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. 2010 Feb 23;107(8):3782-7.
doi: 10.1073/pnas.0913122107. Epub 2010 Feb 8.

Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses

Géraldine Schlecht-Louf  1 , Martial Renard, Marianne Mangeney, Claire Letzelter, Aurélien Richaud, Bertrand Ducos, Isabelle Bouallaga, Thierry Heidmann
Affiliations

Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses

Géraldine Schlecht-Louf et al. Proc Natl Acad Sci U S A. .

Abstract

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins (Envs). The envelope-mediated immunosuppression was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation specifically abolishes IS activity without affecting the "mechanical" fusogenic function of the entire envelope. Here, we genetically "switched off' the envelope-mediated immunosuppression of an infectious retrovirus, the Friend murine leukemia virus, while preserving mutant envelope infectivity both ex vivo and in vivo, thus allowing us to test the functional importance of envelope-mediated immunosuppression in retrovirus physiology. Remarkably, we show, in vivo, that the non-IS mutant virus displays the same propagation kinetics as its WT counterpart in irradiated immunocompromised mice but that it is rapidly and totally cleared from normal immunocompetent mice, which become fully protected against a challenge with the WT retrovirus. Using cell depletion strategies, we further establish that envelope-mediated immunosuppression enables the retrovirus to escape innate (natural killer cells) and adaptive (CD8 T cells) antiviral effectors. Finally, we show that inactivated mutant virions induce higher humoral and cellular responses than their WT counterparts. In conclusion, our work demonstrates the critical role of Env-induced immunosuppression for retrovirus propagation in vivo and identifies a unique definite target for antiretroviral therapies and vaccine strategies, also characterized in the human T-cell leukemia virus (HTLV) and xenotropic murine leukemia virus-related virus (XMRV) retroviruses, opening unprecedented prospects for the treatment of retroviral diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of the fusogenic and IS activities of F-MLV Env and generation of a fusion-positive immunosuppression-negative specific mutant. (A) Schematic representation of the F-MLV Env with the surface (SU) and TM subunits, the furin cleavage site, the hydrophobic fusion peptide, the TM anchor, and the IS domain with its peptide sequence indicated; the E > R and A > F substitutions generated in the single mutant or DM are positioned. (B) Infectivity of Mo-MLV virions pseudotyped with F-MLV Env, WT, single mutants (E14R, A20F), or DM. Infectivity is measured using NIH/3T3 cells as target cells and is expressed as LacZ+ focus-forming units (ffu)/mL supernatant (mean ± SD, n = 3). (C) In vivo IS activity of WT and DM F-MLV Env. IS activity is assayed using F-MLV env-transduced MCA205 tumor cells engrafted into allogenic BALB/c mice and is quantified by an index based on tumor size (mean ± SD, n = 3) (10). (D) Compared in vitro propagation rates of WT (black circles) and DM (gray circles) F-MLV virions using NIH/3T3 cells as target cells. Viral loads in supernatants are measured by quantitative RT-PCR (mean ± SD, n = 4).
Fig. 2.
Fig. 2.
An immunosuppression-positive domain is required for F-MLV propagation in immunocompetent mice. Viremia of untreated (A) or x-ray-irradiated (B) Swiss mice on infection with WT (black circles) or the non-IS DM (gray circles) F-MLV. Mice were i.v. injected at day 0, and viral loads were measured by quantitative RT-PCR from blood samples collected at the indicated times (viral RNA copies/mL). In B, mice were x-ray-irradiated (5 Gy) 2 days before infection. Each circle corresponds to one individual mouse, with the lines connecting the geometric means. No viral RNA was detected in PBS-injected control mice.
Fig. 3.
Fig. 3.
Cellular targets of the Env-mediated immunosuppression effect assayed by immune cell depletions in vivo. Serum viral loads (Lower) after injection of WT (○ and •) or DM (□ and ▪) F-MLV were measured in untreated (• and ▪) or NK.1.1-depleted (○ and □) Swiss (A) or Swiss-Nude (B) mice or in CD8-depleted (• and ▪) or CD8-depleted plus NK1.1-depleted (○ and □) Swiss mice (C). The data correspond to the mean ± SD for five mice and are representative of two to seven independent experiments. Cell depletions (duration of antibody treatment indicated with a bar; Materials and Methods) were controlled by flow cytometry on blood samples (Upper) , with numbers in the dot plot corners indicating the percentage of labeled cells in the corresponding quadrant. NK cell recovery after the last antibody injection was 50% 5 days later and >90% at day 8.
Fig. 4.
Fig. 4.
Mutation within the F-MLV ISD confers protective activity and enhances immunogenicity. (A) Protection of mice immunized with DM F-MLV and challenged with WT F-MLV. Swiss mice were injected with 8 × 108 RNA copies of DM F-MLV (white and gray circles) or with PBS (black circles), and the absence of viremia was checked 4 weeks later (day −35). Nine weeks after infection, mice were challenged with 4 × 107 RNA copies of WT F-MLV (gray and black circles) or injected with PBS (white circles), and postchallenge sera samples were collected at the indicated time points. Each circle corresponds to one individual mouse, with the lines connecting the geometric means of eight mice; data are representative of more than three independent experiments, with no significant departure from full control of viremia by the vaccinated mice in all cases. (B and C) Compared immunogenicity of WT and DM UV-inactivated F-MLV. C57BL/6 mice were injected three times with a 1-week interval with 109 RNA copies of UV-inactivated WT or DM F-MLV in the presence of 50 μg of CpG ODN. (B) One week after the last injection, mice were blood-sampled and serially diluted serum was used to detect TM-specific (Left) and Gag-specific (Right) IgG by ELISA. Results are the mean ± SD of five mice and are representative of three independent experiments. (C) Ten days after the last injection, mice were killed and splenocytes were restimulated in vitro for 72 h in the presence or absence of the I-Ab-restricted H19 Env peptide (Left) or Kb-restricted Gag peptide (Right). Specific IFN-γ secretion by CD4 or CD8 T cells was detected in culture supernatants by standardized sandwich ELISA. Data are the mean ± SD of three independent experiments performed with three to four mice per group.
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