HTLV-1 and -2 envelope SU subdomains and critical determinants in receptor binding
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Human T-cell leukemia virus (HTLV) -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU) of the HTLV envelope glycoprotein (Env) harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV) Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains.
Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD) lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205), that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events.
The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that contain all the determinants required for binding the HTLV receptor.
KeywordsCell Fusion Murine Leukemia Virus Syncytium Formation Cell Surface Binding Amphotropic Murine Leukemia Virus
List of abbreviations used
Human T-cell leukemia virus
envelope extracellular surface component
murine leukemia virus
proline rich region homologue
rabbit immunoglobulin constant fragment
Ampho-MLV Env fused to a carboxy terminal rFc tag
cell fusion interference assay
infectious units per ml
feline leukemia viruses
Hepes buffered saline
sodium dodecyl sulfate
Human T-cell leukemia virus type 1 (HTLV-1) has been found primarily in CD4+ and CD8+ T-lymphocytes in vivo [1, 2, 3], whereas CD8+ T-lymphocytes are thought to be the in vivo reservoir of HTLV-2 . However, the in vitro tropism of HTLV-1 and -2, as determined using HTLV envelope-pseudotyped virions or envelope-induced cell fusion assays, appears to be ubiquitous [5, 6, 7]. Indeed, we recently showed that Glut1, the ubiquitous vertebrate glucose transporter, serves as a receptor for HTLV-1 and -2 envelope glycoprotein (Env) . While the precise organization and properties of the receptor-interacting Env domains has not been reported, we found that the amino terminal two-thirds of the HTLV-1 extracellular surface component (SU) are sufficient to confer HTLV-1 tropism to an ecotropic Friend murine leukemia virus (F-MLV) Env . A cell fusion interference assay performed with this HTLV/F-MLV Env chimera and the parental Env confirmed that this 215 amino acid Env domain, harbors HTLV-1 receptor-binding determinants .
Several studies using synthetic peptides and neutralizing antibodies against the HTLV Env have shown that determinants within this proline rich region homologue (PRRH) are involved in interference to Env-mediated syncytium formation [19, 20, 21]. The PRRH had been thought to encode the receptor-binding domain, as based on cell-to-cell fusion assays [19, 22, 23, 24]. However, although PRRH synthetic peptides can block HTLV Env-mediated syncytia formation, they have no effect on HTLV SU binding  and infection . Indeed, we and others have shown that Env receptor binding per se, as well as interference to receptor-binding, cell-to-cell fusion, syncytium formation, and infection involve several distinct cell surface-associated parameters [27, 28, 29]. In the present report, we produced soluble forms of wild-type and mutant HTLV-1 and 2 SU amino terminal subdomains and tested their receptor-binding abilities. We also tested their ability to specifically interfere with HTLV Env cell surface binding, Env-mediated cell-to-cell fusion, and retroviral infection. By testing these essential parameters of Env-mediated dissemination, we delineated the Env receptor-binding domain (RBD) to the first 160 residues of the mature HTLV-1 and -2 SU, excluding the PRRH, and we identified a conserved tyrosine residue at position 114 of HTLV-1 Env as a critical determinant for HTLV Env receptor binding.
Motif conservation and similar modular organization of HTLV and MLV SU, and identification of a proline-rich region homologue (PRRH) in the HTLV SU
As shown in Figure 1, our alignment of the MLV and HTLV SU reveals several notable motif conservations outlining a similar modular organization of the MLV SU and HTLV SU. A (K/R)LL(T/N)LVQ motif, highly conserved between the F-MLV and HTLV-1 SU, is located immediately downstream of the PRR and its PRRH counterpart, respectively. Another highly conserved motif between MLV and HTLV, GXDP, is found immediately upstream of the PRR/PRRH (Figure 1). These two motifs compelled us to notice the PRRH, between the PSQ and KLLTLVQ sequences in HTLV-1, and between the PTQ and KILKFIQ sequences in HTLV-2 (Figure 1). As counted from the first and last proline in the delineated sequence, the PRRH has a proline content of 30.8% and 30.4% for HTLV-1 and -2, respectively. This is slightly lower than the 35.3%, 36%, 36%, and 35.6% proline content for the ecotropic, polytropic, xenotropic, and amphotropic MLV Env, respectively (Figure 1). The presence of a PRRH in the HTLV SU appeared to be characteristic of their MLV-like modular organization, since HTLV SU average proline content outside of the PRRH does not exceed 11%.
Functional, soluble HTLV Env-receptor binding determinants
HTLV-1 and -2 SU amino terminal subdomains with or without their respective PRRH were constructed as fusion proteins with either an influenza hemagglutinin (HA) or rabbit immunoglobulin Fc (rFc) carboxy terminal tag (Figure 2B). The H1215SU and H2211SU subdomains comprise the first 215 and 211 residues, counting from the first methionine in the signal peptide through the KLLTLVQ of HTLV-1 and KILKFIQ of HTLV-2 Env, respectively (Figure 2B). The H1179SU and H2178SU, comprising the amino terminal 179 and 178 amino acids of the HTLV-1 and -2 Env, respectively, exclude the PRRH sequence (Figure 2B).
Cell lysates and cell culture supernatants were analyzed to evaluate intracellular expression and secretion of functional SU amino terminal domains in transfected-cell cultures, respectively. H1215SU and H2211SU, containing the PRRH sequence, and H2178SU lacking this PRRH were all efficiently expressed in transfected cells (Figure 3B). It is noteworthy, however, that recovery of tagged H1179SU molecules was largely inefficient because the vast majority of this protein was cleaved (data not shown). In contrast, no significant cleavage was observed with the other soluble domains released in the medium (not shown) (Figure 3C). As expected for immunoadhesins, H1215SU, H2211SU, and H2178SU rFc-tagged domains were detected as dimers under non-reducing conditions (not shown). Immunoblots of cell extracts revealed two forms of intracellular H1215SU and H2211SU (Figure 3B); this was likely due to variable glycosylation of these subdomains. However, a single secreted, soluble form of each of these amino terminal subdomains was detected in cell culture supernatants (Figure 3C).
A truncated Ampho-MLV SU-rFc fusion protein that comprises the amino terminal 397 residues of the Ampho-MLV Env fused to a carboxy terminal rFc tag was constructed (A397SU) and used as a heterologous control. A single form of this truncated SU was efficiently expressed in transfected cells (Figure 3B), and abundantly secreted in cell culture medium (Figure 3C).
HTLV-1 and -2 SU subdomains with HTLV receptor binding properties
To determine whether cell surface binding of these soluble SU domains corresponded to bona fide binding to the HTLV receptor, we performed an Env-specific binding interference assay. In this assay, transfection of the above described chimeric Env and SU subdomains into 293T cells resulted in interference to cell surface binding by the soluble HA-tagged H2178SU subdomain (Figure 4B). Indeed, nearly complete interference was observed when cells were transfected with the amino terminal subdomain constructs, in the presence and absence of PRRH sequences (H1215SU and H2211SU versus H1183FEnv and H2178SU) (Figure 4B). This effect was specific as HTLV SU binding was not inhibited by a heterologous A397SU domain (Figure 4B). Therefore, we showed that the first 163 and 158 residues, with a cleaved signal peptide, of the mature HTLV-1 and HTLV-2 SU, respectively, contained the entire HTLV Env RBD. These data also showed that HTLV-1 and 2 cross-interfered, consistent with the fact that they recognize the same cell surface receptor for infection [8, 32].
Interference to HTLV Env-mediated cell-to-cell fusion by HTLV SU amino terminal subdomains
Viral envelope interference occurs when cell surface receptors are occupied by receptor-interacting Env components [33, 34, 35]. Since interference to the different Env-mediated functions involves distinct components [27, 28, 29], we also tested the abilities of the H1183FEnv and the HTLV SU amino terminal subdomains to interfere with HTLV Env-mediated cell fusion. Interference to cell fusion was measured using a quantitative HTLV envelope cell fusion interference assay (CFIA), as previously described .
Interference to HTLV Env-mediated infection by HTLV SU amino terminal subdomains
Interference, as described above, was based on the inhibition of cell-to-cell fusion induced by fusogenic Env expressed in the absence of other viral proteins. We further evaluated the abilities of the Env chimeras and soluble subdomains to specifically interfere with HTLV Env-mediated infection. HTLV Env-pseudotyped MLV virions, MLV(HTLV), were produced to infect 293T target cells. Because these recombinant cell-free virions are not competent for replication, this viral pseudotype infection assay tests a single round of infection, and does not measure replication and subsequent exponential viral dissemination. Therefore, relative infection values are expressed in linear rather than logarithmic scales.
Because HTLV dissemination appears to occur mostly via cell-to-cell contact, we also tested envelope interference to infection by HTLV-1 SU amino terminal domains using a cell-to-cell transmission interference assay. In this assay, cells harboring interfering chimeric Env and soluble subdomains were cocultured with cells producing MLV(HTLV) virions. Transfection of either chimeric Env or soluble subdomains into HeLa target cells decreased MLV(HTLV) infection to levels similar to those observed in the cell fusion interference assay presented in figure 5 (data not shown).
Identification of residues within the HTLV SU amino terminal domain that modulate receptor binding and HTLV Env-mediated interference
We next tested the abilities of these mutants to interfere with HTLV Env-mediated cell fusion and infection, using the assays described above. As mentioned above, all wild-type and mutant HTLV SU subdomains were produced and secreted with a similar efficiency (Figure 7A). Expression of the H1(D106A)SU and H1(Y114A)SU mutants, with decreased capacities to bind the HTLV receptor, correlated with decreased interference to HTLV Env-mediated cell fusion and infection. Indeed, H1(Y114A)SU, which had nearly undetectable level of binding, showed the lowest levels of interference and thus allowed the highest levels of HTLV Env-mediated cell fusion and infection (56% ± 16% and 46% ± 10%, respectively) (Figure 7). Nevertheless, levels of fusion and infection were lower than that observed when the heterologous A397SU was used as a negative control of interference (83% ± 11% and 89% ± 19% for cell fusion and infection, respectively). Thus, overexpression of mutant HTLV SU fragments with highly decreased receptor binding abilities can still exert, albeit to a significantly lesser extent, interference to HTLV Env-mediated cell fusion and infection.
We found that similar levels of interference to HTLV Env-mediated cell fusion and infection were observed when either the parental H1215SU or the mutant H1(S101A)SU were expressed in target cells (Figure 7B and 7C). This is consistent with the capacity of this mutant to bind target cells at levels similar to that of wild type H1215SU. However, interference to HTLV Env-mediated cell fusion and infection did not always correlate with cell surface binding profiles. While the H1(R94A)SU mutant inhibited cell fusion and infection, its effects were significantly lower than those of the wild-type H1215SU (56% ± 8% and 32% ± 2.3%, respectively) (Figure 7B,7C). Thus, although neither Arg94 nor Ser101 of the HTLV-1 SU appears to play a direct role in binding, Arg94 modulates HTLV Env-mediated fusion and infection (Figure 7), likely via post-binding effects rather than binding per se. In conclusion, Tyr114 appeared as the main determinant identified so far for HTLV Env binding, whereas the effects previously described with Arg94 and Ser101 are most likely associated with post-binding events.
Here, we report the generation of MLV Env with chimeric HTLV/MLV SU and truncated HTLV-1 and -2 amino terminal SU subdomains that can be expressed in and secreted from eukaryotic cell lines in functional, soluble form. Using these constructs, we demonstrated that the amino terminal 163 and 158 residues (i.e., expunged of their Env signal peptide) of the mature HTLV-1 and -2 Env SU, respectively, were sufficient to exert both HTLV receptor binding and efficient interference to diverse HTLV Env-mediated functions, including binding, cell-to-cell fusion and cell-free as well as cell-to-cell infection. Although the PRRH sequence comprising amino acid residues 180 to 215 of the HTLV-1 Env and 176 to 211 of the HTLV-2 Env was previously thought to be a receptor binding site, our data preclude a major role for this region in the binding properties described above. Indeed, whereas a synthetic peptide composed of amino acids 197 to 216 and located within the HTLV-1 PRRH, has been reported to interfere with HTLV Env-induced syncytia formation , this peptide was later shown to compete neither with receptor binding of the entire HTLV-1 Env SU , nor with infection . It is therefore likely that the effects reported for PRRH-derived peptides, as measured by syncytia formation, are solely due to post-receptor binding events. However, we identified Tyr114 of the HTLV-1 Env, which corresponds to Tyr110 of the HTLV-2 Env, as a key residue in HTLV Env binding and for all the aforementioned HTLV Env-mediated functional assays. We could not detect binding of H1(Y114A)SU by flow cytometry, while this mutant exerted residual, albeit significantly decreased, interference to HTLV Env-mediated cell fusion and infection. Altered folding outside of the binding domain per se, rather than direct alteration of the receptor-binding site, could also account for the lack of binding of this mutant. However, we favor the latter hypothesis, since the H1(Y114A)SU mutant was properly folded and transported to the plasma membrane and secreted in the medium as efficiently as wild type RBD, thus arguing against gross misfolding of this mutant. Accordingly, Tyr114 appears to be conserved in all known human and simian T cell leukemia viruses strains, which share the same receptor.
The receptor-binding site in MLV RBD is composed of a combination of several cysteine loops located upstream of the PRR [11, 39] which is linked to a conserved anti-parallel β core . The isolation of an F-MLV SU amino terminal subdomain allowed crystallization of MLV RBD and the modeling of the RBD cysteine loop arrangement . The precise organization of cysteine loops, likely to harbor the receptor binding determinants, within the HTLV SU amino terminus remains to be established. Nevertheless, the identification of Tyr114 as a key HTLV-1 RBD residue points at this determinant as a very likely receptor-binding core. This, together with previous works relying on syncytia formation and cell-to-cell transmission [36, 37], will help to distinguish between bona fide receptor binding determinants and determinants involved at a post-binding level.
Another recently identified determinant, the Pro-His-Gln SU motif conserved among gammaretroviruses such as MLV and feline leukemia viruses (FeLV), has been determined to play a major role in viral entry during post-binding events . The mechanism of this effect involves a direct interaction of MLV SU soluble forms with Env attached SU carboxy terminus [41, 42, 43, 44, 45, 46]. This interaction between the SU amino and carboxy termini leads to the T cell-restricted tropism of a natural isolate of FeLV, FeLV T, in which the SU Pro-His-Gln motif is mutated. Indeed, FeLV T is restricted in cat to T cells because they naturally express an endogenous soluble FeLV RBD-related factor called FeLIX that trans-complements the lack of the SU Pro-His-Gln motif in the FeLV T Env and restores its post-binding defect . Despite the HTLV-1 and F-MLV SU homologous modular organization and the assignment of several common motifs between the two latter SU, no obvious Pro-His-Gln motif homologue is present in the HTLV SU amino terminus. Whether a FeLIX-like molecule that interacts with HTLV Env exists in human T cells remains to be addressed. Furthermore, the fact that the Pro-His-Gln has been shown to play a major role in transactivation of viral infection in several gammaretroviruses which are efficiently infectious as cell-free virions [42, 44, 48], raises the question whether the apparent lack of such a motif in the HTLV simple oncovirus-like SU is linked to the relative inefficiency of HTLV Env-mediated infection by cell-free virions. The HTLV SU subdomains described here should prove to be valuable in addressing such questions.
The recent identification of Glut1, the ubiquitous glucose transporter of vertebrates , as a receptor for HTLV Env  adds an additional similarity between the Env of HTLV, a deltaretrovirus, and that of gammaretroviruses. All these virus Env recognize multimembrane-spanning metabolite transporters [50, 51]. This and the common modular organization of the HTLV and MLV SU raise questions regarding the origin of the HTLV Env. It has previously been reported that envelopes of invertebrate retroviruses may have been "captured" from other viruses [52, 53, 54]. As HTLV and MLV have strongly divergent overall genomic organizations, "envelope capture" from related ancestor genes might account for the close relationship between the Env of these phylogenetically distant viruses .
We have generated truncated domains of the HTLV Env amino terminus, upstream of residues 183 and 178 of the HTLV-1 and -2 Env, respectively, that were sufficient to bind target cells of different species through interaction with the HTLV Env receptor. We also identified a tyrosine at position 114 and 110 in HTLV-1 and -2 Env, respectively, as a key determinant for this binding. In addition to their use for further exploration of the mechanisms involved in HTLV entry, the tagged HTLV-1 and -2 RBD subdomains described here are novel tools for the detection of Glut1 cell surface expression and intracellular trafficking. Indeed, we tracked intracellular expression of EGFP-tagged HTLV SU subdomains by time-lapse microscopy, and found that they are preferentially routed toward cell-cell contact areas (unpublished observations), where Glut1 is particularly abundant  and our unpublished observations). Furthermore, those HTLV SU derivatives could be of particular importance in view of the key roles played by Glut1 in various biological processes, including T cell survival and activation [31, 56], tumor genesis [57, 58], and neuronal activity . Interestingly, soluble HTLV SU subdomains inhibit Glut1-mediated glucose transport, and accordingly, expression of mutants with diminished receptor binding ability resulted in less pronounced inhibition  and data not shown). Thus, these HTLV SU derivatives could also be used as glucose transport inhibitors. These data demonstrate the potential for the novel and broad utility of these reagents in the study of HTLV infection as well as biological processes involving glucose transport and metabolism.
Materials and methods
Construction of chimeric Env and HTLV-1 and -2 SU subdomains
To exchange the PRR and PRRH regions, we introduced an allelic Mfe I restriction site in the HTLV-1 and F-MLV Env. Introduction of this site in F-MLV resulted in the substitution of a glutamine and leucine (QL) dipeptide for the parental arginine and valine (RV) residues of the GPRVPIGP motif, at the start of the MLV Env PRR. Introduction of the MfeI site in the PSQL motif of the HTLV-1 SU maintained the parental QL residues, at the start of the HTLV Env PRRH. By exchanging domains at the Mfe I sites, we derived the H1183FEnv chimera containing the amino terminal 183 residues of the HTLV Env followed by the F-MLV PRR. In this chimera, the PSQL/PIGP hybrid sequence is generated at the exchange border, and the PRRH of HTLV is replaced by the F-MLV PRR (Figure 2A). In contrast, the entire PRRH of HTLV-1 is present in the H1215FEnv chimera – this Env chimera has been previously described and designated HHproFc . The H1183FEnv and H1215FEnv chimeras, as well as the parental HTLV-1 and F-MLV Env, were inserted in an allelic fashion into the previously described pCEL retroviral Env expression vector . The HTLV-2 Env expression vector, pCSIX/H2, was constructed by inserting the HindIII – EcoRI fragment from pHTE-2 (a gift from M-C Dokhelar) encompassing the HTLV-2 env gene, the pX region and the 3' LTR into pCSI (CMV promoter, SV-40 intron)  at the HindIII and EcoRI restriction sites.
The H1215SU, H2211SU, H1179SU, and H2178SU subdomains, corresponding to the HTLV-1 and -2 SU amino terminus with and without their respective PRRH, were generated by PCR and subcloned into the pCSI expression vector as fusion proteins harboring a carboxy terminal rFc or HA tag (Figure 2B). The H1(R94A)SU, H1(S101A)SU, H1(D106A)SU, and H1(Y114A)SU substitution mutants were generated by oligonucleotide-directed PCR mutagenesis on the H1215SU vector and subcloned into the pCSI expression vector. All PCR-generated DNA fragments were sequenced using an ABI Prism 310 sequencer. Cloning details are available upon request.
Protein expression and immunoblots
Approximately 5 × 105 293T cells per 35 mm well were transfected with 5 μg of vectors using a calcium-phosphate-Hepes buffered saline (HBS) transfection protocol. Transfection medium was replaced with 3 ml of fresh culture medium twenty hours post-transfection. Forty-eight hours post-transfection cell culture medium (supernatant) was recovered and filtered through a 0.45 μm pore-size membrane to remove cell debris. Twenty μl were directly analyzed by SDS-PAGE (15% polyacrylamide gel), and the rest was aliquoted and stored at -20°C for later use in binding assays (see below). Cell extracts were collected 48 h post-transfection in 1 ml of cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, 0.5% deoxycholate, and a cocktail of mammalian protease inhibitors [Sigma]) and clarified by two successive centrifugations at 13,000 rpm for 10 min at 4°C in a microcentrifuge. Approximately 20 μl of each extract, adjusted after normalization for protein concentration using the Bradford assay (Sigma), were subjected to electrophoresis on SDS-15% acrylamide gels, followed by transfer onto nitrocellulose (Protran; Schleicher & Schuell). Membranes were blocked in phosphate-buffered saline (PBS) containing 5% powdered milk and 0.5% Tween 20, probed with a 1:1000 dilution of a goat anti-RLV gp70 polyclonal antibody (Viromed) followed by a horseradish peroxidase-conjugated anti-goat immunoglobulin (for detection of chimeric Env), or goat anti-rabbit-IgG-horseradish peroxidase-conjugated immunoglobulins (for detection of rFc-tagged SU subdomains). Immunoblots were subsequently washed three times with PBS-0.1% Tween 20 and revealed by chemiluminescence (ECL+, Amersham).
Binding and binding interference assays
Binding assays were performed as previously described . Briefly, 5 × 105 target cells were detached with a PBS-EDTA solution, collected by centrifugation, incubated for 30' at 37°C with 300 μl of rabbit Fc-tagged soluble HTLV-1, HTLV-2, or Ampho-MLV truncated SU, washed, labeled with an anti-rabbit-IgG FITC-conjugated antibody, and analyzed on a FACSCalibur (Becton Dickinson). Data analysis was performed using the CellQuest software (Becton Dickinson). For interference studies, 293T cells were transfected with 4 μg of Env or Env SU subdomain expression vectors (carboxy terminal rFc-tagged forms) using the calcium-phosphate-HBS method. Under these conditions, transfection efficiencies ranged from approximately 80 to 90% of the target cells. Twenty-four and 48 hours post-transfection, cells were collected and transfected 293T cells expressing the different interfering HTLV or Ampho-MLV domains were incubated with a challenging HA-tagged soluble HTLV-2 SU amino terminal subdomain (H2178SU-HA). Cells were stained using a primary 12CA5 anti HA antibody followed by an anti-mouse-IgG FITC-conjugated antibody before detection by flow cytometry.
Envelope interference to cell fusion assay
Briefly, the HTLV/MLV Env chimera, H1183FEnv, was used to interfere with challenging HTLV Env. The interfering non-fusogenic H1183FEnv and truncated HTLV SU subdomains were transiently transfected into HeLaCD4LTRLacZ, a cell line highly susceptible to HTLV Env-induced fusion that contains a stably integrated Tat-dependent LacZ expression vector . These transfectants were cocultured with Tat-expressing NIH3T3(TK-) cells (NIH3T3(TK-)Tat) that were transiently transfected with the challenging HTLV Env. The NIH3T3(TK-)Tat cell line is resistant to HTLV-Env-induced syncytia formation, despite its ability to express the HTLV receptor and to bind HTLV Env, and thus can be used to precisely monitor fusion of the HeLaCD4LTRLacZ target cells [9, 29]. H1183FEnv Env and truncated HTLV SU subdomains plasmid DNA (2 to 3 μg) was transfected into HeLaCD4LTRLacZ cells, while challenging, fusogenic HTLV-1 Env plasmid (1 μg) was transfected into NIH3T3(TK-)Tat. The interfering Env or SU subdomain-presenting cells were detached 24 hours post-transfection and 1–2 × 105 cells were cocultured for 24 hours with 1–2 × 105 challenging HTLV-1 Env-presenting NIH3T3(TK-)Tat cells. Subsequently, the cocultured cells were fixed and stained for β-galactosidase expression as described previously . Transfection efficiencies of the HeLaCD4LTRLacZ target cells were approximately 50%. Mock transfections were performed with similar amounts of control plasmid DNAs. Env interference was measured by the decreased number of blue foci and was expressed as percent blue foci of control fusion (mock-transfected target cells). Data are represented as mean interference (± standard deviation), and statistical significance of interference levels was determined using a pairwise Student's t test.
Envelope interference to infection assay
MLV(Ampho) and MLV(HTLV) pseudotyped virions were produced after transfection of 106 293T cells with 5 μg pCSI/Ampho or pCSIX/H2, respectively, 5 μg pCL/Gag-Pol  and 10 μg of pCLMFG-LacZ , using a calcium-phosphate-HBS transfection protocol. Supernatants were recovered 48 hours post transfection and filtered through 0.45 μm pore-size membrane to remove cell debris, and stored at -80°C. The pCLMFG-LacZ plasmid is a retroviral expression vector that provides a packageable RNA coding for the LacZ gene marker. pCSI/Ampho is an expression vector encoding the Ampho-MLV Env, and the HTLV-2 Env expression vector, pCSIX/H2, is described above.
Virion-containing supernatants were used to infect target 293T cells expressing the chimeric Env or HTLV RBD subdomains. Transfection efficiencies of target 293T cells were >80% in all experiments. Infections were performed 36–48 hours post-transfection on cultures grown in 12 well plates (Costar) at 37°C, medium was changed 24 hours later, and confluent cell monolayers were fixed, stained for β-galactosidase activity before counting blue foci. Interference to infection was determined by infecting transfected target cells with approximately 100 and 1000 iu. Infection was evaluated as described above, and the number of LacZ-positive blue colonies counted was normalized by multiplying by the appropriate dilution factor. The resulting infection values were analyzed as iu/ml of virus containing supernatant. Subsequently the relative infection levels in cells expressing the HTLV SU domains were compared to those of mock transfected cells and were expressed as percentages of control infection (% control).
We thank N. Taylor for helpful discussion and critical reading of the manuscript, G. Labesse for his help in protein sequence analyses, R.K. Naviaux for the gift of pCL-Eco and pMFG-LacZ plasmids, J.A. Young for the rabbit Fc plasmid, J.-C. Dantonel for the anti-HA antibody, F. Carbonell for technical assistance, and all the members of our laboratory for insightful discussion. FJK was supported by an award from the Philippe Foundation and successive fellowships from the Agence Nationale pour la Recherche contre le SIDA (ANRS), the Association pour la Recherche contre le Cancer (ARC), and the Fondation de France. NM is supported by a graduate student fellowship from the MRT. JLB and MS are supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). This work was supported by grants from ARC (ARC Nos. 5989 and 3424), Fondation de France (Nos. 2291 and 2138) and Association Française contre les Myopathies (AFM No.7706) to MS.
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