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HIV infection en route to endogenization: two cases

  • P. Colson
    Affiliations
    Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Université, Marseille, France

    Fondation Institut Hospitalo-Universitaire (IHU) MéiterranéInfection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France
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  • I. Ravaux
    Affiliations
    IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Service de Maladies Infectieuses, Centre Hospitalo-Universitaire Conception, Assistance Publique-Hôpitaux de Marseille, France
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  • C. Tamalet
    Affiliations
    Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Université, Marseille, France

    Fondation Institut Hospitalo-Universitaire (IHU) MéiterranéInfection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France
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  • O. Glazunova
    Affiliations
    Fondation Institut Hospitalo-Universitaire (IHU) MéiterranéInfection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France
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  • E. Baptiste
    Affiliations
    Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Université, Marseille, France
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  • E. Chabriere
    Affiliations
    Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Université, Marseille, France
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  • A. Wiedemann
    Affiliations
    Faculté de Médecine, Université Paris Est, Créteil, France

    Vaccine Research Institute (VRI), Créteil, France
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  • C. Lacabaratz
    Affiliations
    Faculté de Médecine, Université Paris Est, Créteil, France

    Vaccine Research Institute (VRI), Créteil, France
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  • M. Chefrour
    Affiliations
    Laboratoire de Biochimie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France
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  • C. Picard
    Affiliations
    CNRS, EFS, ADES UMR 7268, Aix-Marseille Université, Marseille, France
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  • A. Stein
    Affiliations
    Fondation Institut Hospitalo-Universitaire (IHU) MéiterranéInfection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France

    IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Service de Maladies Infectieuses, Centre Hospitalo-Universitaire Conception, Assistance Publique-Hôpitaux de Marseille, France
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  • Y. Levy
    Affiliations
    Faculté de Médecine, Université Paris Est, Créteil, France

    Vaccine Research Institute (VRI), Créteil, France

    AP-HP, Hôpital H. Mondor – A. Chenevier, Service d'Immunologie Clinique et Maladies Infectieuses, Créteil, France
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  • D. Raoult
    Correspondence
    Corresponding author: Professor D. Raoult, IHU Méditerranée Infection, URMITE, Unité des Rickettsies, Faculté de Médecine, Aix-Marseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
    Affiliations
    Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Université, Marseille, France

    Fondation Institut Hospitalo-Universitaire (IHU) MéiterranéInfection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique-Hôpitaux de Marseille, Marseille, France
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      Abstract

      The long-term spontaneous evolution of humans and the human immunodeficiency virus (HIV) is not well characterized; many vertebrate species, including humans, exhibit remnants of other retroviruses in their genomes that question such possible endogenization of HIV. We investigated two HIV-infected patients with no HIV-related disease and no detection with routine tests of plasma HIV RNA or cell-associated HIV DNA. We used Sanger and deep sequencing to retrieve HIV DNA sequences integrated in the human genome and tested the host humoral and cellular immune responses. We noticed that viruses from both patients were inactivated by the high prevalence of the transformation of tryptophan codons into stop codons (25% overall (3–100% per gene) and 24% overall (0–50% per gene)). In contrast, the humoral and/or cellular responses were strong for one patient and moderate for the other, indicating that a productive infection occurred at one stage of the infection. We speculate that the stimulation of APOBEC, the enzyme group that exchanges G for A in viral nucleic acids and is usually inhibited by the HIV protein Vif, has been amplified and made effective from the initial stage of the infection. Furthermore, we propose that a cure for HIV may occur through HIV endogenization in humans, as observed for many other retroviruses in mammals, rather than clearance of all traces of HIV from human cells, which defines viral eradication.

      Keywords

      Introduction

      Human immunodeficiency virus (HIV) is one of the three ‘big killers’ and causes ≈35 million infections worldwide [
      • De Cock KM
      • Jaffe HW
      • Curran JW
      The evolving epidemiology of HIV/AIDS.
      ]. Despite massive efforts, attempts to cure people infected with this virus have failed [
      • Barre‐Sinoussi F
      • Ross AL
      • Delfraissy JF
      Past, present and future: 30 years of HIV research.
      ,
      • Fauci AS
      • Marston HD
      • Folkers GK
      An HIV cure: feasibility, discovery, and implementation.
      ], except for an adult HIV-positive patient who exhibited long-term aviraemia without antiretroviral therapy following transplantation of stem cells from a donor carrying the protective CCR5 Delta32/Delta32 deletion [
      • Hutter G
      • Nowak D
      • Mossner M
      • et al.
      Long‐term control of HIV by CCR5 Delta32/Delta32 stem‐cell transplantation.
      ]. This failure to reach a cure for HIV was described as primarily due to the absence of eradication of any trace of replication-competent HIV DNA integrated into human cell genomes [
      • Fauci AS
      • Marston HD
      • Folkers GK
      An HIV cure: feasibility, discovery, and implementation.
      ].
      Since the beginning of the HIV pandemic, 5–15% of HIV-infected individuals have been identified as devoid of clinical symptoms and progression in the absence of antiretroviral treatment [
      • Pantaleo G
      • Menzo S
      • Vaccarezza M
      • et al.
      Studies in subjects with long‐term nonprogressive human immunodeficiency virus infection.
      ,
      • Deeks SG
      • Walker BD
      Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy.
      ]. Some of them, representing <1% of HIV-positive persons, exhibit spontaneous control of HIV replication, including the so-called ‘elite controllers’ (ECs), who spontaneously and durably exhibit undetectable or almost undetectable virus in plasma and a normal CD4-cell count [
      • Deeks SG
      • Walker BD
      Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy.
      ,
      • Lambotte O
      • Boufassa F
      • Madec Y
      • et al.
      HIV controllers: a homogeneous group of HIV‐1‐infected patients with spontaneous control of viral replication.
      ]. ECs attracted attention and sparked research to identify the factors associated with spontaneous functional cure of HIV infection. Various potential mechanisms involving viral, host genetic and immunological patterns were identified, but none of them could explain the spontaneous control of HIV replication alone or in all of the cases [
      • Pantaleo G
      • Menzo S
      • Vaccarezza M
      • et al.
      Studies in subjects with long‐term nonprogressive human immunodeficiency virus infection.
      • Deeks SG
      • Walker BD
      Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy.
      • Lambotte O
      • Boufassa F
      • Madec Y
      • et al.
      HIV controllers: a homogeneous group of HIV‐1‐infected patients with spontaneous control of viral replication.
      • Autran B
      • Descours B
      • Avettand‐Fenoel V
      • Rouzioux C
      Elite controllers as a model of functional cure.
      ].
      We investigated here a patient who had been HIV-seropositive since 1985 with asymptomatic infection in the absence of antiretroviral treatment and no detectable infectious virus nor any HIV RNA or DNA genomic material retrieved from the plasma or peripheral blood mononuclear cells (PBMCs), and who thus appeared to be cured. We studied in depth his host response and searched for any trace of HIV DNA integrated into PBMCs. In addition, we searched for other similar cases in our series.

      Methods

       Patients

      Our patient cohort was composed of 1700 HIV-infected patients, including ten elite HIV-1 controllers. Among these ten, two also had undetectable PBMC HIV DNA; they gave their written informed consent to be included in the study.

       Host testing

      Anti-HIV-1 antibody testing was performed using an enzyme-linked immunosorbent assay (Architect; Abbott Diagnostics, Mannheim, Germany) and Western blot testing (Bio-Rad, Stanford, CA, USA). Analysis of plasma tryptophan (W) levels was performed using the 7300 High Performance Amino Acid Analyzer (Beckman Instruments, Inc., Fullerton, CA, USA). APOBEC mRNA quantification in the PBMCs was performed. The sequence of gene encoding the CCR5 chemokine receptor and HLA genotype were determined. Resistance of the PBMCs to HIV super infection was assessed by inoculating these PBMCs with HIV-1 strain NL4.3. To test the inhibitory effect of the two case-patients’ sera on HIV growth, the NL4.3 strain was cultured on control PBMCs in the presence of 100 µL of these sera or control sera (see Supporting information).

       Immunological investigations

      For flow cytometry analyses, PBMCs were stained with the following monoclonal anti-human antibodies used in various combinations: CD3, CD4, CD38, HLADR, CCR7, granzyme B, perforin, TNFα, IFNγ and MIP1β (BD Biosciences, San Jose, CA, USA); CD8 and CD45 RA (eBioscience, San Diego, CA, USA). To study HIV-specific T-cell responses, PBMCs were cultured in the presence of HIV-specific antigens consisting of a mix of 15-mers overlapping 11-amino acid peptides (36 peptides in Gag, Pol and Nef sequences). On day 2, 100 U/mL recombinant IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added, then cells were harvested on day 7, and re-stimulated with the same pool of peptides for 6 hours in the presence of anti-CD28/anti-CD49d co-stimulatory antibodies and Golgi Plug (BD Biosciences). Thereafter, cells were stained for intracytoplasmic cytokines (TNFα, IFNγ and MIP1β) (see Supporting information).

       Virological investigation

       Culture assays

      Culture assays were performed as described previously [
      • Tamalet C
      • Masquelier B
      • Ferchal F
      • et al.
      Short‐term evaluation of zidovudine‐treated patients: decrease in plasma and cellular viraemia titres.
      ] and in the Supporting information.

       HIV nucleic acid and protein detection

      Routine plasma HIV RNA testing was performed using the RealTime HIV-1 assay (Abbott Diagnostics) and the Generic HIV-RNA assay (Biocentric, Bandol, France) (detection limit, 40 and 300 copies/mL, respectively). Total (integrated and unintegrated) cell-associated HIV DNA testing was performed as previously described (detection limit, 20 copies/106 PBMCs) [
      • Avettand‐Fenoel V
      • Prazuck T
      • Hocqueloux L
      • et al.
      HIV‐DNA in rectal cells is well correlated with HIV‐DNA in blood in different groups of patients, including long‐term non‐progressors.
      ]. HIV protein identification in serum was performed by immunoprecipitation with anti-HIV P24 monoclonal mouse antibodies using magnetic beads coupled with protein A (Dynabeads Technology, Invitrogen, Carlsbad, CA, USA). The precipitate was separated by SDS-PAGE and analysed using MALDI-TOF mass spectrometry and a search in viral protein databases (see Supporting information).

       HIV genome sequencing and analyses

      HIV genome sequencing was performed using Sanger population and next-generation sequencing of HIV-1 DNA from the two case-patients’ PBMCs and, to obtain missing fragments of the HIV genome, a new procedure named the ‘Bortsch’ procedure. HIV sequences obtained from the two case-patients are available from GenBank with accession numbers KM878756-810 and KM878811-833. Phylogenetic analyses and subtyping are described in the Supporting information. HIV genomes from ECs and non-ECs were searched for in GenBank (http://www.ncbi.nlm.nih.gov/nuccore/), and retrieved genomes were compared with those from the two case-patients, seeking stop codons, hypermutations, insertions/deletions and other mutations in each HIV-1 protein sequence. For comparison of the W-to-stop mutations in ECs and non-ECs, hypermutated genomes from the Eyzaguirre et al. [
      • Eyzaguirre LM
      • Charurat M
      • Redfield RR
      • Blattner WA
      • Carr JK
      • Sajadi MM
      Elevated hypermutation levels in HIV‐1 natural viral suppressors.
      ] study [
      • Eyzaguirre LM
      • Charurat M
      • Redfield RR
      • Blattner WA
      • Carr JK
      • Sajadi MM
      Elevated hypermutation levels in HIV‐1 natural viral suppressors.
      ] were used, the sequence of each gene being translated then aligned to detect stop codons (see Supporting information).

      Results

      The index case is a 57-year-old patient who heavily used intravenous drugs between 1978 and 1988. He was diagnosed HIV-positive in 1985 and did not suffer from any clinical manifestations linked to HIV infection. He claimed to exhibit a special immunity, presenting no fever or lymphadenopathy during his life. His CD4-cell count was always normal (CD4- and CD8-cell counts were 1576 and 1234, respectively) and no HIV RNA was ever found in his plasma nor HIV DNA in his PBMCs. In contrast, he suffered from active hepatitis C infection. He was continuously exposed to HIV as his wife shared syringes with him until 1988; she was diagnosed HIV-positive in 1985 and developed AIDS with a different virus (see Supporting information). This patient's resistance to HIV justified an in-depth investigation of both his host response and his integrated retrovirus.
      Strong reactivity against all tagged proteins was noted on the Western blot (Fig. 1a). No homozygosity or heterozygosity for the 32-base pair (bp) deletion in the CCR5 co-receptor gene was found. HLA genotype is not associated with HIV protection. The serum of the patient was found to protect HIV-negative PBMCs from the virulent HIV-1 strain NL4.3 (Fig. 1b and Supporting information). This was also observed for three out of eight sera from HIV-positive controls but not for sera from the HIV-negative controls, indicating that NL4-3 can be neutralized by some HIV-positive sera [
      • Wolk T
      • Schreiber M
      N‐Glycans in the gp120 V1/V2 domain of the HIV‐1 strain NL4‐3 are indispensable for viral infectivity and resistance against antibody neutralization.
      ]. Moreover, an attempt to obtain an infection of the PBMCs from this patient with the same virulent strain was unsuccessful (Fig. 1c), although this does not allow speculation on susceptibility to other HIV strains. We conclude that this patient was immune to super infection with HIV. We were unable to grow HIV from this patient but we detected in his serum three viral peptides with similarities to HIV-1 reverse transcriptase (see Supporting information). Sequencing the HIV DNA integrated into the PBMCs of the patient was extremely laborious because of the very low copy number (<20 copies/106 PBMCs), but we obtained a 9302-bp-long subtype B HIV genomic sequence using Sanger population, clonal sequencing and next-generation sequencing (Fig. 2) (see Supporting information). Surprisingly, we found many stop codons, all at W codon (TGG) positions, reaching 25% of the 92 W codons of the whole genome and 100% in three HIV genes (Fig. 2, Table 1 and Supporting information, Table S1). Therefore, we concluded that the virus was inactivated and the patient was cured.
      Figure thumbnail gr1
      FIG. 1(a) Western blot HIV-1 for serum samples from the two case-patients. Values near the Western blot indicate the ratio between the signal obtained for the band compared to that from the positive control (see Supporting information). CA, capside; Env, envelope; gp, glycoprotein; IN, integrase; MA, matrix; Pos., positive control; Neg., negative control; su, subunit; RT, reverse transcriptase. (b) Seroneutralization by the case-patients’ serum of infection of peripheral blood mononuclear cells from HIV-negative donors by HIV-1 strain NL4.3. Ct, PCR cycle threshold; PBMCs, peripheral blood mononuclear cells. (c) Infectability of peripheral blood mononuclear cells from case-patients by the HIV-1 strain NL4.3. Ct, PCR cycle threshold; PBMCs, peripheral blood mononuclear cells.
      Figure thumbnail gr2
      FIG. 2HIV genome retrieved from case-patient no. 1. HIV genes are shown on the outer ring. On the inner rings, blue lines indicate tryptophan (W) codons in genes; red lines indicate W-to-stop mutations. Representation was built using DNAplotter (http://www.sanger.ac.uk/Software/Artemis/circular/).
      TABLE 1Distribution of tryptophan (W)‐to‐stop mutations in HIV genomes from the two case‐patients and natural viral suppressors, at positions devoid of such mutations in two sets of control HIV‐infected patients, one on highly active antiretroviral therapy and the other untreated
      HIV genesgagpolvifvprvpuenvnef
      Codonsgag36gag265pol62pol179pol394pol776pol823pol958vif21vif79vif89vpr38vpu23env840nef57nef183
      Proportion of sequences harbouring a W‐to‐stop mutation for case‐patients (%)Case patient no.101702500001602233790017
      Case patient no.202202525000000001700
      Number of sequences harbouring a W‐to‐stop mutation for natural viral suppressors (n = 23)4611211441206013
      HIV genomes analyzed here are from the article by Eyzaguirre et al.
      • Eyzaguirre LM
      • Charurat M
      • Redfield RR
      • Blattner WA
      • Carr JK
      • Sajadi MM
      Elevated hypermutation levels in HIV‐1 natural viral suppressors.
      The second patient in our cohort was also considered to be an HIV EC with no detectable RNA or DNA using standard methods. This 23-year-old homosexual Chilean man was identified as HIV-infected in 2011 and most likely infected in Chile during the 3 previous years, and his HIV sequence was clustered with South American viral genomes (see Supporting information). His serum showed weak anti-HIV reactivities on the Western blot (Fig. 1a). T-cell phenotypic characteristics of the patient, including frequency of different T-cell subpopulations and the activation or cytotoxic marker expression by CD4 and CD8 T-cells, did not differ from those of an HIV-infected control patient (Fig. 3a). Despite a low CD8+ T-cell response, this patient exhibited high CD4+ T-cell responses in terms of cytokine production (42.8%, 37.9% and 40.1% of CD4+ T-cell-producing IFN-γ, TNF-α and MIP1β, respectively) compared with a group of 22 HIV controls (mean ± standard deviation (SD) = 5.5 ± 14.4%, 4.5 ± 10.2% and 5.9 ± 15.5%) (Y. Levy, personal data) (Fig. 3b ). Serum from this patient did not inhibit HIV infection, and his PBMCs were susceptible to infection by the NL4.3 strain (Fig. 1b-c; see Supporting information). This patient did not harbour homozygosity/heterozygosity for the 32-bp deletion in the CCR5 co-receptor gene and his HLA genotype is not HIV protective. HIV co-culture was unsuccessful. HIV genome sequencing from the patient's PBMCs showed that he exhibited 21 stop codons, with all but two being located at W codons (Tables 1 and S1).
      Figure thumbnail gr3
      FIG. 3Flow cytometric profiles of T cells of case no. 2. (a) Ex vivo phenotype of T cells. Differentiation (CD45RA, CCR7), activation (CD38, HLADR) and cytotoxic (Granzyme B, Perforin) markers were used to analyse CD4 (upper panel) and CD8 (lower panel) T cells. (b) Intracellular cytokine staining after 8 days of PBMC culture with a pool of 36 HIV-1 peptides. HIV-specific CD4+ (upper panel) and CD8+ (lower panel) T cells able to produce IFNγ, TNFα and/or MIP1β. Plots are gated on viable CD3+ CD4+ or CD4+CD8+ T cells, respectively.
      When comparing the distribution of stop codons replacing W, in case-patient 1, the accessory and regulatory genes vif, vpr, vpu and tat were mostly affected (≥50 to 100% of W-to-stop mutations in these genes), whereas in case-patient 2, only the gag, pol, env and nef genes were affected (Supporting information, Table S1). We then compared the HIV genomes from ECs and non-ECs. The median rate of W-to-stop mutations was significantly higher in ECs (including the two present cases) than in non-ECs (20 vs. 9; p < 0.001; Mann-Whitney test) (Supporting information and Table S1). In addition, this mutation was present at 16 particular W codons only in the ECs, and at codons 38 in vpr and 840 in env only in our two case-patients.

      Discussion

      The two case-patients studied here are apparently cured of their HIV infection and one exhibits cells resistant to HIV infection in vitro [
      • Sandler NG
      • Bosinger SE
      • Estes JD
      • et al.
      Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression.
      ]. Functional cure, as defined by spontaneous control of HIV infection without disease progression, was therefore achieved [
      • Shytaj IL
      • Savarino A
      A cure for AIDS: a matter of timing?.
      ]. This occurred despite HIV DNA sequences could be laboriously retrieved from PBMCs. Research into a cure for HIV currently aims to remove any trace of HIV DNA from human cells, including through ‘purging’ of the latent HIV reservoir [
      • Fauci AS
      • Marston HD
      • Folkers GK
      An HIV cure: feasibility, discovery, and implementation.
      ,
      • Shytaj IL
      • Savarino A
      A cure for AIDS: a matter of timing?.
      ]. In contrast, we believe that the persistence of HIV DNA can lead to cure, and protection, from HIV. Globally, protection acquired by populations against viral infections was very commonly through integration of the infecting virus sequences and endogenization, as now also clearly described for bacteria and archaea with the CRISPR-Cas systems [
      • Makarova KS
      • Haft DH
      • Barrangou R
      • et al.
      Evolution and classification of the CRISPR‐Cas systems.
      ]. Retrovirus endogenization corresponds to retroviral DNA integration into germ cells, which allows its vertical transmission [
      • Stoye JP
      Studies of endogenous retroviruses reveal a continuing evolutionary saga.
      ]. Endogenous retroviruses (ERV) are present in genomes from many mammals, including in humans; ≈8% of the genetic sequences of modern man are retroviral sequences, corresponding to ≈100 000 ERV [
      • Liu H
      • Fu Y
      • Xie J
      • et al.
      Widespread endogenization of densoviruses and parvoviruses in animal and human genomes.
      ,
      • Raoult D
      A viral grandfather: genomics in 2010 contradict Darwin's vision of evolution.
      ]. The most famous ERV genes encode syncytins, which allowed syncytiotrophoblast formation in mammalian placentas [
      • Mi S
      • Lee X
      • Li X
      • et al.
      Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis.
      ]. Recently, retrovirus endogenization was highlighted as on-going in koala populations [
      • Tarlinton RE
      • Meers J
      • Young PR
      Retroviral invasion of the koala genome.
      ,
      • Oliveira NM
      • Farrell KB
      • Eiden MV
      In vitro characterization of a koala retrovirus.
      ]. These findings suggest that without therapeutic and prophylactic strategies, after several decades of HIV/host interactions and millions of deaths, it is likely that a few individuals might have endogenized and neutralized the virus and transmitted it to their progeny. In fact, given our difficulties in obtaining HIV sequences from the two case-patients, we speculate that this phenomenon may be much more common than observed in sequence databases, and that HIV cure may have already occurred through endogenization, in default of occurring through HIV ‘reservoir’ eradication [
      • Fauci AS
      • Marston HD
      • Folkers GK
      An HIV cure: feasibility, discovery, and implementation.
      ]. Massive sequencing in the near future of human DNA, including from African native individuals more extensively and anciently exposed to HIV, may reveal traces of such HIV endogenization processes. Duration of endogenization at the individual level is unknown.
      Several mechanisms of resistance to HIV have been described [
      • Deeks SG
      • Walker BD
      Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy.
      ,
      • Lim ES
      • Emerman M
      HIV: going for the watchman.
      ]. Those involved in resistance to HIV super infection observed in case-patient 1 may include resistance conferred by integrated HIV DNA. Thus, resistance to exogenous retroviruses conferred by related ERV has been reported, which can include blocking of entry receptors or cell budding [
      • Viginier B
      • Dolmazon C
      • Lantier I
      • et al.
      Copy number variation and differential expression of a protective endogenous retrovirus in sheep.
      ,
      • Takeda A
      • Matano T
      Inhibition of infectious murine leukemia virus production by Fv‐4 env gene products exerting dominant negative effect on viral envelope glycoprotein.
      ]. Regarding differences between anti-HIV immune responses observed on Western blotting for the two case-patients’ sera, they may be related to different mechanisms of the endogenization process. Reactivities are weaker and fewer for case-patient 2, but this patient exhibits strong CD4+ T-cell responses. In addition, some viral proteins are still produced and may play a role in the maintenance of T-cell immunity detected after cell stimulation in vitro[
      • Sandler NG
      • Bosinger SE
      • Estes JD
      • et al.
      Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression.
      ].
      In our case-patients, we suspect that knock out (KO) of the viral genes by replacing a W codon by a stop codon is the key to understanding the viral neutralization. Indeed, between 16 and 24 W-to-stop mutations, including at positions only observed in ECs, were observed in HIV sequences [
      • Eyzaguirre LM
      • Charurat M
      • Redfield RR
      • Blattner WA
      • Carr JK
      • Sajadi MM
      Elevated hypermutation levels in HIV‐1 natural viral suppressors.
      ]. The reason for these KO mutations is not established (Fig. 4). It may be that the patients’ DNA or RNA polymerases are prone to errors that are not fatal for the patient's health. In any case, these patients had normal W levels in their blood. The best candidates are APOBEC proteins, and specifically APOBEC3G, which enters the virion and changes G to A [
      • Harris RS
      • Liddament MT
      Retroviral restriction by APOBEC proteins.
      ], as observed here, and is thought to represent an ancient strategy of defence against ERV in humans [
      • Lee YN
      • Malim MH
      • Bieniasz PD
      Hypermutation of an ancient human retrovirus by APOBEC3G.
      ]. These proteins may be more expressed or active in the two case-patients, which was not demonstrated here. Otherwise, APOBEC3G activity may have been temporarily boosted by interferon-alpha administered to the index case for his chronic hepatitis C [
      • Sandler NG
      • Bosinger SE
      • Estes JD
      • et al.
      Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression.
      ,
      • Bonvin M
      • Achermann F
      • Greeve I
      • et al.
      Interferon‐inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.
      ]. In addition, a recent study suggests that co-infection with Streptococcus may enhance the activity of these proteins and inhibit HIV growth by generating hypermutability [
      • Wang Z
      • Luo Y
      • Shao Q
      • et al.
      Heat‐stable molecule derived from Streptococcus cristatus induces APOBEC3 expression and inhibits HIV‐1 replication.
      ]. The Vif protein theoretically blocks APOBEC3G activity [
      • Mehle A
      • Strack B
      • Ancuta P
      • Zhang C
      • McPike M
      • Gabuzda D
      Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin‐proteasome pathway.
      ]. As the Vif encoding gene is KO at a functionally critical position (21) [
      • Tian C
      • Yu X
      • Zhang W
      • Wang T
      • Xu R
      • Yu XF
      Differential requirement for conserved tryptophans in human immunodeficiency virus type 1 Vif for the selective suppression of APOBEC3G and APOBEC3F.
      ] in case-patient 1, this is likely to have contributed to the extensive inactivation of the HIV genome.
      Figure thumbnail gr4
      FIG. 4Schematic of hypotheses for tryptophan (W)-to-stop codon mutations, and HIV cure, in the two case-patients. W-to-stop codon mutations may occur due to increased APOBEC3G activity (including that mediated, or boosted, by vif gene knockout) (a), errors of the cellular RNA polymerase (b) or DNA polymerase (c).
      The phenomenon of HIV gene inactivation that precludes the production of replication-competent viruses has been previously reported by several teams, including recently by Eyzaguirre et al., who stated that some of their patients had defective proviral genomes. What we searched for and found here were HIV-infected patients with integrated viral DNA in their genomes but no HIV production. We do not believe that our two case-patients are unique, or the phenomenon described here is new. In contrast, our approach largely differs from earlier ones, as we suggest that persistence of integrated HIV DNA is not a barrier, but on the contrary, may be a prerequisite for HIV cure. Therefore, we propose a new vision of HIV cure through integration, inactivation and potential endogenization of a viral genome into the human genome. Finally, we believe that potential mechanisms of the natural cure of or resistance to HIV through persistence of integrated defective HIV DNA and endogenization are a critical model to develop preventive and curative strategies. We suggest that testing the occurrence of stop codons in the DNA of the integrated HIV (including the two signatures in the vpr and env genes) may predict the control and/or cure of the disease. This is critical because, for all patients being treated, the natural resistance will no longer be apparent. In conclusion, our findings, which warrant further confirmation, are a first step in understanding the resistance to retroviruses. They may allow us to figure out the endogenization of retroviruses and detect resistant patients, as well as to initiate strategies that imitate these patients in order to cure or prevent AIDS.

      Acknowledgements

      We are thankful to Catherine Robert, Said Azza, Natacha Tivoli, Sophie Venaud, Marielle Bedotto, Malgorzata Kowalczewska and Emile Foucat for their technical help.

      Transparency Declaration

      The authors have no conflicts of interest to declare. The study was funded internally.

      Supporting Information

      Additional Supporting Information may be found in the online version of this article:
      • Data S1. Supplementary results.
      • Figure S1. Phylogenetic tree based on HIV pol gene.
      • Figure S2. HIV-1 RNA detection post-stimulation of peripheral blood mononuclear cells from case-patient no. 2 by PHA and antibodies to CD3/CD28, IL2 and IL7.
      • Figure S3. Seroneutralization, by the serum of case-patients (a) and controls (b), of infection by the HIV-1 strain NL4.3 of peripheral blood mononuclear cells from HIV-negative donors.
      • Figure S4. Infectability of peripheral blood mononuclear cells from case-patient no.2 by the HIV-1 strain NL4.3.
      • Table S1. Distribution of tryptophan (W)-to-stop mutations in HIV genomes from the two case-patients, natural viral suppressors, HIV-infected controls on highly active antiretroviral treatment and HIV-infected untreated controls.
      • Table S2. Primers used for Sanger sequencing of HIV DNA from the two case-patients.
      • Table S3. Primers used for Sanger sequencing of patients’ messenger RNA encoding prostaglandin E2 synthase, RNA polymerase and DNA polymerase

      Supplementary Material

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