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• Hospital Pitié-Salpêtrière Paris, University of
  Pierre and Marie Curie Paris VI, France
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• e-mail anne-genevieve.marcelin_at_psl.aphp.fr

Differential impact of APOBEC3-driven mutagenesis
on HIV evolution in diverse anatomical
compartments S. Fourati1, S. Lambert-Niclot1, C.
Soulie1, M. Wirden1, B. Descours2, I. Malet1,
M.A. Valantin1, R. Tubiana1, A. Simon3, C.
Katlama1, G. Carcelain2, V. Calvez1, A.-G.
Marcelin1   1Inserm UMR S943, Paris, France,
2Inserm UMR S945, Paris, France, 3AP-HP, Paris,
France
INTRODUCTION
RESULTS
Within an individual, HIV exists as a population
of related but distinct viral variants termed
viral quasispecies. These variants can be present
in distinct anatomical locations in the same
individual and have the properties to evolve
independently from HIV found in peripheral blood.
Many viral factors contribute to viral
compartmentalization the error proness of HIV
reverse transcriptase, recombination, and rapid
rates of viral replication. In addition, recent
findings suggest that viral restriction factors
APOBEC3 can provide an additional mechanism for
acquiring sequence variation. Despite being
counteracted by HIV-1 Vif protein, APOBEC3
proteins are incompletely neutralized in vivo.
When fixed in viral DNA, APOBEC3-induced
mutations register as guanosine-to-adenosine
(G-to-A) changes in the viral plus strand and are
termed hypermutations when occurring at excessive
levels. Knowledge of organ/tissue specific
impact of APOBEC3 in HIV evolution is important
for investigating viral compartmentalization in
humans. The inhibitory effects of the APOBEC3
proteins could lead to differential accumulation
of defective viruses between reservoirs. In
addition, it is possible that low levels of
activity of these cellular enzymes could be
beneficial to HIV-1 in some compartments and
facilitate immune evasion or accelerate the
development of drug resistance
We first sought to ascertain the population
distribution of HIV-1 G-to-A substitutions (in
APOBEC3 dinucleotide context GG or GA) in each
sample in order to identify APOBEC3-induced
footprint within the context of natural in vivo
sequence variation. Overall, hypermutated
sequences were identified in 33 (11/33) of
subjects in at least one viral compartment.
Figure 1. Population distribution of
APOBEC3-induced hypermutation in each viral
compartment of each patient. In the first group
of patients (n 14, CSF/PBMCS pairs),
hypermutation was detected both in PBMCs and CSF
in 2 patients while 4 other patients exhibited
hypermutated sequences only in CSF. In the second
group (n8, renal tissue/PBMCs pairs), only one
patient exhibited hypermutation detected both in
PBMCs as well as in renal tissue. In the third
group (n8, for rectal tissue/PBMCs),
hypermutation was detected more frequently in
rectal tissue (3 cases) than in PBMCs (one other
case). Red boxes represent hypermutated sequences
in a specific anatomical compartment Blank boxes
represent absence of hypermutation.
Figure 2. Differential impact of APOBEC3-editing
on viral diversification and emergence of drug
resistance mutations in viral anatomical
compartments.
G-to-A changes in each sample sequence was
compared to a consensus sequence and are
indicated by bars (in a specific dinucleotide
context) -APOBEC3 context GG-to-AG (red
bars), GA-to-AA (cyan bars) -Other conexts
GC-to-AC (green bars), GT-to-AT (magenta
bars). when hypermutation was observed in PBMCs
as well as in another compartment (P11, P12,
P22), the hypermutated region (protease or RT)
can differ between compartments. Indeed, for
patient 22 (P22), the viral population was found
hypermutated in the protease region (but not in
RT) in PBMCs whereas in contrast, analyzing renal
tissue in the same patient, viral population
showed clear APOBEC3-induced hypermutation in the
RT region (but not in protease). Such
differential APOBEC3-induced hypermutation
footprint was also observed between CSF and PBMCs
for P11 and P12.
OBJECTIVES
So far, little data is available on the impact of
APOBEC3-induced Guanosine-to-Adenosine (G-to-A)
mutations on viral compartmentalization. We
attempted in this study, to determine the
differential contribution of APOBEC3-editing in
HIV-1 evolution in different anatomical
compartments (Cerebral spinal fluid, rectal
tissue, renal tissue).  
METHODS
Focusing on drug resistance mutations, some
patients (P13, P27, P29) harbored one or several
APOBEC3-induced drug resistance mutations (G73S
in protease, M184I, M230I in RT) in hypermutated
proviruses from sanctuaries (CSF or rectal
tissue) while these mutations were absent from
paired non-hypermutated proviruses in PBMCs,
strongly suggesting that such mutations resulted
from APOBEC3 editing. Consistently with the fact
that differential APOBEC3-induced profile was
detected between compartments (P11, P12, P22),
APOBEC3-induced drug resistance mutations also
varied between compartments. For example, for
patient 11 (P11), PBMCs proviral sequences
harbored E138K and M184I mutations in RT while
viruses in the CSF harbored M184I and M230I
mutations. In contrast, patients 9 and 30 (P9,
P30) showed no evidence of APOBEC3-induced drug
resistance mutations neither in protease nor in
RT.
To evaluate the differential impact of
APOBEC3-editing in HIV-1 compartments, we studied
the level of G-to-A hypermutation in HIV-1
protease and reverse transcriptase bulk sequences
among 30 patients for whom peripheral blood
mononuclear cells (PBMCs) and body tissues or
fluids were collected on the same day (14 paired
PBMCs/Cerebral spinal fluid (CSF) 8 paired
PBMCs/renal tissues 8 paired PBMCs/rectal
tissues). All the study subjects were receiving
HAART and had undetectable viremia (VLlt50
copies/ml) at the study time point. Differences
in the G-to-A mutation frequencies were analyzed
using the Hypermut 2.0 program (http//
www.hiv.lanl.gov/content/sequence/HYPERMUT/
hypermut.html). A sequence was considered
hypermutated if it registered a P value of less
than 0.05 on the Fishers exact test that
compared the number of G-to-A changes in APOBEC3
(GG or GA) versus control contexts (GC or GT).
Conclusion
APOBEC3-induced mutations observed in peripheral
blood may underestimate the overall proportion of
hypermutated viruses in the body as these
mutations seem to be more frequent in sanctuaries
compared to PBMCs in our study. This phenomenon
reinforces the role of APOBEC3 editing in HIV
compartimentalization in vivo. The resulting
mutations may favor escape to antiretrovirals in
these compartments in conjunction with a lower
penetration of drugs in some sanctuaries. On the
other side, because hypermutated sequences often
harbor inactivating mutations, this study
suggests that accumulation of defective viruses
may be more dominant in sanctuaries than in
peripheral blood of patients on effective HAART.
Agence autonome de lInserm
grant agreement n 223131