Transcript Differential impact of APOBEC3-driven mutagenesis on HIV

Differential impact of APOBEC3-driven
mutagenesis on HIV evolution in diverse
anatomical compartments
Pitié-Salpêtrière
Paris, France
Hospital Pitié-Salpêtrière Paris, University of Pierre
and Marie Curie Paris VI, France
Phone:+ 33 1 42 17 75 14
e-mail: [email protected]
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
INTRODUCTION
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.
UMR S943, Paris, France, 2Inserm UMR S945, Paris, France, 3AP-HP, Paris, France
RESULTS
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 (n=8, renal
tissue/PBMCs pairs), only one patient exhibited
hypermutation detected both in PBMCs as well as in renal
tissue. In the third group (n=8, 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.
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
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 APOBEC3induced 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
To evaluate the differential impact of APOBEC3editing 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
(VL<50 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 Fisher’s exact test that compared the
number of G-to-A changes in APOBEC3 (GG or GA)
versus control contexts (GC or GT).
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.
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 NATIONALE DE RECHERCHES
SUR LE SIDA ET LES HÉPATITES VIRALES
Agence autonome de l’Inserm
– grant agreement n° 223131 –