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

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.

Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins.

We have previously demonstrated that the envelope proteins of a murine and primate retrovirus are immunosuppressive in vivo. This property was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to have the env-expressing cells escape (at least transiently) immune rejection. Here, we analyzed the immunosuppressive activity of the human and murine syncytins. These are envelope genes from endogenous retroviruses independently coopted by ancestral hosts, conserved in evolution, specifically expressed in the placenta, and with a cell-cell fusogenic activity likely contributing to placenta morphogenesis. We show that in both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is immunosuppressive and, rather unexpectedly, the other (human syncytin-1 and mouse syncytin-A) is not (albeit able to induce cell-cell fusion). Delineation of the immunosuppressive domain by deletion analysis, combined with a comparison between immunosuppressive and nonimmunosuppressive sequences, allowed us to derive a mutation rule targeted to specific amino acids, resulting in selective switch from immunosuppressive to nonimmunosuppressive envelope proteins and vice versa. These results unravel a critical function of retroviral envelopes, not necessarily "individually" selected for in the retrovirus endogenization process, albeit "tandemly" conserved in evolution for the syncytin pairs in primates and Muridae. Selective inactivation of immunosuppression, under conditions not affecting fusogenicity, should be important for understanding the role of this function in placental physiology and maternofetal tolerance.

Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo.

Tumor development is a multistep process in which both genetic and epigenetic events cooperate for the emergence of a malignant clone. The possibility that endogenous retroviruses promote the expansion of a neoplastic clone by subverting immune surveillance has been proposed, but remained elusive. Here we show that knocking down-by RNA interference-an endogenous retrovirus spontaneously induced in the B16 murine melanoma results in the rejection of the tumor cells in immunocompetent mice, under conditions where control melanoma cells grow into lethal tumors. The knockdown does not modify the transformed phenotype of the cells, as measured both in vitro by a soft agar assay and in vivo by tumor cell proliferation in immunoincompetent (X-irradiated and severe combined immunodeficiency) mice. Tumor rejection can be reverted upon adoptive transfer of regulatory T cells from control melanoma-engrafted mice, as well as upon reexpression of the sole envelope gene of the endogenous retrovirus in the knocked down cells. These results show that endogenous retroviruses can be essential for a regulatory T-cell-mediated subversion of immune surveillance and could be relevant to human tumors where such elements-and especially their envelope gene-are induced.

Crystal structure of a pivotal domain of human syncytin-2, a 40 million years old endogenous retrovirus fusogenic envelope gene captured by primates.

HERV-FRD is a human endogenous retrovirus that entered the human genome 40 million years ago. Its envelope gene, syncytin-2, was diverted by an ancestral host most probably because of its fusogenic property, for a role in placenta morphogenesis. It was maintained in a functional state in all primate branches as a bona fide cellular gene, submitted to a very low mutation rate as compared to infectious retrovirus genomes. The structure of the syncytin-2 protein thus provides a good insight into that of the oldest mammalian retroviral envelope. Here, we report the crystal structure of a central fragment of its "fossil" ectodomain, allowing a remarkable superposition with the structures of the corresponding domains of present-day infectious retroviruses, in spite of a more than 60% divergent sequence. These results suggest the existence of a unique structural solution selected by these proteins for their fusogenic function.

Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors.

The possibility of obtaining, from any antibody, a fluorescent conjugate which responds to the binding of the antigen by a variation of fluorescence, would be of great interest in the micro- and nano-analytical sciences. This possibility was explored with antibody mAb4E11, which is directed against the dengue virus and for which no structural data is available. Three rules of design were developed to identify residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. (i) The target residue belonged to the hypervariable loops of the antibody. (ii) It was adjacent, along the amino acid sequence of the antibody, to a residue which was functionally important for the interaction with the antigen. (iii) It was not important in itself for the interaction with the antigen. Eight conjugates between a single chain variable fragment of mAb4E11 and an environment-sensitive fluorophore were constructed. Three of them showed an increase in their fluorescence intensity by 1.5-2.8-fold on antigen binding, without loss of affinity. This increase allowed the titration of the antigen in serum above a threshold concentration of 10nM. Experiments of quenching with potassium iodide suggested that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the binding of the antigen, and that therefore its mechanism is general.

Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies.

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.

Harnessing malE for the study of antigen/antibody recognitions.

The construction of hybrids between the variable fragment (Fv) of antibodies and protein MalE of Escherichia coli at the genetic level makes possible their preparation in a functional state, independently of any interaction with the antigen. We used such hybrids and a mutagenesis approach to study the recognition between antibody D1.3 and its antigen lysozyme, and its maturation. We subsequently transformed D1.3 into a reagentless fluorescent biosensor by knowledge-based design.

Improving the sensitivity and dynamic range of reagentless fluorescent immunosensors by knowledge-based design.

The variable fragment (Fv) of an antibody can be transformed into a reagentless fluorescent biosensor by mutating a residue into a cysteine in the neighborhood of the paratope (antigen-binding site) and then coupling an environment-sensitive fluorophore, e.g., N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester), to the mutant cysteine. For some residues, named operational, the formation of the conjugate does not affect the affinity of the Fv fragment for the antigen, and the binding of the antigen generates a measurable variation in the fluorescence intensity of the conjugate. We tested if this signal variation could be increased by coupling several molecules of fluorophores to the same molecule of Fv. Seven operational residues have been previously identified in the single-chain Fv (scFv) of monoclonal antibody D1.3 (mAbD1.3), directed against lysozyme. Ten double mutants of scFvD1.3, involving these residues, were constructed and coupled to the IANBD ester. The fluorescence of the double conjugates revealed a transfer of resonance energy between the two identical fluorescent groups. This homotranfer could be more important in the free state of the conjugate than in its antigen-bound state and increase its sensitivity for the detection of the antigen by up to 2.9-fold. A poorly sensitive conjugate could be improved by coupling a second molecule of fluorophore to residues located far from the paratope. Mutations altering the affinity of scFvD1.3 for lysozyme were introduced into one of its fluorescent conjugates. Using a mixture of three mutant derivatives of this unique conjugate, we could titrate lysozyme with precision in a concentration range encompassing 3 orders of magnitude.