Differential impact of APOBEC3-driven mutagenesis on HIV evolution in diverse anatomical compartments S. Fourati 1, S. Lambert-Niclot 1, C. Soulie 1, M. Wirden 1, B. Descours 2, I. Malet 1, M.A. Valantin 1, R. Tubiana 1, A. Simon 3, C. Katlama 1, G. Carcelain 2, V. Calvez 1, A.-G. Marcelin 1 1 Inserm UMR S943, Paris, France, 2 Inserm UMR S945, Paris, France, 3 AP-HP, Paris, France Piti é -Salpêtri è re Paris, France Hospital Pitié-Salpêtrière Paris, University of Pierre and Marie Curie Paris VI, France Phone: METHODS 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 (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 ( 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). INTRODUCTION OBJECTIVES 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 AGENCE NATIONALE DE RECHERCHES SUR LE SIDA ET LES HÉPATITES VIRALES Agence autonome de l’Inserm Conclusion 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. 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). – grant agreement n° – 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. 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. 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. 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. Figure 2. Differential impact of APOBEC3-editing on viral diversification and emergence of drug resistance mutations in viral anatomical compartments.