, reinforced the role
of integration sites in the expansion and persistence of
HIV1 (REFS7,8082). Although the majority of viruses
are defective, some clonally expanded cells contain
replication-competent viruses. A study recently identi
fied a substantially expanded cellular clone in a patient
with HIV1 and squamous cell carcinoma82. The clone
carried a replication-competent provirus that was inte
grated into a repetitive region of the host DNA; hence,
the integration site could not be precisely localized.
Under current drug-treatment regimens, replication-
competent viruses persist within resting memory CD4+
Tcells1,2,5 and can further be expanded through the
clonal amplification of these cells7,8082,136. An additional
level of complexity was introduced by the finding that
only a small proportion of the intact, non-defective pro
viruses in individuals undergoing ART were induced fol
lowing reactivation6. Out of 213 non-induced proviral
clones, around 12% had intact genomes and were inte
grated into active transcriptional units, which suggests
that these replication-competent non-induced proviruses
could become activated invivo. However, not all of these
intact proviruses were induced to release virus follow
ing cellular activation in the standard viral outgrowth
assay, which is often used to measure the latent reser
voir 6. Repeated stimulation of infected cells from patients
with HIV1 resulted in more viruses being reactivated
from latency than in the first round, which implies that
reactivation from latency is a stochasticevent 6.
The mechanisms that maintain HIV1 invivo were
recently explored in a detailed genetic analysis of viral
sequences from individuals who have natural control
of the infection (so-called HIV1 controllers). Marked
differences were identified between populations of
HIV1infected CD4+ Tcells in blood and lymphoid
tissues. Ongoing infection was reported to occur in
follicular helper Tcells (TFH) and other memory CD4+
subsets in lymph nodes. As some of these cells survive
and return to the circulation, containing an inducible
HIV1 genome, it was proposed that these cells link
the pool of infected cells from lymphoid tissues to the
pool of infected cells in blood. The latter pool of cells
is represented mostly by highly differentiated, clon
ally expanded CD4+ Tcells of the memory phenotype
that harbour archival viral sequences that usually are
refractory to activation137.
Proliferative self-renewal of the viral sequences in
clonally expanded cells can indeed represent a selective
advantage for the provirus. However, it is unclear how
clonal proliferation affects transcriptional processes and,
in particular, how it affects expression of the provirus.
These studies also point to the fact that integrated
viruses in resting CD4+ Tcells are heterogeneous in
their nature and can indeed be divided into those that
are induced to release replication-competent virus
after one round of activation and those that are not
(non-induced). Most of the non-induced proviruses
are defective, but a portion is intact and can be induced
through additional rounds of activation. A minimal esti
mate of the inducible latent viral reservoir is provided
by the viral outgrowth assay (VOA)6, whereas PCR-
based approaches detect all proviruses and thus vastly
overestimate the size of the latent reservoir 138.
Although studies on material from patients infected
with HIV represent the most valuable source of informa
tion, an interesting insight into the correlation between
integration sites and latency could also be obtained from
experimental approaches. In this regard, the develop
ment of a dual colour (HIV Duo-Fluo I) virus system,
which detects latently infected cells following infection,
represents an interesting tool139,140.
Understanding the relevance of integration sites for
the outcome of viral gene expression and infection may
have much broader implications. It might be relevant for
the development of gene therapy tools (vectors) that are
applicable in clinics and can have also important con
notations for understanding the functions and networks
in the nucleus that are related to stem cell properties
andcancer.
Therapeutic targeting of integration
Antiretroviral drugs that are designed to target the
three essential viral enzymes, reverse transcriptase,
protease and integrase, have a crucial role in ART. The
first ART regimens that were able to suppress viraemia
to below the limit of detection in clinical assays con
sisted of two nucleoside analogue reverse transcriptase
inhibitors (NRTIs) in addition to a non-nucleoside
reverse transcriptase inhibitor or an inhibitor of HIV1
protease141,142. Although regimens of this kind were the
mainstay of ART for many years, they have been largely
replaced by regimens that consist of two NRTIs and an
integrase inhibitor 143 Thus, the discovery of HIV1 inte
grase inhibitors has had a major effect on treatment.
A novel screen for compounds that blocked the strand
transfer reaction led to the discovery of this class of
drugs144. Raltegravir (RAL) is a prototypical integrase
strand transfer inhibitor (INSTI). In combination with
two NRTIs, RAL causes a very rapid decrease in viral
load145. The rapid decrease in viraemia results from
the fact that INSTIs act later in viral life cycle than
other antiretrov iral drugs rather than from excep
tional antiviral activity 146. INSTIs lack the cooperative
doseresponse curves that are responsible for the high
antiviral activity of the HIV1 protease inhibitors147.
The clinical success of INSTIs results mainly from
favourable synergistic interactions with other antiretro
viral drugs and from the fact that they have few side
effects148. The lack of side effects promotes adherence,
which is crucial to the success of ART. If adherence is
suboptimal, patients develop resistance to RAL, indi
cating an urgent need to develop new generations of
integrase inhibitors or find an alternative approach to
target integration.
Elvitegravir (EVG) and dolutegravir (DTG) were
developed as a new generation of INSTIs. ELV shows
some cross-resistance with RAL149. Resistance to DTG has
not yet proven to be a clinical problem, possibly because
of the high fitness cost of resistancemutations150,151.

10 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Cre recombinase
A tyrosine recombinase
enzyme that catalyses a
site-specific recombination
event between two DNA
recognition sites; these sites
consist of 13bp palindromic
sequences that flank an 8bp
spacer region. The unique and
specific recombination system
of the enzyme is used to
manipulate genes and
chromosomes in several
applications.
Zinc-finger nucleases
(ZFNs). Artificial restriction
enzymes that are created
through the fusion of a
zinc-finger DNA-binding
domain and a DNA-cleavage
domain. These enzymes
facilitate targeted editing of
thegenome by creating
double-strand breaks in the
DNA at a specific (desired)
location.
Transcription activator-like
effector nucleases
(TALENs). Restriction nucleases
that are engineered to cut
specific DNA sequences.
Efforts have also been made to design inhibitors
that have a different mode of action than INSTIs. One
such approach is to target integrase multimerization,
which is a highly dynamic process of interactions
between different subunits. Several molecules that
bind to theintegrase CCD dimer interface promote
aberrantmultimerization152.
Another target is LEDGF interaction, which occurs
through the IBD of LEDGF and the CCD of integrase153.
The proteinprotein interaction surface involves a
well-defined pocket, is limited in size and is composed
of multiple hydrophobic interactions and hydrogen bond
interactions; it thus represents an excellent starting point
for the development of small-molecule inhibitors. The
first small-molecule quinoline-based inhibitors, which
were reported to target the binding site in the CCD, were
termed integraseLEDGF inhibitors (LEDGINs)154 or
allosteric integrase inhibitors (ALLINIs); they simul
taneously block the multimerization and allosterically
inhibit the enzymatic activity of integrase154. LEDGINs
are not cross-resistant with INSTIs: the mode of action of
INSTIs requires the assembly of intasomes on the 3 ends
of LTRs, whereas LEDGINs must be present before this
assembly to reach full efficacy. LEDGINs not only block
integration but also decrease the replicative capacity of
HIV1 by increasing the multimerization of integrase
during virion assembly 155. Efforts are being made to eval
uate LEDGINs for clinical use, but clinical trials have not
yet been carriedout.
Another alternative approach is focused on targeting
the integrated provirus rather than blocking the inte
gration reaction. Strategies that are based on cleavage
of the integrated provirus have gained a lot of atten
tion in the past few years. These strategies are based on
programmable nucleases that specifically bind to, and
cleave, integrated HIV1 DNA. Among the first enzy
matic candidates is a Cre recombinase (Tre) that binds to,
and cuts, the LTR156,157; more recently, engineered zinc-
finger nucleases (ZFNs) or transcription activator-like effector
nucleases (TALENs) were developed as promising new
tools for nuclease-based HIV1 therapy. These nucle
ases were designed with the goal of decreasing off-target
mutations and obtaining higher nuclease activity 158,159.
Despite the great potential of nuclease-based approaches,
off-target effects, as well as technical challenges that are
related to their delivery to target cells, prevent their
clinical translation at this presenttime.
The newest entry in the field of gene editing, the
CRISPRCas9 system, has advantages over ZFNs and
TALENs. DNA target specificity is determined by a 1723
nucleotide guide RNA that binds to Cas9 and directs it
to a complementary DNA for cleavage; the formed ribo
nucleoprotein complex shows higher specificity than the
proteinDNA complexes that are formed in ZFN and
TALEN systems and can be targeted to any sequence in
the genome. The system has already been used by differ
ent research groups to cleave and inactivate HIV1 DNA
in human cells160163. These pioneering studies will possi
bly lead to future clinical applications with the purpose
of either cleaving integrated latent HIV1 or inhibiting
viral replication once the problem of delivery issolved.
Conclusions
A huge effort has been made in the past few decades
towards a better understanding of the molecular mecha
nisms that govern the integration of HIV1 into the cellu
lar genome. HIV1 preferentially targets transcriptionally
active genes and open regions of chromatin. Inside the
nuclear space not all active genes are targeted in the same
way, because HIV1 prefers active genes that are spatially
distributed in the outer shell of the nucleus. Cellular pro
teins that are involved in nuclear entry have implications
for integration that, although controlled by the viral inte
grase, depend on the chromatin-tethering factor LEDGF,
as well as on nuclear pore proteins. Despite the fact that
productive transcription usually follows integration,
silencing of the viral genome can occur and persistent
viral reservoirs, which are a major hurdle in curing infec
tion with HIV1, are formed. Very intriguing questions
that still need to be answered are to what extent are inte
gration and viral transcription connected and how do sites
of integration define the transcriptional activity of the
provirus. Most importantly, is there a direct connection
between integration and silencing of the viral genome?
Although there are indications that HIV integration might
be tightly connected with the establishment and mainte
nance of latency this requires further and more definitive
confirmation, and will be a subject of future studies.

1. Chun,T.W. etal. Quantification of latent tissue
reservoirs and total body viral load in HIV1 infection.
Nature 387, 183188 (1997).
2. Finzi,D. etal. Identification of a reservoir for HIV1
inpatients on highly active antiretroviral therapy.
Science 278, 12951300 (1997).
3. Wong,J.K. etal. Recovery of replication-competent
HIV despite prolonged suppression of plasma viremia.
Science 278, 12911295 (1997).
4. Chun,T.W. etal. Presence of an inducible HIV1
latentreservoir during highly active antiretroviral
therapy. Proc. Natl Acad. Sci. USA 94, 1319313197
(1997).
5. Finzi,D. etal. Latent infection of CD4+ Tcells provides
a mechanism for lifelong persistence of HIV1, even in
patients on effective combination therapy. Nat. Med.
5, 512517 (1999).
6. Ho,Y.C. etal. Replication-competent noninduced
proviruses in the latent reservoir increase barrier
toHIV1 cure. Cell 155, 540551(2013).
This study demonstrates that the majority of
proviruses in resting CD4+ T cells from treated
patients are non-inducible because they are
highlydefective. A portion of these non-induced
proviruses have intact genomes and can be induced
after several rounds of stimulation.
7. Cohn,L.B. etal. HIV1 integration landscape
duringlatent and active infection. Cell 160, 420432
(2015).
Together with references 80 and 81, this work
shows that HIV1 integrates into genes that are
associated with cellular proliferation and clonal
expansion. It also suggests that most of the
proviruses in dividing clonally expanded Tcells
aredefective.
8. Coffin,J., Hughes,S. & Varmus,H. (eds) Retroviruses.
(Cold Spring Harbor Laboratory Press, 1997).
9. Suzuki,Y. & Craigie,R. The road to chromatin
nuclear entry of retroviruses. Nat. Rev. Microbiol. 5,
187196 (2007).
10. Matreyek,K.A. & Engelman,A. Viral and cellular
requirements for the nuclear entry of retroviral
preintegration nucleoprotein complexes. Viruses 5,
24832511 (2013).
11. Yamashita,M. & Emerman,M. Capsid is a
dominantdeterminant of retrovirus infectivity in
nondividing cells. J.Virol. 78, 56705678 (2004).
12. Yamashita,M., Perez,O., Hope,T.J. & Emerman,M.
Evidence for direct involvement of the capsid protein
in HIV infection of nondividing cells. PLoS Pathog. 3,
15021510 (2007).
13. Lee,K. etal. Flexible use of nuclear import pathways
by HIV1. Cell Host Microbe 7, 221233 (2010).
A study that identifies truncated CPSF6 as a
dominant negative inhibitor of HIV1 infection
and in which CPSP6 is shown to have a role in
targeting HIV1 to use specific cofactors.
14. Schaller,T. etal. HIV1 capsid-cyclophilin
interactions determine nuclear import pathway,
integration targeting and replication efficiency. PLoS
Pathog. 7, e1002439 (2011).
This work connects, for the first time, the capsid of
HIV1 with the selection of integration site by
showing that capsid mutants that do not recruit
CPSF6 or CYPA integrate into different target
regions of the genome.

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 11

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

15. Peng,K. etal. Quantitative microscopy of functional HIV
post-entry complexes reveals association of replication
with the viral capsid. eLife 3, e04114 (2014).
16. Koh,Y. etal. Differential effects of human
immunodeficiency virus type1 capsid and cellular
factors nucleoporin 153 and LEDGF/p75 on the
efficiency and specificity of viral DNA integration.
J.Virol. 87, 648658 (2013).
17. Bukrinsky,M.I. etal. A nuclear localization signal
within HIV1 matrix protein that governs infection of
non-dividing cells. Nature 365, 666669 (1993).
18. Reil,H., Bukovsky,A.A., Gelderblom,H.R.
&Gottlinger,H.G. Efficient HIV1 replication can occur
in the absence of the viral matrix protein. EMBO J. 17,
26992708 (1998).
19. Guenzel,C.A., Herate,C. & Benichou,S. HIV1 Vpr
astill enigmatic multitasker. Front. Microbiol. 5, 127
(2014).
20. Popov,S. etal. Viral protein R regulates nuclear import
of the HIV1 pre-integration complex. EMBO J. 17,
909917 (1998).
21. Fouchier,R.A. etal. Interaction of the human
immunodeficiency virus type1Vpr protein with the
nuclear pore complex. J.Virol. 72, 60046013 (1998).
22. McDonald,D. etal. Visualization of the intracellular
behavior of HIV in living cells. J.Cell Biol. 159,
441452 (2002).
23. Brass,A.L. etal. Identification of host proteins required
for HIV infection through a functional genomic screen.
Science 319, 921926 (2008).
24. Konig,R. etal. Global analysis of hostpathogen
interactions that regulate early-stage HIV1 replication.
Cell 135, 4960 (2008).
Together with reference 23, this work describes a
high-throughput screening that identifies several
cellular factors that are involved in HIV1 infection.
25. Bushman,F.D. etal. Host cell factors in HIV
replication: meta-analysis of genome-wide studies.
PLoS Pathog. 5, e1000437 (2009).
26. Di Nunzio,F. etal. Nup153 and Nup98 bind the HIV1
core and contribute to the early steps of HIV1
replication. Virology 440, 818 (2013).
27. Matreyek,K.A., Yucel,S.S., Li,X. & Engelman,A.
Nucleoporin NUP153 phenylalanine-glycine motifs
engage a common binding pocket within the HIV1
capsid protein to mediate lentiviral infectivity.
PLoSPathog. 9, e1003693 (2013).
28. Gamble,T.R. etal. Crystal structure of human
cyclophilin A bound to the amino-terminal domain of
HIV1 capsid. Cell 87, 12851294 (1996).
29. Ocwieja,K.E. etal. HIV integration targeting:
apathway involving Transportin3 and the nuclear pore
protein RanBP2. PLoS Pathog. 7, e1001313 (2011).
30. Bosco,D.A., Eisenmesser,E.Z., Pochapsky,S.,
Sundquist,W.I. & Kern,D. Catalysis of cis/trans
isomerization in native HIV1 capsid by human
cyclophilin A. Proc. Natl Acad. Sci. USA 99,
52475252 (2002).
31. Christ,F. etal. TransportinSR2 imports HIV into the
nucleus. Curr. Biol. 18, 11921202 (2008).
32. Kataoka,N., Bachorik,J.L. & Dreyfuss,G.
TransportinSR, a nuclear import receptor for SR
proteins. J.Cell Biol. 145, 11451152 (1999).
33. Lee,K. etal. HIV1 capsid-targeting domain of cleavage
and polyadenylation specificity factor 6. J.Virol. 86,
38513860 (2012).
34. Bhattacharya,A. etal. Structural basis of HIV1 capsid
recognition by PF74 and CPSF6. Proc. Natl Acad.
Sci.USA 111, 1862518630 (2014).
35. Rasheedi,S. etal. The cleavage and polyadenylation
specificity factor 6 (CPSF6) subunit of the capsid-
recruited pre-messenger RNA cleavage factor I (CFIm)
complex mediates HIV1 integration into genes.
J.Biol.Chem. 291, 1180911819 (2016).
36. Chin,C.R. etal. Direct visualization of HIV1
replication intermediates shows that capsid and
CPSF6 modulate HIV1 intra-nuclear invasion and
integration. Cell Rep. 13, 17171731 (2015).
37. Ruegsegger,U., Blank,D. & Keller,W. Human
pre-mRNA cleavage factor Im is related to
spliceosomal SRproteins and can be reconstituted
invitro from recombinant subunits. Mol. Cell 1,
243253 (1998).
38. De Iaco,A. etal. TNPO3 protects HIV1 replication
from CPSF6mediated capsid stabilization in the host
cell cytoplasm. Retrovirology 10, 20 (2013).
39. Hilditch,L. & Towers,G.J. A model for cofactor use
during HIV1 reverse transcription and nuclear entry.
Curr. Opin. Virol. 4, 3236 (2014).
40. Sowd,G.A. etal. A critical role for alternative
polyadenylation factor CPSF6 in targeting HIV1
integration to transcriptionally active chromatin.
Proc.Natl Acad. Sci. USA 113, E1054E1063
(2016).
This work shows, for the first time, that CPSF6, as a
capsid-binding protein, has a role in directing HIV1
integration to transcriptionally active chromatin
regions, whereas LEDGF, as an integrase partner,
directs integration into genes.
41. Arhel,N.J. etal. HIV1 DNA flap formation promotes
uncoating of the pre-integration complex at the
nuclear pore. EMBO J. 26, 30253037 (2007).
42. Craigie,R. & Bushman,F.D. HIV DNA integration.
Cold Spring Harb. Perspect. Med. 2, a006890
(2012).
43. Craigie,R. & Bushman,F.D. Host factors in retroviral
integration and the selection of integration target
sites. Microbiol. Spectr. 2, MDNA300262014
(2014).
44. Engelman,A., Bushman,F.D. & Craigie,R.
Identification of discrete functional domains of HIV1
integrase and their organization within an active
multimeric complex. EMBO J. 12, 32693275
(1993).
45. van Gent,D.C., Vink,C., Groeneger,A.A.
&Plasterk,R.H. Complementation between HIV
integrase proteins mutated in different domains.
EMBO J. 12, 32613267 (1993).
46. Dyda,F. etal. Crystal structure of the catalytic domain
of HIV1 integrase: similarity to other polynucleotidyl
transferases. Science 266, 19811986 (1994).
47. Craigie,R. HIV integrase, a brief overview from
chemistry to therapeutics. J.Biol. Chem. 276,
2321323216 (2001).
48. Carayon,K. etal. A cooperative and specific DNA-
binding mode of HIV1 integrase depends on the
nature of the metallic cofactor and involves the zinc-
containing Nterminal domain. Nucleic Acids Res. 38,
36923708 (2010).
49. Kulkosky,J., Jones,K.S., Katz,R.A., Mack,J.P.
&Skalka,A.M. Residues critical for retroviral
integrative recombination in a region that is highly
conserved among retroviral/retrotransposon
integrases and bacterial insertion sequence
transposases. Mol. Cell. Biol. 12, 23312338
(1992).
50. Leavitt,A.D., Shiue,L. & Varmus,H.E. Site-directed
mutagenesis of HIV1 integrase demonstrates
differential effects on integrase functions invitro.
J.Biol. Chem. 268, 21132119 (1993).
51. Manganaro,L. etal. Concerted action of cellular JNK
and Pin1 restricts HIV1 genome integration to
activated CD4+ T lymphocytes. Nat. Med. 16,
329333 (2010).
52. Lutzke,R.A., Vink,C. & Plasterk,R.H.
Characterization of the minimal DNA-binding domain
of the HIV integrase protein. Nucleic Acids Res. 22,
41254131 (1994).
53. Eijkelenboom,A.P. etal. The solution structure of the
amino-terminal HHCC domain of HIV2 integrase: a
three-helix bundle stabilized by zinc. Curr. Biol. 7,
739746 (1997).
54. Cereseto,A. etal. Acetylation of HIV1 integrase by
p300 regulates viral integration. EMBO J. 24,
30703081 (2005).
55. Savarino,A. In silico docking of HIV1 integrase
inhibitors reveals a novel drug type acting on an
enzyme/DNA reaction intermediate. Retrovirology 4,
21 (2007).
56. Hare,S., Gupta,S.S., Valkov,E., Engelman,A.
&Cherepanov,P. Retroviral intasome assembly and
inhibition of DNA strand transfer. Nature 464,
232236 (2010).
Together with reference 57, this study resolves the
Xray crystal structure of the PFV intasome, which
has substantially contributed to our understanding
of the mechanisms of retroviral-DNA integration.
57. Maertens,G.N., Hare,S. & Cherepanov,P. The
mechanism of retroviral integration from Xray
structures of its key intermediates. Nature 468,
326329 (2010).
Similar to reference 56, this study shows the
Xraycrystal structure of the PFV intasome in
complex with target DNA before, and following,
strand transfer.
58. Bushman,F.D., Fujiwara,T. & Craigie,R. Retroviral
DNA integration directed by HIV integration protein
invitro. Science 249, 15551558 (1990).
59. Craigie,R., Fujiwara,T. & Bushman,F. The IN protein
of Moloney murine leukemia virus processes the viral
DNA ends and accomplishes their integration invitro.
Cell 62, 829837 (1990).
60. Lewinski,M.K. etal. Retroviral DNA integration: viral
and cellular determinants of target-site selection.
PLoSPathog. 2, e60 (2006).
61. Krishnan,L. etal. Structure-based modeling of the
functional HIV1 intasome and its inhibition. Proc. Natl
Acad. Sci. USA 107, 1591015915 (2010).
62. Vink,C. etal. Analysis of the junctions between human
immunodeficiency virus type1 proviral DNA and
human DNA. J.Virol. 64, 56265627 (1990).
63. Muller,H.P. & Varmus,H.E. DNA bending creates
favored sites for retroviral integration: an explanation
for preferred insertion sites in nucleosomes. EMBO J.
13, 47044714 (1994).
64. Pryciak,P.M. & Varmus,H.E. Nucleosomes, DNA-
binding proteins, and DNA sequence modulate
retroviral integration target site selection. Cell 69,
769780 (1992).
65. Serrao,E. etal. Integrase residues that determine
nucleotide preferences at sites of HIV1 integration:
implications for the mechanism of target DNA binding.
Nucleic Acids Res. 42, 51645176 (2014).
66. Demeulemeester,J. etal. HIV1 integrase variants
retarget viral integration and are associated with
disease progression in a chronic infection cohort.
CellHost Microbe 16, 651662 (2014).
67. Holman,A.G. & Coffin,J.M. Symmetrical base
preferences surrounding HIV1, avian sarcoma/leukosis
virus, and murine leukemia virus integration sites.
Proc. Natl Acad. Sci. USA 102, 61036107 (2005).
68. Brady,T. etal. HIV integration site distributions in
resting and activated CD4+ Tcells infected in culture.
AIDS 23, 14611471 (2009).
69. Maskell,D.P. etal. Structural basis for retroviral
integration into nucleosomes. Nature 523, 366369
(2015).
This work shows, through the use of single-particle
cryoEM, how the PFV intasome as a viral DNA
recombination machinery captures nucleosomes to
enable integration.
70. Demeulemeester,J., De Rijck,J., Gijsbers,R.
&Debyser,Z. Retroviral integration: site matters:
mechanisms and consequences of retroviral
integration site selection. Bioessays 37, 12021214
(2015).
71. Bushman,F. etal. Genome-wide analysis of retroviral
DNA integration. Nat. Rev. Microbiol. 3, 848858
(2005).
72. Schroder,A.R. etal. HIV1 integration in the human
genome favors active genes and local hotspots. Cell
110, 521529 (2002).
The first study to define HIV1 integration sites
genome-wide through the use of whole-genome
sequencing. This study shows that HIV1 favours
integration into active transcriptional units.
73. Mitchell,R.S. etal. Retroviral DNA integration: ASLV,
HIV, and MLV show distinct target site preferences.
PLoS Biol. 2, E234 (2004).
74. Han,Y. etal. Resting CD4+ Tcells from human
immunodeficiency virus type1 (HIV1)-infected
individuals carry integrated HIV1 genomes within
actively transcribed host genes. J.Virol. 78,
61226133 (2004).
75. Barr,S.D. etal. HIV integration site selection:
targeting in macrophages and the effects of different
routes of viral entry. Mol. Ther. 14, 218225
(2006).
76. Sherrill-Mix,S. etal. HIV latency and integration site
placement in five cell-based models. Retrovirology
10, 90 (2013).
77. Wang,G.P., Ciuffi,A., Leipzig,J., Berry,C.C.
&Bushman,F.D. HIV integration site selection:
analysis by massively parallel pyrosequencing
revealsassociation with epigenetic modifications.
Genome Res. 17, 11861194 (2007).
78. Ferris,A.L. etal. Lens epithelium-derived
growthfactor fusion proteins redirect HIV1
DNA integration. Proc. Natl Acad. Sci. USA 107,
31353140 (2010).
79. Singh,P.K. etal. LEDGF/p75 interacts with mRNA
splicing factors and targets HIV1 integration to highly
spliced genes. Genes Dev. 29, 22872297 (2015).
80. Wagner,T.A. etal. HIV latency. Proliferation of cells
with HIV integrated into cancer genes contributes to
persistent infection. Science 345, 570573 (2014).
81. Maldarelli,F. etal. HIV latency. Specific HIV integration
sites are linked to clonal expansion and persistence of
infected cells. Science 345, 179183 (2014).
References 80 and 81 connect HIV1 integration
with the clonal expansion of target cells and propose
that this could contribute to the aberrant expansion
of viral genomes and latent reservoirs.

12 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

82. Simonetti,F.R. etal. Clonally expanded CD4+ Tcells
can produce infectious HIV1 invivo. Proc. Natl Acad.
Sci. USA 113, 18831888 (2016).
This work provides evidence that clonally expanded
HIV1infected cells contain replication-competent
viruses.
83. Ikeda,T., Shibata,J., Yoshimura,K., Koito,A.
&Matsushita,S. Recurrent HIV1 integration at the
BACH2 locus in resting CD4+ Tcell populations during
effective highly active antiretroviral therapy. J.Infect. Dis.
195, 716725 (2007).
84. Kobayashi,S. etal. Identification of IGHCBACH2
fusion transcripts resulting from cryptic chromosomal
rearrangements of 14q32 with 6q15 in aggressive
Bcell lymphoma/leukemia. Genes Chromosomes
Cancer 50, 207216 (2011).
85. Flucke,U. etal. Presence of C11orf95MKL2 fusion is
a consistent finding in chondroid lipomas: a study of
eight cases. Histopathology 62, 925930 (2013).
86. Liu,H. etal. Integration of human immunodeficiency
virus type1 in untreated infection occurs preferentially
within genes. J.Virol. 80, 77657768 (2006).
87. Marini,B. etal. Nuclear architecture dictates HIV1
integration site selection. Nature 521, 227231
(2015).
This work shows that HIV1 positions its genome in
regions in proximity to the NPC at the nuclear
periphery.
88. Albanese,A., Arosio,D., Terreni,M. & Cereseto,A.
HIV1 pre-integration complexes selectively target
decondensed chromatin in the nuclear periphery. PloS
ONE 3, e2413 (2008).
89. Wu,X., Li,Y., Crise,B. & Burgess,S.M. Transcription
start regions in the human genome are favored
targetsfor MLV integration. Science 300, 17491751
(2003).
90. Ge,H., Si,Y. & Roeder,R.G. Isolation of cDNAs
encoding novel transcription coactivators p52 and p75
reveals an alternate regulatory mechanism of
transcriptional activation. EMBO J. 17, 67236729
(1998).
91. Singh,D.P. etal. Lens epithelium-derived growth
factor: effects on growth and survival of lens epithelial
cells, keratinocytes, and fibroblasts. Biochem. Biophys.
Res. Commun. 267, 373381 (2000).
92. Cherepanov,P. etal. HIV1 integrase forms stable
tetramers and associates with LEDGF/p75 protein in
human cells. J.Biol. Chem. 278, 372381 (2003).
93. Ciuffi,A. etal. A role for LEDGF/p75 in targeting
HIVDNA integration. Nat. Med. 11, 12871289
(2005).
94. Llano,M. etal. An essential role for LEDGF/p75 in HIV
integration. Science 314, 461464 (2006).
95. Maertens,G. etal. LEDGF/p75 is essential for nuclear
and chromosomal targeting of HIV1 integrase in
human cells. J.Biol. Chem. 278, 3352833539 (2003).
96. Schrijvers,R. etal. LEDGF/p75independent HIV1
replication demonstrates a role for HRP2 and remains
sensitive to inhibition by LEDGINs. PLoS Pathog. 8,
e1002558 (2012).
97. Shun,M.C. etal. LEDGF/p75 functions downstream
from preintegration complex formation to effect gene-
specific HIV1 integration. Genes Dev. 21, 17671778
(2007).
98. Debyser,Z., Christ,F., De Rijck,J. & Gijsbers,R.
Hostfactors for retroviral integration site selection.
Trends Biochem. Sci. 40, 108116 (2015).
99. Pradeepa,M.M., Sutherland,H.G., Ule,J.,
Grimes,G.R. & Bickmore,W.A. Psip1/Ledgf p52 binds
methylated histone H3K36 and splicing factors and
contributes to the regulation of alternative splicing.
PLoS Genet. 8, e1002717 (2012).
100. Eidahl,J.O. etal. Structural basis for high-affinity
binding of LEDGF PWWP to mononucleosomes.
NucleicAcids Res. 41, 39243936 (2013).
101. Van Steensel,B. & Henikoff,S. Identification of invivo
DNA targets of chromatin proteins using tethered dam
methyltransferase. Nat. Biotechnol. 18, 424428
(2000).
102. De Rijck,J., Bartholomeeusen,K., Ceulemans,H.,
Debyser,Z. & Gijsbers,R. High-resolution profiling of
the LEDGF/p75 chromatin interaction in the ENCODE
region. Nucleic Acids Res. 38, 61356147 (2010).
103. Gijsbers,R. etal. Role of the PWWP domain of lens
epithelium-derived growth factor (LEDGF)/p75 cofactor
in lentiviral integration targeting. J.Biol. Chem. 286,
4181241825 (2011).
104. Farnet,C.M. & Bushman,F.D. HIV1 cDNA
integration: requirement of HMG I(Y) protein for
function of preintegration complexes invitro. Cell 88,
483492 (1997).
105. Lesbats,P. etal. Functional coupling between HIV1
integrase and the SWI/SNF chromatin remodeling
complex for efficient invitro integration into stable
nucleosomes. PLoS Pathog. 7, e1001280 (2011).
106. Allouch,A. etal. The TRIM family protein KAP1 inhibits
HIV1 integration. Cell Host Microbe 9, 484495
(2011).
107. Quercioli,V. etal. Comparative analysis of HIV1 and
murine leukemia virus three-dimensional nuclear
distributions. J.Virol. 90, 52055209 (2016).
108. Cremer,T. etal. Chromosome territories a functional
nuclear landscape. Curr. Opin. Cell Biol. 18, 307316
(2006).
109. Cavalli,G. & Misteli,T. Functional implications of genome
topology. Nat. Struct. Mol. Biol. 20, 290299 (2013).
110. Burdick,R.C., Hu,W.S. & Pathak,V.K. Nuclear import
of APOBEC3Flabeled HIV1 preintegration complexes.
Proc. Natl Acad. Sci. USA 110, E4780E4789 (2013).
111. Guelen,L. etal. Domain organization of human
chromosomes revealed by mapping of nuclear lamina
interactions. Nature 453, 948951 (2008).
112. Ibarra,A. & Hetzer,M.W. Nuclear pore proteins and
the control of genome functions. Genes Dev. 29,
337349 (2015).
113. Raices,M. & DAngelo,M.A. Nuclear pore complex
composition: a new regulator of tissue-specific and
developmental functions. Nat. Rev. Mol. Cell Biol. 13,
687699 (2012).
114. Lelek,M. etal. Chromatin organization at the nuclear
pore favours HIV replication. Nat. Commun. 6, 6483
(2015).
115. Krull,S. etal. Protein Tpr is required for establishing
nuclear pore-associated zones of heterochromatin
exclusion. EMBO J. 29, 16591673 (2010).
116. Wong,R.W., Mamede,J.I. & Hope,T.J. Impact of
nucleoporin-mediated chromatin localization and
nuclear architecture on HIV integration site selection.
J.Virol. 89, 97029705 (2015).
117. Van Lint,C., Bouchat,S. & Marcello,A. HIV1
transcription and latency: an update. Retrovirology 10,
67 (2013).
118. Lusic,M. & Giacca,M. Regulation of HIV1 latency
bychromatin structure and nuclear architecture.
J.Mol.Biol. 427, 688694 (2014).
119. Dahabieh,M.S., Battivelli,E. & Verdin,E.
Understanding HIV latency: the road to an HIV cure.
Annu. Rev. Med. 66, 407421 (2015).
120. du Chene,I. etal. Suv39H1 and HP1 are responsible
for chromatin-mediated HIV1 transcriptional silencing
and post-integration latency. EMBO J. 26, 424435
(2007).
121. Imai,K., Togami,H. & Okamoto,T. Involvement of
histone H3 lysine 9 (H3K9) methyltransferase G9a in
the maintenance of HIV1 latency and its reactivation
by BIX01294. J.Biol. Chem. 285, 1653816545
(2010).
122. Friedman,J. etal. Epigenetic silencing of HIV1 by the
histone H3 lysine 27 methyltransferase enhancer of
Zeste 2. J.Virol. 85, 90789089 (2011).
123. Kauder,S.E., Bosque,A., Lindqvist,A., Planelles,V.
&Verdin,E. Epigenetic regulation of HIV1 latency by
cytosine methylation. PLoS Pathog. 5, e1000495
(2009).
124. Blazkova,J. etal. CpG methylation controls
reactivation of HIV from latency. PLoS Pathog. 5,
e1000554 (2009).
125. Marcello,A. etal. Recruitment of human cyclin T1 to
nuclear bodies through direct interaction with the PML
protein. EMBO J. 22, 21562166 (2003).
126. Sabo,A., Lusic,M., Cereseto,A. & Giacca,M.
Acetylation of conserved lysines in the catalytic core of
cyclin-dependent kinase 9 inhibits kinase activity and
regulates transcription. Mol. Cell. Biol. 28,
22012212 (2008).
127. Williams,S.A., Kwon,H., Chen,L.F. & Greene,W.C.
Sustained induction of NFB is required for efficient
expression of latent human immunodeficiency virus
type1. J.Virol. 81, 60436056 (2007).
128. Lenasi,T., Contreras,X. & Peterlin,B.M.
Transcriptional interference antagonizes proviral gene
expression to promote HIV latency. Cell Host Microbe
4, 123133 (2008).
129. Han,Y. etal. Orientation-dependent regulation of
integrated HIV1 expression by host gene
transcriptional readthrough. Cell Host Microbe 4,
134146 (2008).
130. Shan,L. etal. Influence of host gene transcription level
and orientation on HIV1 latency in a primary-cell
model. J.Virol. 85, 53845393 (2011).
131. Siliciano,R.F. & Greene,W.C. HIV latency. Cold Spring
Harb. Perspect. Med. 1, a007096 (2011).
132. Dieudonne,M. etal. Transcriptional competence of
the integrated HIV1 provirus at the nuclear periphery.
EMBO J. 28, 22312243 (2009).
133. Lusic,M. etal. Proximity to PML nuclear bodies
regulates HIV1 latency in CD4+ Tcells. Cell Host
Microbe 13, 665677 (2013).
134. Jordan,A., Bisgrove,D. & Verdin,E. HIV reproducibly
establishes a latent infection after acute infection of
Tcells invitro. EMBO J. 22, 18681877 (2003).
This work describes the generation of latently
infected Jurkat cells (JLats), which, to date, remain
one of the most widely used clonal models of
latency. Integration sites were proposed to
correlate with the silencing of the viral genome.
135. Bruner,K.M. etal. Defective proviruses rapidly
accumulate during acute HIV1 infection. Nat. Med.
22, 10431049 (2016).
136. Kim,M. & Siliciano,R.F. Reservoir expansion by Tcell
proliferation may be another barrier to curing HIV
infection. Proc. Natl Acad. Sci. USA 113, 16921694
(2016).
137. Boritz,E.A. etal. Multiple origins of virus persistence
during natural control of HIV infection. Cell 166,
10041015 (2016).
This study shows that, in individuals that have
natural control of HIV1 replication, three
mechanisms contribute to HIV replication in
anatomically and functionally distinct compartments.
138. Eriksson,S. etal. Comparative analysis of measures
of viral reservoirs in HIV1 eradication studies.
PLoSPathog. 9, e1003174 (2013).
139. Calvanese,V., Chavez,L., Laurent,T., Ding,S. &
Verdin,E. Dual-color HIV reporters trace a population
of latently infected cells and enable their purification.
Virology 446, 283292 (2013).
140. Dahabieh,M.S., Ooms,M., Simon,V. & Sadowski,I.
A doubly fluorescent HIV1 reporter shows that the
majority of integrated HIV1 is latent shortly after
infection. J.Virol. 87, 47164727 (2013).
141. Gulick,R.M. etal. Treatment with indinavir,
zidovudine, and lamivudine in adults with human
immunodeficiency virus infection and prior
antiretroviral therapy. N. Engl. J.Med. 337,
734739 (1997).
142. Perelson,A.S. etal. Decay characteristics of
HIV1infected compartments during combination
therapy. Nature 387, 188191 (1997).
143. Gunthard,H.F. etal. Antiretroviral drugs for
treatment and prevention of HIV infection in adults:
2016 recommendations of the International
Antiviral SocietyUSA Panel. JAMA 316, 191210
(2016).
144. Hazuda,D.J. etal. Inhibitors of strand transfer that
prevent integration and inhibit HIV1 replication in
cells. Science 287, 646650 (2000).
One of the first screenings of chemical compounds
that block the strand transfer activity of
HIV1integrase, which led to the identification of
raltegravir.
145. Christ,F. & Debyser,Z. HIV1 integrase inhibition:
looking at cofactor interactions. Future Med. Chem.
7, 24072410 (2015).
146. Sedaghat,A.R., Dinoso,J.B., Shen,L., Wilke,C.O.
& Siliciano,R.F. Decay dynamics of HIV1 depend on
the inhibited stages of the viral life cycle. Proc. Natl
Acad. Sci. USA 105, 48324837 (2008).
147. Shen,L. etal. Dose-response curve slope sets class-
specific limits on inhibitory potential of anti-HIV
drugs. Nat. Med. 14, 762766 (2008).
148. Jilek,B.L. etal. A quantitative basis for antiretroviral
therapy for HIV1 infection. Nat. Med. 18, 446451
(2012).
149. Zolopa,A.R. etal. Activity of elvitegravir, a once-
daily integrase inhibitor, against resistant HIV type1:
results of a phase2, randomized, controlled, dose-
ranging clinical trial. J.Infect. Dis. 201, 814822
(2010).
150. Garrido,C. etal. Resistance associated mutations to
dolutegravir (S/GSK1349572) in HIV-infected patients
impact of HIV subtypes and prior raltegravir
experience. Antiviral Res. 90, 164167 (2011).
151. Brenner,B.G. & Wainberg,M.A. Clinical benefit of
dolutegravir in HIV1 management related to the high
genetic barrier to drug resistance. Virus Res. http://
dx.doi.org/10.1016/j.virusres.2016.07.006 (2016).
152. Kessl,J.J. etal. An allosteric mechanism for inhibiting
HIV1 integrase with a small molecule. Mol.
Pharmacol. 76, 824832 (2009).
153. Cherepanov,P. etal. Solution structure of the HIV1
integrase-binding domain in LEDGF/p75. Nat. Struct.
Mol. Biol. 12, 526532 (2005).

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 13

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

154. Christ,F. etal. Rational design of small-molecule
inhibitors of the LEDGF/p75integrase interaction
and HIV replication. Nat. Chem. Biol. 6, 442448
(2010).
This study details a structure-based rational design
of LEDGINs, integrase and LEDGF interaction
inhibitors with antiviral activity.
155. Desimmie,B.A. etal. LEDGINs inhibit late stage HIV1
replication by modulating integrase multimerization in
the virions. Retrovirology 10, 57 (2013).
156. Sarkar,I., Hauber,I., Hauber,J. & Buchholz,F. HIV1
proviral DNA excision using an evolved recombinase.
Science 316, 19121915 (2007).
157. Hauber,I. etal. Highly significant antiviral activity of
HIV1 LTR-specific tre-recombinase in humanized mice.
PLoS Pathog. 9, e1003587 (2013).
158. Qu,X. etal. Zinc-finger-nucleases mediate specific and
efficient excision of HIV1 proviral DNA from infected
and latently infected human Tcells. Nucleic Acids Res.
41, 77717782 (2013).
159. Strong,C.L. etal. Damaging the integrated HIV
proviral DNA with TALENs. PloS ONE 10, e0125652
(2015).
160. Ebina,H., Misawa,N., Kanemura,Y. & Koyanagi,Y.
Harnessing the CRISPR/Cas9 system to disrupt latent
HIV1 provirus. Sci. Rep. 3, 2510 (2013).
161. Hu,W. etal. RNA-directed gene editing specifically
eradicates latent and prevents new HIV1 infection.
Proc. Natl Acad. Sci. USA 111, 1146111466
(2014).
162. Liao,H.K. etal. Use of the CRISPR/Cas9 system as an
intracellular defense against HIV1 infection in human
cells. Nat. Commun. 6, 6413 (2015).
163. Wang,G., Zhao,N., Berkhout,B. & Das,A.T.
CRISPRCas9 can inhibit HIV1 replication but
NHEJ repair facilitates virus escape. Mol. Ther. 24,
522526 (2016).
164. Dekker,J. & Misteli,T. Long-range chromatin
interactions. Cold Spring Harb. Perspect. Biol. 7,
a019356 (2015).
165. Dixon,J.R., Gorkin,D.U. & Ren,B. Chromatin
domains: the unit of chromosome organization.
Mol.Cell 62, 668680 (2016).
166. Bonev,J. & Cavaalli,G. Organization and function of
the 3D genome. Nat. Rev. Genet. 17, 661678
(2016).
References 164166 are comprehensive recent
reviews of the genomic regulatory landscapes and
nuclear architecture.
167. Dundr,M. Nuclear bodies: multifunctional companions
of the genome. Curr. Opin. Cell Biol. 24, 415422
(2012).
168. Kind,J. etal. Single-cell dynamics of genome-nuclear
lamina interactions. Cell 153, 178192 (2013).
169. Harr,J.C. etal. Directed targeting of chromatin to
the nuclear lamina is mediated by chromatin state
and Atype lamins. J.Cell Biol. 208, 3352 (2015).
170. Schermelleh,L. etal. Subdiffraction multicolor
imaging of the nuclear periphery with 3D structured
illumination microscopy. Science 320, 13321336
(2008).
Acknowledgements
M.L. is funded by grants from the German Centre for Infectious
Research (Deutsche Zentrum fur Infektion Forschung, DZIF)
and by the Hector Foundation for AIDS and Cancer Research.
Competing interests statement
The authors declare no competing interests.

14 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.