Previous Article | Next Article 
Journal of Virology, September 2001, p. 8478-8486, Vol. 75, No. 18
Vectorologie Rétrovirale et
Thérapie Génique, Ecole Normale Supérieure de Lyon,
INSERM U412, 69364 Lyon Cedex 07, France
Received 27 November 2000/Accepted 13 June 2001
We have previously reported a set of Moloney murine leukemia virus
derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insertion of the Pit-2 binding domain (BD) at the N
terminus of the ecotropic retroviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2059-2064, 1996). The resulting chimeric envelopes share two BDs: an additional N-terminal BD
(Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 BD). By
inserting a variety of different amino acid spacers between the two
binding domains, we showed that retroviruses can potentially use the
targeted cell surface receptor Pit-2, the ecotropic retroviral receptor
mCAT-1, or both receptors cooperatively for entry into target cell (S. Valsesia-Wittmann et al., EMBO J 6:1214-1223, 1997). An extreme
example of receptor cooperativity was encountered when envelopes with
specific proline-rich interdomain spacers (PRO spacers) were tested:
both receptors had to be coexpressed at the surface of the targeted
cells to cooperatively allow infection. Here, we characterized the role
of PRO spacer in the cooperation of receptors. We have shown that the
particular organization of the PRO spacer 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 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 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.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8478-8486.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Chimeric Murine Leukemia Virus
env
-Turn Polyproline Spacers in Receptor
Cooperation
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
a 
-turn polyprolinewas
responsible for the cooperative effect. In the native configuration of
the viruses, the structure masked the regions located downstream of the
PRO spacer, thus the mCAT-1 BD. After interaction with the targeted
Pit-2 receptor, the BD of the backbone envelope became accessible, and
we demonstrated that interaction between the mCAT-1 BD and the mCAT-1
receptor is absolutely necessary. This interaction leads to natural
fusion triggering and entry of viruses into targeted cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-turn polyproline. This
particular structure has uncommon properties, such as the ability to
oligomerize in quaternary structure and also to develop elastomeric
forces (20).
-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
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, AMO
PRO2, AMOPRO3,
AMOPRO4, and AMOPRO chimeric envelopes were as described elsewhere
(21, 22).
PRO3,
5'-TATGTGCGGCCGCGTCTGGCAGAACGGGGTTTGG) located downstream of
the BspEI site of the FBMOSALF env gene in
combination with an oligonucleotide providing a BamHI site (UpGS3, 5'-CATCGGATCCGGTGGTGGCGGATCGGCCGCACCTCATCAAGTCTAT)
to generate a 623-bp fragment also encompassing the GS3 spacer
domain on the FBAMOSALF env gene template. These two
PCR-amplified fragments were digested, respectively, with
XhoI-BamHI and BamHI-BspEI
and cloned into XhoI-BspEI-digested FBAMOSALF plasmid.
MOD84K plasmid was generated by two PCR amplifications. (i) The first
used oligonucleotide Xba-ATG (5'-ACCATCCTCTGGACGACATG) located on the XbaI site and an ATG initiation codon
of FBMOSALF env gene in combination with an oligonucleotide
providing the substitution of D to K and creating an XhoI
site (Low XhoD84K, 5'-ATTTTCTCGAGCAGCCTGGGCTGCTGCCCC). The
PCR amplification product of 360 bp was then digested by
XbaI and XhoI. (ii) The second used
oligonucleotide LMOAPRO3 in combination with an oligonucleotide providing the substitution of D to K and creating an XhoI
(Up XhoD84K, 5'-AGGCTGCTCGAGAAAATGCGAAGAACCTTTAACCTCCC). The
470-bp PCR product was then digested with
XhoI-BamHI. The two digested PCR fragments were
cloned into the XbaI-BamHI FBMOSALF plasmid expressing the MO envelope.
To generate the AMOD84K mutants, the same strategy was used with
oligonucleotide 805FC instead of Xba-ATG in combination with the
oligonucleotide Low XhoD84K to generate a 430-bp fragment and also the
same 470-bp PCR fragment resulting from LMOAPRO3-Up XhoD84K
amplification. Both fragments were digested by XhoI and XhoI-BamHI, respectively, and cloned into an
XhoI-BamHI FBAMOSALF plasmid.
To generate the AMO3PROD84K and AMOPROD84K constructs, the
NotI-BamHI fragment from AMOD84K plasmid sharing
the D-to-K mutation was cloned into the
NotI-BamHI AMO3PRO and AMOPRO plasmids.
To generate AMOGIXD84K, the 200-bp XhoI-BstEII
fragment from the AMOG1X plasmid, corresponding to the G1X spacer, was
cloned into the XhoI-BstEII AMOD84K plasmid.
To generate expression plasmids for MOAPRO, an upper oligonucleotide
(UpMOAPRO, 5'-ACTGGGGCTTACGTTTGT-3-') located just upstream of the BamHI site of the MMLV env gene was used
in combination with a lower oligonucleotide providing a NotI
site (LMOAPRO,
5'-ATCGAGGTCACCGCGGCCGCGGGACCCCGAGTCCCCATAGGGCCC-3') to
generate by PCR a 200-bp fragment encompassing the PRO
interdomain spacer on the FBMOSALF env gene as a
template. This PCR fragment was double digested with BamHI
and NotI. In parallel, a 2-kbp NdeI-BamHI fragment from the FBMOSALF plasmid was
purified, providing the 5' end of the MO env gene. These two
inserts were then cloned into the
NdeI-NotI-digested FBEASALF plasmid
(7).
The MOAPRO plasmid was generated by performing a PCR amplification
with UpMOAPRO oligonucleotide in combination with the LMOAPRO3 lower
oligonucleotide, providing a shorter PRO spacer. After
BamHI-NotI digestion, this insert was cloned into
the BamHI-NotI-linearized MOAPRO plasmid.
TELCeB6 cells were transfected with envelope expression plasmids by
calcium phosphate precipitation as previously described (6). Transfected cells were selected with phleomycin (50 µg/ml), and phleomycin-resistant colonies were pooled.
Virus-containing supernatants were collected after an overnight
production from freshly confluent env-transfected TELCeB6 cells in regular medium.
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 |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
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.
|
|
-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).
-spirals of bovine elastin, such a
repetitive -turn motif in the MLV env proline-rich region
may display unusual physical properties, such as self-assembly into oligomeric quaternary structures, increasing structural order with
increasing temperature, and the development of elastomeric forces
coincident with molecular ordering (20).
In order to see if the properties of the polyproline -turn helices
can account for the behavior of retroviruses coated with AMOPRO and
AMOPRO envelopes, we tried to replace PRO spacers by a nonviral
-spiral peptide from bovine elastin (4, 20). Three
synthetic peptides were chosen for this approach: (i) an 18-amino-acid
spacer corresponding to three repeats of the hexapeptide APGVGV, named
EL3VA (APGVGVAPGVGVAPGVGV), or (ii) a 15-amino-acid peptide
corresponding to three repeats of a pentapeptide VPGVG, named EL3V
(VPGVGVPGVGVPGVG), which were both shown to correspond to the main
repeating sequences identified in the tropoelastin, the precursor of
fibrous elastin, or (iii) three repeats of the pentapeptide APGVG
motif, called EL3 (APGVGAPGVGAPGVG).
These peptides were predicted to have propensities to form a -spiral
and can be classified from weakest to strongest in -turn helix
propensity as EL3, EL3VA, and EL3V.
In order to check if the size of the spacer might influence receptor
cooperation efficiency, a length control unfolded spacer of 15 amino
acids was designed: GS3, which corresponds to three repeats of four
glycine residues and one serine residue. This spacer was cloned into
the AMO env to generate the AMOGS3 envelope.
The resulting envelopes, AMOEL3VA, AMOEL3V, AMOEL3, and AMOGS3, were
transfected into TelCeB6 cells that provide MLV core and an nls-LacZ
vector (6).
By Western blot analysis of cell lysates of transfected cells or
supernatants of these cells, we demonstrated that chimeric EL3
envelopes were processed and incorporated into MLV particles (Fig. 3A
and B). We also showed that they were all
able to bind to the Pit-2 receptor (Fig.
4). To address the question of the infectivity of retroviruses generated, cells expressing either Pit-2 or
mCAT-1 receptor alone (CHO-Pit-2 and CerD9, respectively) or Pit-2 and
mCAT-1 (CeAR13) were infected. When AMOGS3 envelopes were used as a
control (Fig. 2), minimum or no significant cooperation of receptors
was seen.
|
|
-spiral peptide inserted. We first showed that substitution of the PRO spacer
by the less-stable -spiral peptide (EL3) leads to a cooperation of
receptors similar to the one obtained for AMOPRO3 envelope: chimeric
virions can infect cells only if the target Pit-2 receptor was
expressed, with very low efficiency when mCAT-1 receptor was present
and with increased efficiency when both receptors are expressed. Once
more, when the intermediate-strength -spiral peptide (EL3VA) was
used as a spacer, the infection of target cells was strictly dependent
on the presence of the two receptors, a result surprisingly similar to
that seen with the 60-amino-acid PRO spacer. These results demonstrated
the critical role of the nature of the -turn helix in the
cooperation of receptors.
However, with the EL3V spacer, which had the strongest propensity to
form a -spiral (whereas the chimeric protein is normally and
correctly processed), no infection was observed, even in the presence
of both receptors, thus demonstrating that insertion of very stable
-spiral spacers blocks the transmission of conformational changes of
the protein after binding to the Pit-2 receptor.
These data suggested that the substitution of PRO spacers by -turn
polyproline helix from bovine elastin leads to a cooperation of
receptors and that the efficiency of cooperation depends on the
stability of the helix.
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).
-turn polyproline
helix is crucial for receptor cooperation efficiency.
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).
|
PRO3, and AMOPRO, AMOPRO3D84K and
AMOPROD84K env-bearing viruses remained unable to infect
cells which only expressed mCAT-1. The D84K mutation did not affect the
entry of viruses through Pit-2, since AMOD84K, AMOPRO3D84K, and
AMOG1XD84K were able to infect cells expressing Pit-2 receptors.
AMOPROD84K, like the control AMOPRO env-bearing viruses,
remained unable to infect cells when only Pit-2 is expressed.
|
PRO3D84K, and AMOG1XD84K a 1-log reduction of viral titers
compared to the corresponding wild-type chimeric envelopes.
These results are consistent with an infection occurring, in the normal
configuration, in a ratio of 1 log through mCAT-1 receptors and a range
of 103 to 104 through Pit-2, depending on the
chimeric envelope. This suggests that the mechanisms of viral entry
through these two receptors are different and that mCAT-1 is less
efficient at allowing fusion when the mCAT-1 BD is not separated from
the N-terminal domain of env by PRO spacers.
However, for AMOPRO viruses, for which infection was dependent on the
presence of both receptors, inhibition of the interaction between
mCAT-1 BD and its receptor completely prevented infection, thus
demonstrating the crucial role of this second-step interaction for the
entry of chimeric viruses into cells.
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.
PRO, the MOAPRO virions could only
infect cells that express the targeted receptor alone (in that case
CeRD9 cells expressing mCAT-1) and the CeAR13 cells expressing
both receptors (Table 1). However,
although AMOPRO virions were unable to infect cells that only
expressed the receptor corresponding to the backbone envelope
(CeRD9), MOAPRO virions remained able to infect, even with
low efficiency, cells through Pit-2 receptors (CHO-Pit-2).
|
| |
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.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by Agence Nationale pour la Recherche contre le SIDA (ANRS), the European Community, AFIRST, Association Française contre les Myopathies (AFM), Association pour la Recherche contre le Cancer (ARC), SIDACTION, and Institut National de la Santé et de la Recherche Médicale (INSERM).
I thank Michèle Ottmann-Terrangle, David Camerini, and Fabian Wild for critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* Present address: Laboratoire des Virus Gamma Herpès Humain, Ecole Normale Supérieure de Lyon, INSERM U412, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. Phone: 334-72-72-81-75. Fax: 334-72-72-87-77. E-mail: sandrinewittmann{at}yahoo.fr.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Andersen, K. B.
1994.
A domain of murine retrovirus surface protein gp70 mediates cell fusion, as shown by a novel SC-1 cell fusion system.
J. Virol.
68:3175-3182 |
| 2. | Bae, Y., S. M. Kingsman, and A. J. Kingsman. 1997. Functional dissection of the Moloney murine leukemia virus envelope protein gp70. J. Virol. 71:2092-2099[Abstract]. |
| 3. | Battini, J. L., O. Danos, and J. M. Heard. 1995. Receptor-binding domain of murine leukemia virus envelope glycoproteins. J. Virol. 69:713-719[Abstract]. |
| 4. | Bhandary, K., S. E. Senadhi, K. U. Prasad, K. Prasad, D. W. Urry, and S. Vijay-Kumar. 1990. Conformation of a cyclic decapeptide analog of a repeat pentapeptide sequence of elastin: cyclo-bis(valyl-prolyl-alanyl-valyl-glycyl). Int. J. Peptide Protein Res. 36:122-127[Medline]. |
| 5. | Chou, P. Y., and G. D. Fasman. 1978. Empirical predictions of protein conformation. Annu. Rev. Biochem. 47:45-148. |
| 6. | Cosset, F.-L., F. J. Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell. 1995. Retroviral retargeting by envelopes expressing an N-terminal binding domain. J. Virol. 69:6314-6322[Abstract]. |
| 7. | Cosset, F.-L., Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. L. Collins. 1995. High-titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69:7430-7436[Abstract]. |
| 8. |
Evans, L. H.,
R. P. Morrison,
F. G. Malik,
J. Portis, and W. Britt.
1990.
A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses.
J. Virol.
64:6176-6183 |
| 9. | Fontenot, J. D., N. Tjandra, C. Ho, P. C. Andrews, and R. C. Montelaro. 1994. Structure and self-assembly of a retrovirus (FeLV) proline-rich neutralization domain. J. Biomol. Struct. Dyn. 11:821-837[Medline]. |
| 10. |
Gray, K. D., and M. J. Roth.
1993.
Mutational analysis of the envelope gene of Moloney murine leukemia virus.
J. Virol.
67:3489-3496 |
| 10a. | Hatziioannou, T., E. Delahaye, F. Martin, S. J. Russell, and F. L. Cosset. 1999. Retroviral display of functional binding domains fused to the amino terminus of influenza hemagglutinin. Hum. Gene Ther. 10:1533-1544[CrossRef][Medline]. |
| 11. | Kabat, D. 1989. Molecular biology of Friend viral erythroleukemia. Curr. Top. Microbiol. Immunol. 148:1-42[Medline]. |
| 12. | Kozak, S. L., D. C. Siess, M. P. Kavanaugh, A. D. Miller, and D. Kabat. 1995. The envelope glycoprotein of an amphotropic murine retrovirus binds specifically to the cellular receptor/phosphate transporter of susceptible species. J. Virol. 69:3433-3440[Abstract]. |
| 13. |
Lavillette, D.,
M. Maurice,
C. Roche,
S. J. Russell,
M. Sitbon, and F.-L. Cosset.
1998.
A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes.
J. Virol.
72:9955-9965 |
| 14. | MacKrell, A. J., N. W. Soong, C. M. Curtis, and W. F. Anderson. 1996. Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding. J. Virol. 70:1768-1774[Abstract]. |
| 15. | Matsushima, C. N., E. Creutz, and R. H. Kretsinger. 1990. Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten. Proteins 7:125-155[CrossRef][Medline]. |
| 16. |
Nussbaum, O.,
A. Roop, and W. F. Anderson.
1993.
Sequences determining the pH dependence of viral entry are distinct from the host range-determining region of the murine ecotropic and amphotropic retrovirus envelope proteins.
J. Virol.
67:7402-7405 |
| 17. |
Ott, D., and A. Rein.
1992.
Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.
J. Virol.
66:4632-4638 |
| 18. |
Pinter, A.,
T.-E. Chen,
A. Lowy,
N. G. Cortez, and S. Siligari.
1986.
Ecotropic murine leukemia virus-induced fusion of murine cells.
J. Virol.
57:1048-1054 |
| 19. |
Takeuchi, Y.,
F. L. Cosset,
P. J. Lachmann,
H. Okada,
R. A. Weiss, and M. K. L. Collins.
1994.
Type C retrovirus inactivation by human complement is determined by both the viral genome and the producer cell.
J. Virol.
68:8001-8007 |
| 20. | Urry, D. W. 1988. Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces. J. Protein Chem 7:1-34[CrossRef][Medline]. |
| 21. | Valsesia-Wittmann, S., F. J. Morling, B. H. K. Nilson, Y. Takeuchi, S. J. Russell, and F.-L. Cosset. 1996. Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU. J. Virol. 70:2059-2064[Abstract]. |
| 22. | Valsesia-Wittmann, S., F. J. Morling, B. H. K. Nilson, Y. Takeuchi, S. J. Russell, and F.-L. Cosset. 1997. Receptor cooperation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding. EMBO J. 6:1214-1423[CrossRef]. |
| 23. |
Zavorotinskaya, T., and L. M. Albritton.
1999.
Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein.
J. Virol.
73:5034-5042 |
Journal of Virology, September 2001, p. 8478-8486, Vol. 75, No. 18
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8478-8486.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Qian, Z., Albritton, L. M.
(2004). An Aromatic Side Chain Is Required at Residue 8 of SU for Fusion of Ecotropic Murine Leukemia Virus. J. Virol.
78: 508-512
[Abstract] [Full Text] -
Martin, F., Chowdhury, S., Neil, S. J., Chester, K. A., Cosset, F.-L., Collins, M. K.
(2003). Targeted Retroviral Infection of Tumor Cells by Receptor Cooperation. J. Virol.
77: 2753-2756
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»