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J Virol, June 1998, p. 4956-4961, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Entry of Amphotropic Murine Leukemia Virus Is
Influenced by Residues in the Putative Second Extracellular
Domain of Its Receptor, Pit2
Betsy D.
Leverett,1
Karen B.
Farrell,2
Maribeth V.
Eiden,2 and
Carolyn A.
Wilson1,*
Division of Cellular and Gene Therapies,
Center for Biologics Evaluation and Research, Food and Drug
Administration,1 and
Laboratory of
Cellular and Molecular Regulation, National Institute of Mental
Health, National Institutes of Health,2
Bethesda, Maryland
Received 9 September 1997/Accepted 2 March 1998
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ABSTRACT |
Human cells express distinct but related receptors for the gibbon
ape leukemia virus (GALV) and the amphotropic murine leukemia virus
(A-MuLV), termed Pit1 and Pit2, respectively. Pit1 is not able to
function as a receptor for A-MuLV infection, while Pit2 does not confer
susceptibility to GALV. Previous studies of chimeric receptors
constructed by interchanging regions of Pit1 and Pit2 failed to clarify
the determinants unique to Pit2 which correlate with A-MuLV receptor
function. In order to identify which regions of Pit2 are involved in
A-MuLV receptor function, we exchanged the putative second and third
extracellular domains of Pit1, either individually or together, with
the corresponding regions of Pit2. Our functional characterization of
these receptors indicates a role for the putative second extracellular
domain (domain II) in A-MuLV infection. We further investigated the
influence of domain II with respect to A-MuLV receptor function by
performing site-specific mutagenesis within this region of Pit2. Many
of the mutations had little or no effect on receptor function. However, the substitution of serine for methionine at position 138 (S138M) in a
Pit1 chimera containing domain II of Pit2 resulted in a 1,000-fold reduction in A-MuLV receptor function. Additional mutations made within
domain II of the nonfunctional S138M mutant restored receptor function
to nearly wild-type efficiency. The high degree of tolerance for
mutations as well as the compensatory effect of particular substitutions observed within domain II suggests that an element of
secondary structure within this region plays a critical role in the
interaction of the receptor with A-MuLV.
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INTRODUCTION |
Most retroviruses initiate infection
of host cells through a specific interaction with a cell surface
receptor. Among the murine leukemia viruses (MuLVs) there are now six
different classes identified based on receptor usage, including
ecotropic (E-MuLV), amphotropic (A-MuLV), xenotropic, dualtropic
(mink cell focus-forming virus), 10A1-MuLV, and the recently identified
(4) Mus dunni endogenous virus. MuLVs belonging
to each receptor class use discrete receptors for viral entry into
mouse cells except 10A1-MuLV, which can use two different receptors
(14, 19), one of which is also used by A-MuLVs. The E-MuLV
receptor was the first of the murine type C retrovirus receptors to be
identified and has been shown to be a multimembrane-spanning amino acid
transporter (2). The human cDNAs encoding the receptors for
a primate type C retrovirus, the gibbon ape leukemia virus (GALV) and a
second type C retrovirus, A-MuLV, have also been identified (16,
23). These two receptors, originally named Glvr-1 and Glvr-2,
respectively, have recently been renamed Pit1 and Pit2,
respectively, to reflect their normal function as transporters of
inorganic phosphate (10, 17). The Pit receptors not only
have comparable cellular functions but also have the same proposed
membrane topology and 62% amino acid identity (23). Despite
the similarities of these receptors, they exhibit distinct virus
recognition properties: Pit1 functions for GALV infection but does not
confer susceptibility to A-MuLV, while the reverse is true for Pit2
(14, 20).
The current structural model for the Pit receptors is based on
hydropathy analysis (16) and features 10 transmembrane
domains, internal N and C termini, a large cytoplasmic region, and five extracellular loops. Although most of the proposed membrane topology has yet to be verified, the location of sections of the cytosolic loop
has been confirmed (6). A number of studies using chimeric receptors constructed by interchanging regions of Pit1 and Pit2 (Pit1-Pit2 receptors) (8, 18, 22) have indicated the
importance of the putative fourth extracellular domain for GALV
receptor function. Indeed, a single mutation in this region of Pit2
rendered it functional for GALV infection (8). However,
recent studies have raised the possibility of involvement by other
domains in the interaction with GALV (5, 21). A similarly
ambiguous picture has emerged with respect to A-MuLV permissivity.
Initial results from the assessment of Pit1-Pit2 chimeric receptors
with either the first three or the last two extracellular domains of the Pit1 receptor replaced with the corresponding regions of Pit2 suggested that the determinants of A-MuLV receptor function do not
reside exclusively in any single region of the Pit2 receptor (8). In an effort to obtain a more complete understanding of A-MuLV-receptor interaction, we have constructed a series of Pit1-Pit2 chimeric and mutant receptors and have assessed their abilities to
confer sensitivity to A-MuLV infection on CHO K1 cells. We have
observed not only an important role for the putative second extracellular domain in A-MuLV receptor function but also the ability
of certain combinations of mutations within the second domain to
compensate for other substitutions in this domain that abolish receptor
function.
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MATERIALS AND METHODS |
Cells.
CHO K1 cells, Chinese hamster ovary cells, were
obtained from the American Type Culture Collection (CCL 61). M. dunni tail fibroblast (MDTF) cells, which were derived from the
feral mouse M. dunni, were provided by Olivier Danos (and
are also available from the American Type Culture Collection [CRL
2017]). MDTF cells expressing Pit1 or Pit2 receptors have been
previously described (8). The PA317/G1BgSvN (11, 12,
25) and PG13/G1BgSvN (11, 12) retrovirus packaging
cell lines have been described previously. CHO K1 cells were maintained
in alpha minimal essential medium supplemented with 5% fetal bovine
serum, 4 mM glutamine, 1 mM sodium pyruvate, 100 U of penicillin per
ml, and 100 µg of streptomycin per ml. All other cells were
maintained in Dulbecco's modified Eagle's medium supplemented in a
similar manner.
Construction of chimeric and mutant receptor cDNA plasmids.
The Pit1-Pit2 chimeras (Fig. 1) are named
by using five letters which designate the origin of each of the five
extracellular domains, A for those derived from the A-MuLV receptor
(Pit2) and G for those derived from the GALV receptor (Pit1). Wherever
nucleotide or amino acid positions are indicated, the numbers given
correspond to the Pit2 sequence unless otherwise noted (23).
The chimeras were constructed in the pSP72 plasmid (Promega, Madison,
Wis.) as follows: GAGGG was made by exchange of the cDNA for the
putative second extracellular domain of Pit1 with that of Pit2 between the NheI and AccI sites (nucleotides [nt] 151 to 597); GAAGG was made by replacing the cDNA for both the second and
third putative extracellular regions of Pit1 between the
NheI and BglII sites (nt 151 to 1065) with that
for the corresponding regions of Pit2; GGAGG was made by the exchange
of the cDNA for the putative third extracellular domain of Pit1 with
that of Pit2 between the AccI and BglII sites (nt
597 to 1065). Pit1 cDNA (pSP72-OJ9) and the chimeric and mutant
receptor cDNAs constructed in pSP72 were subcloned from pSP72, using
the HindIII and EcoRV sites at either end of the receptor sequence, into the pLNSX retroviral vector (13) prepared with HindIII and ClaI (filled in
with T4 DNA polymerase [Boehringer Mannheim, Indianapolis, Ind.]) for
expression analysis. Pit2 cDNA was subcloned from pSP72 (pSP72-Pit2),
using the EcoRI sites at either end of the receptor
sequence, into the pLNSX retroviral vector prepared with
EcoRI, and the cohesive ends were dephosphorylated with
alkaline phosphatase (Boehringer GmbH, Mannheim, Germany). Retroviral
expression vectors (pLNSX) encoding the various receptors are
generically referred to here as pLNSR.
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FIG. 1.
Schematic representation of chimeric receptor cDNAs used
to examine virus receptor function. Numbered boxes represent regions
encoding the five predicted extracellular domains (left to right, 5' to
3'). The regions derived from Pit1 are shown in black; those derived
from Pit2 are shown in white. Chimeric receptors are named by using
single letters to designate the source for each of the five
extracellular domains (A for Pit2, G for Pit1). Restriction enzyme
sites are given in their appropriate locations in the receptor cDNAs;
nucleotide positions of the restriction sites are given in the
Materials and Methods section. The NheI and BglII
sites are given in parentheses because they were originally introduced
as silent mutations in either Pit1 or Pit2 (8).
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Mutations were introduced into the second extracellular domain of
either Pit1 or GAGGG by using one of two methods of site-directed mutagenesis. In the first method, synthetic oligonucleotides containing nucleotide changes appropriate either to introduce a specific amino
acid residue change or to create a restriction site without affecting
the encoded amino acid were designed. PCRs were performed in a two-step
process described previously (9), and the resulting products
were cloned directly into the TA pCRII vector (Invitrogen, San Diego,
Calif.). Mutant versions of the Pit1 and GAGGG cDNAs generated in this
way were constructed in the pSP72 plasmid by replacing the region
between the NheI and AccI sites (nt 151 to 597)
of either Pit1 or GAGGG cDNA with the corresponding region containing
the mutant sequence from the TA PCRII plasmid. Final subcloning into
pLNSX was accomplished as described above for the Pit1 and GAGGG
constructs. In the second method, pLNSR plasmid constructs of Pit1 and
the chimeric receptor GAGGG were used as templates for mutagenesis
according to the QuikChange method (Stratagene, La Jolla, Calif.), with
appropriately designed mutant oligonucleotides. Plasmids constructed by
either method were sequenced to confirm the presence of desired
mutations and to verify the absence of unscheduled mutations.
Expression of chimeric and mutant receptor cDNAs.
Calcium
phosphate-mediated gene transfer of mutant and control receptor cDNA
plasmids for both transient and stable expression in CHO K1 cells was
carried out by the Profection method (Promega). For transient
expression, calcium phosphate precipitate containing 3 µg of pLNSR
plasmid DNA was prepared and applied to each of three individual wells
of CHO K1 cells in a 12-well dish as described previously
(8). For stable receptor cDNA expression, cells transfected
with chimeric and mutant receptor pLNSR plasmids were selected for 10 to 14 days in appropriate growth medium supplemented with G418 (450 µg per ml of active substance). Following selection, pooled
populations of G418-resistant cells were assayed for receptor function
as described below.
Stable expression of receptor cDNAs in MDTF cells was achieved by
transfecting each of the pLNSR plasmid DNAs into PA317 packaging
cells
by the Profection method and selecting transfected cells
in medium
containing 450 µg of active G418 per ml for 10 to 14
days. The
supernatants from each of the pLNSR vector-producing
cell lines were
then used to infect MDTF cells, as previously
described (
8).
MDTF cells were selected for 7 to 10 days at
600 µg of active G418
per ml. Pooled populations of G418-resistant
cells were used in GALV
infection assays as described below.
Assays for receptor function.
The recombinant retrovirus
genome G1BgSvN, which carries the bacterial lacZ and
neomycin resistance genes (11), was used in all infection
assays. For the assay of A-MuLV infection of CHO K1 cells, cells were
infected with 2 ml of filtered (0.45-µm-pore-size filter) supernatant
from PA317/G1BgSvN retrovirus packaging cells containing 3 µg of
Polybrene per ml, either 48 h after transfection for transient
experiments or 24 h after seeding at 3 × 104
cells/well in 12-well dishes for stable expression. At 48 h
postexposure to the vector, cells were fixed and stained with X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) (24) and the numbers of blue foci per well were quantified. The GALV infection assay and histochemical staining of control and
pLNSR-expressing MDTF cells were carried out in a manner similar to
that described for A-MuLV with supernatant from PG13/G1BgSvN retrovirus
packaging cells.
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RESULTS |
Functional analysis of Pit1-Pit2 chimeric receptors.
Chimeric
receptors were constructed to determine whether a minimum domain in the
N-terminal half of Pit2 plays a role in A-MuLV receptor function.
Chimeras GAAGG, GAGGG, and GGAGG were constructed by replacement of
either or both of the second and third extracellular domains of Pit1
with the corresponding regions of Pit2 (Fig. 1). As shown in Table
1, CHO K1 cells expressing chimera GAAGG,
containing the Pit2 sequence in both the second and third extracellular
domains, are susceptible to infection by vectors with the A-MuLV
envelope, whereas CHO K1 cells expressing GGAGG, featuring only the
third domain of Pit2, are not susceptible. The GAGGG construct contains the Pit2 sequence in only the second extracellular domain and functions as a receptor for A-MuLV, demonstrating that the presence of
the second extracellular domain (domain II) of Pit2 correlates with
A-MuLV receptor function.
Mutations in extracellular domain II influence A-MuLV receptor
function.
To elucidate which, if any, specific residues within the
second extracellular domain are responsible for its apparent role in
A-MuLV receptor function, site-specific mutagenesis was performed in
this region of both native Pit1 and the chimeric GAGGG receptor. These
receptors represent nonfunctional (Pit1) and functional (GAGGG) A-MuLV
receptors. A comparative analysis of the amino acid sequences of Pit1
and Pit2 in the proposed second extracellular domain (Fig.
2) reveals eight positions occupied by
different residues in the two receptors. Seven of the eight differences in the second extracellular domain are clustered in the C-terminal portion of the domain. The goal of mutagenesis in this region was to
make changes at each of these seven positions to determine if either a
loss of A-MuLV receptor function in the GAGGG backbone or a gain of
function in native Pit1 could be achieved. Mutant receptors were tested
for their abilities to render CHO K1 cells susceptible to PA317/G1BgSvN
in transient transfection-infection experiments. Both Pit1 and GAGGG
function as receptors for GALV. Therefore, in order to verify the
expression and integrity of receptor proteins, all mutant receptor cell
lines were tested for GALV receptor function. All mutant receptors
reported here retain GALV receptor function at efficiencies close to
that of the wild type,
Pit1
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FIG. 2.
Comparison of Pit1 and Pit2 sequences in the predicted
second extracellular domain (domain II; amino acids 107 to 141 [based
on Pit2 numbering]).
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The series of Pit1 mutant receptors shown in Table
2 begins with the replacement of the
three charged amino acids in domain
II of Pit1 with the corresponding
Pit2 residues (Pit1-1); progressive
substitutions with corresponding
Pit2 residues (Pit1-2 and Pit1-3)
have been introduced until the
sequence in domain II resembles
that of Pit2 (Pit1-4). All the Pit1
mutants differ from Pit2 in
this region by a single lysine (position
108; arginine in Pit2).
Mutant Pit1-4, which is otherwise identical to
Pit2 in domain
II is functional for A-MuLV infection. The Pit1-3 mutant
was found
not to function as an A-MuLV receptor when assessed in the
transient
assay, even though it differs from the functional Pit1-4
mutant
by a single residue (the Q132T change). A number of other
mutational
combinations in this series of mutants have no detectable
effect
on A-MuLV receptor function (data not shown). Three changes
involving
charged residues, K130I, E133K, and K136Q, together with
mutation
S138M, do not appear to affect receptor function (mutant
Pit1-1).
However, single and double mutations involving only S138M and
I141V result in functional receptors, Pit1-5, Pit1-6, and Pit1-7.
Mutagenesis of the seven residues that differ between Pit1 and Pit2 in
the second extracellular domain of functional GAGGG
was used to
identify receptors that fail to function for A-MuLV
infection (Table
3). The two large hydrophobic residues in
the
domain, F125 and W137, were replaced with alanine in mutants C1-1
and C1-2 without an effect on A-MuLV receptor function. Differences
in
charge were explored with mutants C1-3, C1-4, and C1-5; the
T132Q
change was made in C1-6; and a group of three mutations,
I130K, T132Q,
and K133E, was introduced in mutant C1-7. None of
these mutations
produced any significant change in the efficiency
of A-MuLV receptor
function. The single mutation M138S present
in receptor C1-8 results in
a loss of A-MuLV receptor function,
as shown by a transient CHO K1
assay.
An analysis of GAGGG mutants indicated that receptor function is not
compromised in spite of a number of nonconservative amino
acid changes,
suggesting that receptor function can tolerate a
high degree of
flexibility in this region. To complement these
findings, the
nonfunctional C1-8 mutant was used as a receptor
template on which to
make other mutations in the second extracellular
domain which could
restore A-MuLV receptor function. The trio
of changes, I130K, T132Q,
K133E, did not affect the function of
the GAGGG receptor (C1-7) yet
were able to overcome the effect
of the M138S mutation, yielding
functional receptor C1-9. To determine
which, if any, of the three
residues is primarily responsible
for the observed compensation, each
mutation was made individually
in the C1-8 receptor (Table
4). Mutants C1-10 and C1-11, featuring
the I130K and K133E mutations, respectively, encode functional
A-MuLV
receptors, rendering CHO K1 cells permissive to A-MuLV,
while the T132Q
mutation in the C-12 mutant has no observable
effect on A-MuLV receptor
function.
To improve the sensitivity of the A-MuLV infection assay and thereby
fully characterize the receptors which give negative
results in the
transient transfection-infection experiments, stable
expression of
selected mutant receptors was established in CHO
K1 cells. For
transient experiments, the upper limit of detection
in the Pit2
positive control wells ranges from 2 × 10
2 to 8 × 10
2 per well, compared with 8 × 10
5 to
1 × 10
6 per well for assays of the stable Pit2
receptor cell line. In
the functional analysis of stable
receptor-bearing cell lines,
several of the mutant receptors which
appeared nonfunctional for
A-MuLV infection in transient experiments
revealed limited receptor
function in established cell lines.
Specifically, established
CHO K1 cell lines expressing Pit1-3, C1-8,
and C1-10 (Fig.
3 and
Table
2) demonstrated susceptibility to A-MuLV
but the titer
for each was 1,000-fold less than that for the
positive-control
Pit2 cell line. These results are consistent with the
diminished
A-MuLV receptor function observed with these receptors in
the
transient assay. Receptors which scored lower than 50% of the
Pit2
control in the transient infection assay typically produced
titers
approximately 100-fold lower than those produced by Pit2
in the assay
of stable cell lines. Three receptors, GGAGG, Pit1-1,
and Pit1-2, do
not confer susceptibility to PA317/G1BgSvN when
tested in either
transient- or stable-expression experiments.
All of these mutant
receptors efficiently mediate GALV entry when
expressed in MDTF cells,
demonstrating that the mutations have
not caused improper folding or
cell membrane targeting (Fig.
3 and Table
2).
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FIG. 3.
Analysis of A-MuLV and GALV receptor function after
stable expression in either CHO K1 or MDTF cells, respectively. CHO K1
cells expressing receptor cDNAs were exposed to PA317/G1BgSvN
retroviral vectors at dilutions of supernatant ranging from 1:2 to
1:1,000 (2 ml per well). MDTF cells expressing receptor cDNAs were
exposed to PG13/G1BgSvN retroviral vectors at two dilutions of
supernatant, 1:100 and 1:1,000 (2 ml per well). Infection assay
procedures for both vectors are described in the Materials and Methods
section. Mean titers are represented graphically for PA317/G1BgSvN
infection of receptor-expressing CHO K1 cells (black bars) and
PG13/G1BgSvN infection of receptor-expressing MDTF cells (gray bars).
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DISCUSSION |
Chimeric receptors have been invaluable in mapping the important
regions of virus-receptor interaction for several retroviral receptors,
including those for E-MuLV (1) and avian leukosis virus
(26), as well as the human immunodeficiency virus (HIV) receptor (3, 20). However, the results obtained using
chimeric receptors have been less revealing for both A-MuLV and the HIV coreceptors (3, 20). In the case of A-MuLV, two reciprocal chimeras with either the first three or last two domains of Pit2, AAAGG
and GGGAA, are functional for A-MuLV infection (8, 18), indicating that neither the N-terminal nor the C-terminal domains of
Pit2 can exclusively account for A-MuLV receptor function. Supporting
these findings is the observation that similar reciprocal chimeras
constructed by interchanging regions either of Pit1 and HaPit2, the
Pit2 homolog expressed in E36 hamster cells (25), or of Pit1
and RaPit2 (rat homolog of Pit2; formerly termed Ram-1) (15), are also functional for A-MuLV infection
(8). The finding that both GAGGG and GAAGG chimeras function
as receptors for A-MuLV and feature domain II of Pit2, while GGAGG
lacks the second extracellular domain of Pit2 and is nonfunctional for
A-MuLV infection, is the first unambiguous indication of influence by
any particular domain of Pit2 on the A-MuLV receptor interaction.
Although it remains evident that no single domain is both necessary and
sufficient, the putative second extracellular domain clearly plays a
critical role in the interaction with A-MuLV.
We have used a transient assay system for an initial assessment of
A-MuLV receptor function, followed by stable expression of the
receptors in CHO K1 cells. Evaluation of infection in stable cell lines
provides an expanded range with which to more accurately distinguish
impaired receptor function from a complete loss of receptor function,
and is therefore necessary for accurate quantitation of relative A-MuLV
receptor efficiency. Several receptors which routinely failed to
function for A-MuLV infection in the transient assay are functional
when assayed in established cell lines, although consistently
1,000-fold less efficient than the positive control, Pit2. None of the
mutant receptors tested exhibit any significant change in GALV receptor
function relative to Pit1, indicating that all mutant and chimeric
receptors are capable of being appropriately expressed in cells. The
results with stable receptor-bearing cell lines confirm that receptors
which were less than 5% of the Pit2 control in the transient assay
have impaired A-MuLV receptor function. The infection experiments
reported here rely on viral entry and expression. Our system does not
distinguish whether a 1,000-fold decrease in A-MuLV infection
efficiency correlates with defective entry or with a decrease in
receptor binding affinity.
In examining the potential role of the second extracellular domain in
A-MuLV infection, several possibilities can be considered unlikely
based on the current results. First, the dramatic effects involving
charged residues that have been reported for the interaction between
GALV and the fourth extracellular domain of Pit1 (5, 8) are
not evident as part of the interaction between the second extracellular
domain of Pit2 and A-MuLV, as mutations of charged residues alone in
this domain neither restored nor abrogated function. In addition, the
impact of large hydrophobic residues upon virus-receptor interaction,
observed by Zingler and Young (27) with the avian leukosis
virus and its receptor, does not appear to be a factor in the loss of
A-MuLV receptor function (mutants C1-1 and C1-2). Finally, the presence
of a linear virus recognition sequence in the second extracellular
domain is unlikely, considering both the large number of different
mutations and the combinations of mutations tolerated in this domain
without a loss of A-MuLV receptor function.
The amino acid at position 138, methionine in Pit2 and serine in Pit1,
appears to have a pivotal influence on A-MuLV receptor function. The
single mutation of M138S, or its reverse, affects receptor function
significantly in both Pit1 and GAGGG: the Pit1-5 mutant is functional,
although at reduced efficiency relative to Pit2, and the C1-8 mutant is
1,000-fold less efficient than Pit2. The apparent importance of this
single difference between the receptors is perhaps less surprising when
viewed in terms of its possible structural implications. The occurrence
of serine in protein sequences is most often associated with turn
structures, while methionine is found more often in -sheets
(7). Mutation of the amino acid at position 141 from
isoleucine to valine resulted in marginal receptor function in both the
transient assay and the assay of the stable Pit1-6 cell line. While the
I141V change seems a relatively conservative mutation, the difference
in the size of the side chains, owing to the additional methyl group in
isoleucine relative to valine, and the statistically significant association of valine with -sheet structure (7) are
possible explanations for the apparent effect on A-MuLV receptor
function. The observation that the S138M and I141V changes, either
together or alone, confer A-MuLV receptor function on Pit1 may indicate that a specific element of secondary structure, such as a -sheet, is
required in this portion of domain II for proper interaction with
A-MuLV or with some other part of the receptor. Whether it is because
the residues at positions 138 and 141 of domain II participate in
direct interaction with the viral SU or with elements elsewhere in the
receptor or because these residues strongly influence secondary
structure in this region of the receptor, which in turn affects the
interaction with A-MuLV, these residues seem to be an integral
part of the role played by the second extracellular domain in A-MuLV
receptor function.
The flexibility of the putative second extracellular domain with
respect to A-MuLV receptor function is evidenced not only by toleration
of a significant number of mutations in the region but also by
compensation involving specific combinations of residues in this
domain. The type and arrangement of mutational combinations which do
not alter A-MuLV receptor function in either native Pit1 or GAGGG
suggest that the topology of this domain may have more impact on A-MuLV
receptor function than specific interactions involving amino acid side
chains and argue against the possibility that a linear epitope of viral
interaction exists in this region of the receptor. A compensatory
effect on C1-8 mutant receptor function has been observed with mutants
C1-9, C1-10, and C1-11, in which the negative effect of the M138S
(C1-8) mutation is overcome by other mutations in the domain, and with
Pit1-1 and Pit1-5, in which the positive effect of the S138M change is
not evident when it is introduced in combination with other changes.
Compensation within this region of the second extracellular domain, in
the absence of any other obvious correlations between charge or
hydrophobicity and A-MuLV receptor function, is further support for the
possible importance of the domain II secondary structure in the
interaction with A-MuLV rather than a requirement for specific
residues. This finding is not unexpected considering the results from
chimeric receptor studies, in which C-terminal domains derived from
Pit2 are able to compensate in terms of A-MuLV receptor function when N-terminal domains have been derived from Pit1 and vice versa (Table
1), indicating compensation on the domain level.
Although the involvement by domain II in A-MuLV infection has been
effectively demonstrated, the nature of its involvement remains
unclear. It is reasonable to speculate that neither a concise viral
recognition sequence nor a lock-and-key mechanism which involves the
second extracellular domain is in operation. The interaction between
domain II and A-MuLV might instead be more of a "loose fit," with a
minimum number of interchangeable contact points required.
Alternatively, domain II may not be involved in a direct interaction
with A-MuLV but rather may participate either as one of several domains
required to create the necessary topological features for A-MuLV
binding or as a stabilizing element in an interaction between A-MuLV
and other regions of the Pit2 receptor. Studies with other retroviral
systems have provided similar conclusions. The discovery that HaPit2,
the hamster homolog of Pit2, functions as a receptor for GALV even
though it differs from Pit1 in seven of the nine residues in the
proposed GALV binding site (25) suggests that overall
conformational determinants rather than a particular sequence of amino
acid residues are acting to influence GALV entry. Our chimera and
mutational results with A-MuLV are in accord with these findings,
indicating both a high degree of tolerance for sequence variation and
the capacity for compensation within the second extracellular domain. A
recent mutational analysis of the GALV binding site, in which mutant forms were substituted for the corresponding sequence in Pit1 and
several chimeric receptors, has demonstrated that the ability of
mutations in the binding site to alter GALV receptor function is
dependent upon the receptor into which they are introduced (5). In support of these conclusions, chimeric receptor
studies have indicated that more than one domain of the Pit receptors is involved in both GALV (21) and A-MuLV infection (8,
21). Involvement of regions other than domain II of the Pit2
protein in A-MuLV entry is consistent with the observed effects of
domain II on viral infection efficiency presented here.
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ACKNOWLEDGMENTS |
We thank Keith Peden for critical review of the manuscript and
the Protein and Nucleic Acid Lab Core facility in the Center for
Biologics Evaluation and Research for synthesis of oligonucleotides.
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FOOTNOTES |
*
Corresponding author. Mailing address: FDA, CBER,
HFM-530, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301)
827-0481. Fax: (301) 827-0449. E-mail:
wilsonc{at}A1.cber.fda.gov.
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REFERENCES |
| 1.
|
Albritton, L. M.,
J. W. Kim,
L. Tseng, and J. M. Cunninghan.
1993.
Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.
J. Virol.
67:2091-2096[Abstract/Free Full Text].
|
| 2.
|
Albritton, L. M.,
L. Tseng,
D. Scadden, and J. M. Cunningham.
1989.
A putative murine ecotropic receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.
Cell
57:659-666[Medline].
|
| 3.
|
Atchison, R. E.,
J. Gosling,
F. S. Monteclaro,
C. Franci,
L. Digilio,
I. F. Charo, and M. A. Goldsmith.
1996.
Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines.
Science
274:1924-1926[Abstract/Free Full Text].
|
| 4.
|
Bonham, L.,
G. Wolgamot, and A. D. Miller.
1997.
Molecular cloning of Mus dunni endogenous virus: an unusual retrovirus in a new murine viral interference group with a wide host range.
J. Virol.
71:4663-4670[Abstract].
|
| 5.
|
Chaudry, G. J., and M. V. Eiden.
1997.
Mutational analysis of the proposed gibbon ape leukemia virus binding site in Pit1 suggests that other regions are important for infection.
J. Virol.
71:8078-8081[Abstract].
|
| 6.
|
Chien, M.-L.,
J. L. Foster,
J. L. Douglas, and J. V. Garcia.
1997.
The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion.
J. Virol.
71:4564-4570[Abstract].
|
| 7.
|
Chou, P. Y., and G. D. Fasman.
1974.
Prediction of protein conformation.
Biochemistry
13:222-245[Medline].
|
| 8.
|
Eiden, M. V.,
K. B. Farrell, and C. A. Wilson.
1996.
Substitution of a single amino acid residue is sufficient to allow the human amphotropic murine leukemia virus receptor to also function as a gibbon ape leukemia virus receptor.
J. Virol.
70:1080-1085[Abstract].
|
| 9.
|
Higuchi, R.,
B. Krummel, and R. K. Saiki.
1988.
A general method of in vitro preparation and specific mutagenesis of DNA fragments: a study of protein and DNA interactions.
Nucleic Acids Res.
16:7351-7367[Abstract/Free Full Text].
|
| 10.
|
Kavanaugh, M. P.,
D. G. Miller,
W. Zhang,
W. Law,
S. L. Kozak,
D. Kabat, and A. D. Miller.
1994.
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.
Proc. Natl. Acad. Sci. USA
91:7071-7075[Abstract/Free Full Text].
|
| 11.
|
McLachlin, J. R.,
N. Mittereder,
M. B. Daucher,
M. Kadan, and M. A. Eglitis.
1993.
Factors affecting retroviral vector function and structural integrity.
Virology
195:1-5[Medline].
|
| 12.
|
Miller, A. D.,
J. V. Garcia,
N. von Suhr,
C. M. Lynch,
C. Wilson, and M. V. Eiden.
1991.
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J. Virol.
65:2220-2224[Abstract/Free Full Text].
|
| 13.
|
Miller, A. D., and G. J. Rosman.
1989.
Improved retroviral vectors for gene transfer and expression.
BioTechniques
7:980-990.
[Medline] |
| 14.
|
Miller, A. D., and G. Wolgamot.
1997.
Murine retroviruses use at least six different receptors for entry into Mus dunni cells.
J. Virol.
71:4531-4535[Abstract].
|
| 15.
|
Miller, D. G., and A. D. Miller.
1994.
A family of retroviruses that utilize related phosphate transporters for cell entry.
J. Virol.
68:8270-8276[Abstract/Free Full Text].
|
| 16.
|
O'Hara, B.,
S. V. Johann,
H. P. Klinger,
D. G. Blair,
H. Rubinson,
K. J. Dunne,
P. Sass,
S. M. Vitek, and T. Robins.
1990.
Characterization of a human gene conferring sensitivity to infection by gibbon ape leukemia virus.
Cell Growth Differ.
1:119-127[Abstract].
|
| 17.
|
Olah, Z.,
C. Lehel,
W. B. Anderson,
M. V. Eiden, and C. A. Wilson.
1994.
The cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium-dependent phosphate transporter.
J. Biol. Chem.
269:25426-25431[Abstract/Free Full Text].
|
| 18.
|
Pederson, L.,
S. V. Johann,
M. van Zeijl,
F. S. Pedersen, and B. O'Hara.
1995.
Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus.
J. Virol.
69:2401-2405[Abstract].
|
| 19.
|
Rein, A.
1982.
Interference grouping of murine leukemia viruses: a distinct receptor for the MCF-recombinant viruses in mouse cells.
Virology
120:251-257[Medline].
|
| 20.
|
Rucker, J.,
M. Samson,
B. J. Doranz, et al.
1996.
Regions in beta-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity.
Cell
87:437-446[Medline].
|
| 21.
|
Tailor, C. S., and D. Kabat.
1997.
Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.
J. Virol.
71:9383-9391[Abstract].
|
| 22.
|
Tailor, C. S.,
Y. Takeuchi,
B. O'Hara,
S. V. Johann,
R. A. Weiss, and M. K. L. Collins.
1993.
Mutation of amino acids within the gibbon ape leukemia virus (GALV) receptor differentially affects feline leukemia virus subgroup B, simian sarcoma-associated virus, and GALV infections.
J. Virol.
67:6737-6741[Abstract/Free Full Text].
|
| 23.
|
van Zeijl, M.,
S. V. Johann,
E. Closs,
J. Cunningham,
R. Eddy,
T. B. Shows, and B. O'Hara.
1994.
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family.
Proc. Natl. Acad. Sci. USA
91:1168-1172[Abstract/Free Full Text].
|
| 24.
|
Wilson, C., and M. Eiden.
1991.
Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses.
J. Virol.
65:5975-5982[Abstract/Free Full Text].
|
| 25.
|
Wilson, C. A.,
K. B. Farrell, and M. V. Eiden.
1994.
Properties of a unique form of the murine amphotropic leukemia virus receptor expressed on hamster cells.
J. Virol.
68:7697-7703[Abstract/Free Full Text].
|
| 26.
|
Young, J. A. T.,
H. E. Varmus, and P. F. Bates.
1994.
A protein related to the LDL receptor is a cellular receptor specific for subgroup A avian leukosis and sarcoma viruses, p. 61-73.
In
E. Wimmer (ed.), Cellular receptors for animal viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 27.
|
Zingler, K., and J. A. Young.
1996.
Residue Trp-48 of Tva is critical for viral entry but not for high-affinity binding to the SU glycoprotein of subgroup A avian leukosis and sarcoma viruses.
J. Virol.
70:7510-7516[Abstract].
|
J Virol, June 1998, p. 4956-4961, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
