INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Most type C mammalian retrovirus envelopes share proline-rich regions located in the middle of their surface proteins (SUs). These regions provide a hinge separating two functional domains of the SU (10): the N-terminal receptor binding domain (BD) (3) and the C-terminal domain involved in post-binding entry events (16, 18). Several reports suggest that the proline-rich domain is not just a flexible linker but rather a functional domain; it was found (i), in some cases, to influence receptor recognition (3, 17); (ii) to be important for stabilization of SU-transmembrane protein (TM) interaction (10), and (iii) to affect virus fusiogenicity possibly by altering glycosylation (1). A direct role of the natural proline-rich region of the SU in stabilization of the conformation and fusiogenicity of the murine leukemia virus (MLV) envelope (13, 23) was recently demonstrated. Predictive structure analysis indicated that the proline-rich peptides present in these contructs may be organized as a -turn polyproline. This particular structure has uncommon properties, such as the ability to oligomerize in quaternary structure and also to develop elastomeric forces (20).

When such a proline-rich domain was inserted between two BDs of a chimeric MLV envelope glycoprotein, generated by fusion of a Pit-2 BD at the N terminus of Moloney retrovirus envelope protein mCAT-1 BD, we showed that the entry of retroviruses into targeted cells required the presence of both receptors Pit-2 and mCAT-1 at the surface of the cells (21, 22). Binding assays indicated that the mCAT-1 BD of the virion-associated chimeric envelopes is partially or completely hidden (22). Moreover, substitution of proline-rich intergenic spacer (PRO spacer) by a characterized -turn polyproline fragment from bovine elastin gave the same efficiency of cooperation of the receptors. Finally, we demonstrated that, after unmasking, secondary interaction between mCAT-1 and the backbone envelope is a crucial prerequisite for induction of the fusion process involving the TM subunit. These data are consistent with a model for receptor cooperativity in which binding to the targeted (Pit-2) receptor triggers conformational rearrangements of the envelope that lead to unmasking of the hidden mCAT-1 BD, thereby facilitating its interaction with the viral (mCAT-1) receptor, which leads to fusion.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell lines. The human fibroblastic TE671 cell line (ATCC CRL8805) was grown in Dulbecco modified Eagle medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (Gibco-BRL). These cells express Pit-2 receptor and are susceptible to amphotropic MLV.

Plasmids, transfection, and virus production. Plasmids encoding the ecotropic (FBMOSALF), amphotropic (FBASALF), and AMO chimeric (FBAMOSALF) envelopes were described elsewhere (6). Expression plasmids for AMOFx, AMO1, AMO1Fx, AMOG1Fx, AMOG2, AMOG2Fx, AMOG3Fx, AMOPRO2, AMOPRO3, AMOPRO4, and AMOPRO chimeric envelopes were as described elsewhere (21, 22).

Immunoblots. Virus producer cells were lysed in a 20 mM Tris-HC1 buffer (pH 7.5) containing 1% Triton X-100, 0.05% sodium dodecyl sulfate (SDS), 5 mg of sodium deoxycholate per ml, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated for 10 min at 4°C and then centrifuged for 10 min at 10,000 × g to pellet the nuclei. Supernatants were then frozen at 70°C until further analysis. Virus samples were obtained by ultracentrifugation of viral supernatants (10 ml) in a SW41 Beckman rotor (30,000 rpm, 1 h, 4°C). Pellets were suspended in 100 µl of phosphate-buffered saline (PBS) and frozen at 70°C. Samples (30 µg for cell lysates or 10 µl for purified viruses) were mixed 5:1 (vol/vol) in a 375 mM Tris-HCl (pH 6.8) buffer containing 6% SDS, 30% -mercaptoethanol, 10% glycerol, and 0.06% bromophenol blue; boiled for 3 min, and then run on SDS-10% acrylamide gels. After protein transfer onto nitrocellulose filters, immunostaining was performed in Tris base saline (pH 7.4) (TBS) with 5% milk powder and 0.1% Tween 20. The Antibodies (Quality Biotech, Inc.) were goat antisera raised against either Rausher leukemia virus (RLV) gp70-SU protein or RLV p30-CA protein and were diluted 1/1,000 and 1/10,000, respectively. Blots were developed using horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin antibodies (Dako) and an enhanced chemiluminescence kit (Amersham Life Science).

Binding assays. Target cells were washed in PBS and detached by a 10-min incubation at 37°C with 0.02% EDTA in PBS. Cells were washed in PBA (PBS with 2% fetal calf serum and 0.1% sodium azide). Then, 5 × 10⁵ cells were incubated in PBA with viruses for 45 min at 37°C. Cells were then washed with PBA and incubated for 45 min at 4°C with monoclonal antibody 83A25, which recognizes a common epitope present on the ecotropic and amphotropic MLV surface unit (SU) and allows the detection of SU proteins that are either soluble or associated with the virus (8), or 9E8, which is specific of the transmembrane unit of the MLV envelope (TM), and allows the detection of MLV envelope only when associated with the virus (virus binding). Cells were washed twice with PBA and incubated with anti-rat immunoglobulin fluorescein isothiocyanate-conjugated antibodies (Dako). At 5 min before the two final washes in PBA, cells were counterstained with 20 µg of propidium iodide per ml. The specific fluorescence of the cells was analyzed with a flow cytometer (FACSCalibur; Becton Dickinson).

Infection assays. Target cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well. Viral supernatant dilutions containing 5 µg of Polybrene per ml were added, and cells were incubated for 3 to 5 h at 37°C. Viral supernatant was then removed, and cells were incubated in regular medium for 48 h. X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining and viral titer determination as lacZ infectious units (iu)/milliliter were performed as previously described (6).

	RESULTS

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We have previously reported a set of chimeric MMLV-derived envelopes designed to retarget the Pit-2 phosphate transporter molecule by insertion into MMLV envelope of the first 205 amino acids of the amphotropic MLV 4070A SU corresponding to the BD (4070A BD) (Fig. 1) (22). We have shown that the infection of target cells by virions bearing these envelopes is strictly dependent on the nature of the interdomain spacer separating the 4070A BD from the MMLV backbone envelope. When a proline-rich motif of 60 amino acids was inserted as a spacer, generating the AMOPRO envelope, infection was dependent on the presence of both receptors (Pit-2 and mCAT-1) at the surface of the target cells, suggesting that these receptors cooperate during virus entry (Fig. 2). In contrast, when truncated forms of the proline-rich motif (AMOPRO2, AMOPRO3, or AMOPRO4 chimeric envelopes) were used, infection occurred only when the targeting receptor was expressed at the surface of cells but not when receptor corresponding to the backbone envelope was expressed alone. These data are consistent with a dynamic role of the proline-rich motif inserted into chimeric MLV-derived env in receptor cooperation during retroviral entry.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Schematic representation of envelope chimeras. The plasmid construct coding for chimeric envelope glycoproteins in which the Pit-2 BD was fused to MMLV SU is displayed. The general format is shown diagrammatically, and the amino acid sequence of the interdomain spacer is shown in detail. Vertical arrows, protein cleavage sites; SU, surface protein; TM, transmembrane protein; SP, envelope signal peptide; T, transmembrane domain; Pit-2 ligand, MLV-A receptor BD; black boxes, MMLV-derived envelope sequences; gray boxes, MLV-A-derived envelope sequences; white boxes, other MLV-derived envelope sequence. All env genes were expressed using the same promoter (LTR) and polyadenylation sites (polyA) from a subgenomic mRNA using retroviral splice donor (SD) and acceptor (SA) sites.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Infection by virions expressing targeting envelopes. Virions were used to infect CHO cells expressing Pit-2 receptor (CHO-Pit-2 cells; white bars), mCAT-1 receptor (CeRD9; gray bars), or both receptors (CeAR13; striped bars). Data correspond to the average of 20 repeats (each duplicate), and no significant difference from one experiment to another was observed (standard deviation of 0.25 log, 99% confidence interval of the mean).

-Turn helix plays a critical role in receptor cooperation. The proline-rich region of 4070A MLV is predicted by the Chou-Fasman analysis (5) to form a regular arrangement of 11 -turns induced by the majority of the prolines in this region (13, 22). This highly ordered structure, consisting of multiple reverse turns, is consistent with the polyproline -turn helix form of a secondary structure (15).

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Viral incorporation of chimeric envelope glycoproteins. Immunoblots of pellets of viral particles from TELCeB6 cells transfected with the envelopes. The blot was stained with an SU antiserum and cut at 46 kDa in order to stain the lower part with a p30 antiserum to detect the CA protein as a viral production control.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Binding assays on Pit-2 receptor. Human TE671 cells expressing Pit-2 receptors were used as a target. The background of fluorescence was provided by incubating the cells with DMEM only (white histograms). Binding assays (black histograms) were performed with some of the envelopes shown in Fig. 1. The envelope glycoprotein content of the different samples was normalized by immunoblot on a viral supernatant.

Deletion of proline residue in the -turn helix abolishes cooperation. In order to confirm the dynamic role of the env structure in receptor cooperation, we replaced the repeated proline residues with isoleucine, which prevents -turn helix formation. By PCR mutation we replaced the three proline residues present in the EL3 spacer of AMOEL3 and AMOEL3-VA and derived envelopes named AMOEL3-I and AMOEL3-AI, respectively. As expected, these mutations had no effect on the processing and virion incorporation of the corresponding envelopes (Fig. 3B).

Interaction with both receptors is required for fusion transduction. Our results are consistent with a model of two-step interaction for the entry of the targeted virions into cells. To address the requirement, in the second step, of MMLV backbone envelope interaction with the mCAT-1 receptor, we generated a set of mCAT-1 binding-defective mutants. It has been previously demonstrated (14) that an Asp residue in position 84 in the MMLV wild-type env plays a critical role in binding to receptor. When Asp-84 is replaced with a Lys residue, the infection is blocked. By PCR amplification, we created the Asp-to-Lys mutation at position 84 of the mCAT-1 BD of AMO, AMOPRO3, AMOPRO, and AMOG1X, resulting in AMOD84K, AMOPRO3D84K, AMOPROD84K, and AMOG1XD84K, respectively. The resulting envelopes were tranfected into TELCeB6 cells, and we showed by Western blot analysis that the production, maturation, and incorporation into viral particles of each env glycoprotein were not significally affected by the mutation (Fig. 3C).

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Envelope binding assays. The background fluorescence was provided by incubating the cells with DMEM only (dotted line histograms). (A) Pit-2 binding control of the D84K mutant. Human TE671 cells expressing Pit-2 receptors were used as a target. Binding assays of D84K mutant envelopes (white histograms) were compared to chimeric envelope unmutated on residue 84 (black histograms). (B) mCAT-1 binding test of D84K mutant. CeRD9 cells expressing mCAT-1 receptor alone were used as a target. Binding assays of D84K mutant envelope binding (white histograms) were compared to unmutated recombinant envelope (black histograms). The envelope glycoprotein content of the different samples was normalized by immunoblot on a viral supernatant.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Infection by virions expressing mCAT-1-deficient binding envelopes. Virions were used to infect CHO cells expressing Pit-2 receptor (CHO-Pit-2 cells; white bars), mCAT-1 receptor (CeRD9; gray bars), or both receptors (CeAR13; striped bars). Data correspond to the average of 20 repeats, and no significant difference from experiment to another was observed (standard deviation of 0.2 log, 99% confidence interval of the mean).

Reciprocal receptor cooperation. To test if there is a preferential order of interaction with receptors, we used as the backbone envelope the 4070A amphotropic envelope. The ecotropic mCAT-1 BD was fused at the N terminus of the 4070A Env protein. These two BD were spaced either with PRO3 or PRO spacers. The resulting plasmids, named MOAPRO3 and MOAPRO, were transfected into TELCeB6 cells as previously described.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Proline-rich regions are known to play a critical role in several pro- and eucaryotic systems (9, 15, 20). Depending on their structure, they contribute directly or indirectly to the binding properties, the structure and stability, or the functionality of proteins that contain such proline-rich regions. Thus, the proline-rich region seems to be a functional domain rather than a spacing region. We showed that insertion of a 60-residue PRO peptide between two BDs of chimeric MMLV-derived retroviral glycoproteins limited their ability to infect cells: infection became strickly dependent on the presence of both receptors, corresponding to the two BDs of the chimeric envelope glycoproteins at the surface of the cells.

Predictive structure analysis indicates that this PRO spacer may be organized as a -turn helix. We showed that this structure can account for the behavior of the chimeric envelope since the substitution of PRO spacers by nonviral peptides, shown to be organized as a -turn helix, induces receptor cooperation. Further, we demonstrated that the efficiency of receptors cooperation is modulated (i) by the number of turns forming the -helix and (ii) within one -turn of the helix by the number and nature of the amino acid spacing the PRO residues.

We showed that the most efficient cooperation was observed with the chimeric envelope containing the whole PRO region and that, at least, three to four -turns are required to mask the backbone ecotropic envelope. Even for an equal given number of -turns, while three repeats of PGVGAP corresponding to EL3 spacer "cooperates" as well as three turns of the PRO spacer (PRO3), three repeats of PGVGVP (corresponding to EL3V) completely blocked cooperation. We speculate that the greater stability of the -turn arrangement is not compatible with the refolding of the spacer after interaction between the additional N-terminal binding domain and its receptor. These data seem to indicate that spacers need to share a stability not too strong in order to transmit "signal" after interaction trough changes of conformation.

We also showed that the 18-amino-acid EL3VA spacer (APGVGV motif) presents the same "cooperative" properties as the 60-residue PRO spacers, and we can assume that this spacer has an improved arrangement of the -turns in terms of structure and stability.

Taken together, these data suggest that the composition, length, and strength that determines the stability of the -turn helix is crucial for the efficiency of receptor cooperation. As in the naive viral SU, the BD located downstream of the PRO spacer is masked by the presence of PRO spacers (22); these data are consistent with two-step receptor cooperation as follows: (i) binding to a targeted surface molecule induces conformational changes in the chimeric glycoprotein envelope via the -turn helix and (ii) this, in turn, allows interaction with the fusion-competent retroviral receptor.

It is interesting to draw a parallel between the masking of the receptor binding region located downstream of the PRO spacer in the chimeric envelope and the masking of the fusion peptide by the proline-rich region naturally present between the BD of the SU and TM subunits in wild-type MLV envelope glycoproteins. Viruses have been selected to develop efficient and specific systems to block the activity of their fusion peptide until they bind specifically to the right receptor, and we can argue that the natural proline-rich region of wild-type MLV glycoproteins share a similar function.

This two-step entry of a virus into a cell, in a natural, efficient way, is attractive for the design of a new generation of retroviral vectors suitable for gene therapy. Indeed, we previously demonstrated that "one-step targeting", by insertion of new BDs at the N terminus of the MMLV glycoprotein (6, 21) is not efficient in terms of the transduction of human cells. So, depending on the cells to be targeted, the next strategy will be to design a chimeric targeting envelope based on the backbone envelope for which human cells express the most efficient receptors in terms of transduction of the fusion signal. This might be performed with (i) an amphotropic envelope for which the ubiquitous receptor Pit-2 is expressed at the surface of nearly all human cells, (ii) a gibbon ape leukemia virus envelope for which Pit-1 receptors are largely expressed at the surface of hematopoietic precursors, or (iii) a hemagglutinin envelope of influenza viruses which recognizes the ubiquitous sialic acid (10a).

We demonstrated that reciprocal Pit-2-mCAT-1 cooperation with MOAPRO virions is possible, although infection still occurs partially directly through the targeted receptor. These observations suggested that even if ecotropic and amphotropic envelopes share similar topologies and properties, their exploitation of receptor potential seem to be slightly different.

In the MOAPRO construct, the PRO spacer inserted between the two BDs was derived from ecotropic strain, whereas for the AMOPRO construct the PRO spacer was derived from amphotropic ones.

The efficiency of masking of the PRO spacer appears to be lower for the amphotropic backbone envelope compared to the ecotropic one. This observation can be correlated with the predicted structure of these two regions: the amphotropic helix shares more stable and more numerous -turns than ecotropic ones. We speculate that amphotropic envelopes are more fusogenic than ecotropic ones and, as a prerequisite, the inhibition of the fusion domain is more effective in order to restrict the fusion process unless specific binding to the receptor occurs.

A new goal will be to generalize this two-step targeting phenomena to the design of vectors suitable for gene therapy purposes. First, the backbone envelope has to be improved depending on the human cells to be targeted. We have to choose the most suitable backbone envelope for which the target cells express the most efficient retroviral receptors. Second, the spacer has to be optimized in order to design the most efficient -turn helix polyproline in terms of masking and also unmasking. Lastly, the most specific ligand able to target the virus to specific cell surface molecules will have to be developed.

The improvement of these three components will give rise to a new concept for gene therapy retroviral vector design.