the genes is ~10−10. In prior studies, a limited
number of HIV integration sites were identified in patients (14–17), and HIV proviruses
were found in intron 6 of MKL2 and intron 5
of BACH2, in the same orientation that the
genes are transcribed. However, in the published studies the integration sites in these
genes were not linked to the clonal expansion
of the infected cells (table S5). Both BACH2
and MKL2 are involved in the growth and
development of cells, and BACH2 is known to
play a key role in T cell development (18). Both
genes [and the MKL2-related gene MKL1, in
which there were four independent integration
sites, some of which were associated with clonally expanded cells in patient 1 (table S4)]
have been implicated in human cancers (19–21),
in which they were activated by DNA rearrangements that created gene fusions. The pattern of multiple integrations in MKL2 and BACH2
found in the patients cannot be the result of
preferential integration because HIV integration
is neither intron-specific nor orientation-specific
(22). Thus, the only plausible explanation for
the data that is in accord with the rules for HIV
integration is that the cells with the integrations
in MKL2 and BACH2 were selected after integration because the integrations in these genes contributed to the expansion and persistence of the
host cells. This interpretation is supported, for
BACH2, by a report showing that this gene is a
target for retroviral insertional activation in
mice infected with mouse leukemia virus (23).
Most of our analyses were performed by using
cells from patients on long-term cART, which
blocks the infection of additional cells but has
no effect on cells that have already been in-
fected (9). During untreated HIV infections,
~109 cells are infected daily. The vast majority
(99%) of the newly infected cells die within 24
to 48 hours, and a substantial proportion of
the remaining cells die within 2 to 4 weeks
(24–26). Viremia decreases by 4 to 5 logs when
patients undergo cART; however, the number
of cells containing HIV DNA decreases by ap-
proximately 1 log (9), indicating that a sub-
stantial fraction (~10%) of the cells that were
infected before the initiation of cART persist.
Most of these long-lived infected cells contain
proviruses that are obviously defective; how-
ever, ~12% of the proviruses appear to be func-
tional, although only a small fraction of these
apparently functional proviruses can be in-
duced to make virus in ex vivo experiments
(27). Cells infected with highly defective or fully
latent proviruses that produce little or no viral
protein may have a survival advantage rela-
tive to cells that produce virions because cells
that express viral proteins are more likely to
be lysed by HIV-specific cytotoxic T lympho-
cyte or be subject to cytopathic effects of the
viral proteins. This same logic applies to HIV-
infected cells that undergo clonal expansion.
Although we have not yet shown that clonally
expanded cells produce replication-competent
HIV, we have shown that a highly expanded
clone of cells does produce HIV virions in suf-
ficient quantity to cause viremia, which means
that the selection against cells that produce viral
proteins is not so strong that it prevents ex-
tensive clonal expansion of cells that express
the viral proteins required to produce virions.
Our data show that many of the infected cells
that persist have undergone clonal expansion;
these clones were revealed but not created by
cART. For some infected cell clones, it is
likely that the integration site is only a pas-
sive marker of clonal expansions that are driven
by another factor or factors, such as antigen
stimulation or homeostatic proliferation sig-
nals (28). In contrast, we show here that some
cells with HIV integration sites in specific genes
are strongly selected because these integrations
promote the survival and expansion of the in-
fected cells. Although there are obvious similarities
in the integration sites seen in the five patients,
there is considerable heterogeneity from one pa-
tient to another, both in the extent of clonal ex-
pansion and in the genes in which proviruses
are integrated in the clonally expanded cells
(fig. S5). This complexity highlights the diffi-
culty in attempting to extrapolate, from bulk
HIV DNA quantification, the size and nature
of the population of HIV proviruses that make
up the reservoir that gives rise to HIV rebound
after cessation of cART (28).
Our findings have relevance for three important areas. (i) To effectively target HIV persistence with the goal of achieving a cure, it will be
important not only to suppress any replication
of the virus, but also to block the expansion of
infected cells. (ii) Although the HIV vectors used
in gene therapy have safety features that the parental virus lacks, we now know that like many
other retroviruses, HIV integration can lead to
clonal expansion and persistence of infected
cells. This discovery suggests that persons treated
with HIV-based vectors should be carefully monitored for evidence of clonal expansion of vector-infected cells. (iii) We also suggest that it is time
to reexamine the question of whether HIV integration can contribute to the development of
malignancies. Although there are well-defined
cancers in HIV-infected patients that are the
result of uncontrolled expression of herpes viruses,
there are reports of a small number of lymphomas
with HIV proviruses integrated at defined sites;
one lymphoma had a provirus integrated in
BACH2 (15, 29, 30). Despite these published
reports, it is widely believed that HIV DNA is
not detectable in most cancers from HIV-infected
patients; however, the experiments supporting
this belief are not well-documented in the literature. It is possible that prior attempts to detect
HIV DNA in cancers examined only a very small
portion of the HIV genome and, as such, missed
HIV proviruses having large deletions; large deletions are a characteristic of the proviruses that
cause mouse and avian tumors. Thus, our findings have important implications for designing
and implementing strategies to eliminate persistent HIV infection, for the use of lentiviral vectors
for gene therapy in human patients and, possibly,
for the origin of some HIV-related malignancies.
REFERENCES AND NOTES
1. S. G. Deeks et al., Nat. Rev. Immunol. 12, 607–614 (2012).
2. S. Palmer, L. Josefsson, J. M. Coffin, J. Intern. Med. 270,
3. J. R. Bailey et al., J. Virol. 80, 6441–6457 (2006).
4. M. F. Kearney et al., PLOS Pathog. 10, e1004010 (2014).
5. C. C. Berry et al., Bioinformatics 28, 755–762 (2012).
6. N. A. Gillet et al., Blood 117, 3113–3122 (2011).
7. S. S. De Ravin et al., J. Virol. (2014).
8. Materials and methods are available as supplementary
materials on Science Online.
9. J. Coffin, R. Swanstrom, Cold Spring Harb. Perspect. Med. 3,
10. H. Imamichi et al., AIDS (2014).
11. L. Josefsson et al., Proc. Natl. Acad. Sci. U.S.A. 110,
12. N. Rosenberg, P. Jolicoeur, in Retroviruses, J. M. Coffin,
S. H. Hughes, H. E. Varmus, Eds. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1997),
13. A. R. Schröder et al., Cell 110, 521–529 (2002).
14. T. Ikeda, J. Shibata, K. Yoshimura, A. Koito, S. Matsushita,
J. Infect. Dis. 195, 716–725 (2007).
15. K. D. Mack et al., J. Acquir. Immune Defic. Syndr. 33, 308–320
16. Y. Han et al., J. Virol. 78, 6122–6133 (2004).
17. H. Katano et al., Microbes Infect. 9, 1581–1589 (2007).
18. G. Hu, J. Chen, Nat. Commun. 4, 2830 (2013).
19. S. Kobayashi et al., Genes Chromosomes Cancer 50, 207–216
20. U. Flucke et al., Histopathology 62, 925–930 (2013).
21. S. Muehlich et al., Oncogene 31, 3913–3923 (2012).
22. R. Craigie, F. D. Bushman, Cold Spring Harb. Perspect. Med. 2,
23. J. Liu et al., BMC Mol. Biol. 10, 2 (2009).
24. D. D. Ho et al., Nature 373, 123–126 (1995).
25. X. Wei et al., Nature 373, 117–122 (1995).
26. J. M. Coffin, Science 267, 483–489 (1995).
27. Y. C. Ho et al., Cell 155, 540–551 (2013).
28. N. Chomont et al., Nat. Med. 15, 893–900 (2009).
29. B. Shiramizu, B. G. Herndier, M. S. McGrath, Cancer Res. 54,
30. B. G. Herndier et al., Blood 79, 1768–1774 (1992).
The authors are indebted to the study participants and
to the clinical staff of the National Institute of Allergy and
Infectious Diseases/Critical Care Medicine Department
clinic who cared for them. We thank C. Lane, H. Malech,
H. Imamichi, S. Matsushita, and L. Frenkel for stimulating
discussions. We are grateful to J. Meyer and A. Kane
for help with the figures and T. Burdette for help in
preparing the manuscript. The data presented in this
work is tabulated in the main paper and in the
supplementary materials. The integration sites are
compiled in table S3; the data can also be accessed
using the National Center for Biotechnology Information
accession no. PRJNA241020. Funding for this research
was provided with Federal funds from the National Cancer
Institute, an NIH Bench to Bedside award (F.M.), and
by funds from the National Cancer Institute under
contract HSSN261200800001E (X. W. and L.S.). J.M.C.
was supported by a Research Professorship from the
American Cancer Society with additional support from
the F. M. Kirby Foundation and by funding from the
National Cancer Institute (Leidos contract 25XS119).
J. W.M. was supported by funding from the National Cancer
Institute (Leidos contract 25XS119). The content of this
publication does not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does mention of
trade names, commercial products, or organizations imply
endorsement by the U.S. government.
Materials and Methods
Figs. S1 to S5
Tables S1 to S5
1 April 2014; accepted 13 June 2014
Published online 26 June 2014;