Comparison of cDNAs encoding the gibbon ape leukaemia virus

Journal of General Virology (1994), 75, 1901-1908.
1901
Printed in Great Britain
Comparison of cDNAs encoding the gibbon ape leukaemia virus receptor
from susceptible and non-susceptible murine cells
Carolyn A. W i l s o n , * ~ Karen B. Farrell and Maribeth V. Eiden
Laboratory of Cell Biology, National Institute of Mental Health, Building 36, Room 2D22, 9000 Rockville Pike,
Bethesda, Maryland 20892, U.S.A.
The gibbon ape leukaemia virus (GaLV) family of type C
retroviruses consists of five closely related viral isolates,
GaLV SF, GaLV SEATO, GaLV Br, GaLV H and
simian sarcoma-associated virus. The eDNA encoding
the human receptor for GaLV SEATO had previously
been isolated. We now demonstrate that all of the above
GaLVs can use the human form of the GaLV receptor to
infect cells. All murine cells analysed to date have been
found to be resistant to infection by GaLVs owing to the
absence of a functional GaLV receptor. We have now
identified a murine cell line which is unique in its
susceptibility to GaLV infection. This cell line was
established from a Japanese feral mouse, Mus musculus
molossinus. We cloned and sequenced the cDNA for the
receptor expressed in these cells and compared it to the
cDNA for the GaLV receptor expressed in resistant
murine cells such as NIH 3T3 (derived from M. m.
musculus) and MDTF (derived from M. dunni tail
fibroblasts). The crucial region for GaLV infection (the
fourth extraceUular domain) from the functional M. m.
molossinus GaLV receptor is quite divergent from the
same region of the M. m. musculus and M. dunni
proteins, but similar to that of the functional human
GaLV receptor. These results confirm the importance of
the amino acids of this region in GaLV receptor
function.
Introduction
(Kawakami & Holmberg, 1980). A third GaLV was
obtained from a gibbon ape with B cell leukaemia
housed in a colony on Hall's Island off the coast of
Bermuda and is designated GaLV H (Gallo et al., 1978;
Reitz et al., 1979). Brain tissue from three tumour-free
gibbon apes was the source of the GaLV Br isolates.
Interestingly, two of these gibbon apes had been injected
with brain extracts from kuru patients (Todaro et al.,
1975). The last member of the GaLV family, simian
sarcoma-associated virus (SSAV) was isolated from a
woolly monkey with multiple fibrosarcomas; this woolly
monkey had been housed with a gibbon ape (Theilen et
al., 1971). Although clearly related to each other on
structural and genetic bases, it has not been determined
whether all GaLVs use the same receptor to infect cells.
Furthermore, the xenotropic murine leukaemia virus (XMuLV) isolated from the Asian feral mouse Mus caroli
interferes with superinfection by GaLV SEATO and
SSAV in mink cells (Lieber et al., 1975). This finding
suggests that some X-MuLVs and GaLVs may use a
common receptor. X-MuLVs constitute a class of
MuLVs in which not all members have the same host
range and interference properties, indicating that specific
X-MuLVs may use different receptors to infect cells.
The human receptor for GaLV SEATO has recently
been identified to be a protein with multiple membranespanning domains (O'Hara et at., 1990). Murine NIH
The members of the gibbon ape leukaemia virus (GaLV)
family of primate type C retroviruses have been grouped
together on the basis of nucleic acid hybridization studies
and immunological cross-reactivity (Benveniste &
Todaro, 1973; Rangan, 1974; Thiel et al., 1978).
Members of the GaLV family have been isolated from
primates in a variety of different disease states. The first
isolate of GaLV, known as GaLV SF, was obtained from
a gibbon ape with lymphocytic leukaemia in a colony in
San Francisco (Kawakami et al., 1972; Snyder et al.,
1973). To date, other members include a virus isolated
from a gibbon in the SEATO Laboratory gibbon ape
colony in Thailand where five spontaneous cases of
granulocytic leukaemia had been reported (DePaoli et
al., 1973; Kawakami & Buckley, 1974). The GaLV
SEATO virus is the only GaLV that has been demonstrated to be a causative agent of leukaemia in gibbons.
Kawakami and co-workers were able to induce chronic
granulocytic leukaemia in 8 to 9 month old gibbon apes
after the injection of concentrated GaLV SEATO virus
t Present address : Division of Cellular and Gene Therapies, Center
for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20892, U.S.A.
0001-2317 © 1994 SGM
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1902
C. A . W i l s o n a n d o t h e r s
3T3 cells, like other murine cell lines such as SC-1 and
MDTF, are resistant to GaLV infection (Eglitis et at.,
1993). NIH 3T3 cells express a non-functional form of
the GaLV receptor (Johann et al., 1992). Transfer of
human DNA encoding the GaLV receptor into NIH 3T3
fibroblasts renders them sensitive to infection by GaLV
SEATO (O'Hara et al., 1990). Mutagenesis studies of the
human and murine forms of the GaLV receptor have
allowed identification of a region in the fourth extracellular domain which is critical for susceptibility to the
virus (Johann et al., 1993; Tailor et al., 1993).
In this study we used NIH 3T3 cells containing human
DNA encoding the GaLV SEATO receptor to determine
whether all members of the GaLV family of viruses can
use this receptor to infect cells. In addition, we identified
a murine cell line which differs from other murine cells in
its susceptibility to GaLV SEATO infection. We have
cloned and sequenced the GaLV receptor cDNA from
this GaLV-susceptible murine cell line and compared its
sequence to other murine-derived cell cDNAs encoding
non-functional GaLV receptors.
Methods
Cell lines and viruses. The following cell lines were used in this
study: Rat-2 embryo fibroblasts (ATCC CRL 1764); TblLu , bat lung
fibroblasts (ATCC CCL 88); HOS, human osteosarcoma cells (ATCC
CRL 1543); Mv-l-Lu, mink lung fibroblasts (ATCC CCL 64); NIH
3T3 murine fibroblasts (ATCC CRL 1658); NIH 3T3/GRT-5, N[H
3T3 cells expressing the human GaLV receptor (O'Hara et al., 1990);
MDTF, M. dunni tail fibroblasts (provided by Dr Janet Hartley,
National Institute of Allergy and Infectious Diseases, Bethesda, Md.,
U.S.A.); MMK, M. musculus molossinus kidney cells (ATCC CRL
6439); PA317 (ATCC CRL 9078); CRIP/BAG and CRE/BAG
[provided by Dr C. Cepko, Harvard Medical School, Boston, Mass.,
U.S.A. (Danos & Mulligan, 1988; Price et al., 1987)]; and PG13/BAG
(ATCC CRL 10683; Miller et al., 1991). All cell lines were maintained
in Dulbecco's modified Eagle's medium (DMEM, Whittaker Bioproducts) supplemented with 5 % fetal bovine serum (FBS), 100 U/ml
of penicillin, 100 gg/ml of streptomycin and 40 mM-glutamine.
GaLV wild-type viruses were obtained from the following productively infected cell lines: SEATO-88, GaLV SEATO-infected bat
lung fibroblasts; GaLV-4-88, GaLV-Br-infected bat lung fibroblasts;
71-AP-1, SSAV-infected marmoset fibroblasts; UCD-144, GaLV SFinfected primate T cells; 6G1-PB, GaLV-H-infected lymphocytes.
Xenotropic MuLV Cas No. 1 (Cloyd et al., 1979, 1985), X-MuLV(C),
viral supernatant and BALB/c-IU#1 xenotropic MuLV (Cloyd et al.,
1979), X-MuLV(B)-infected mink lung fibroblasts were obtained from
Dr Christine Kozak (National Institute of Allergy and Infectious
Diseases, Bethesda, Md., U.S.A.).
Virus pseudotypes and GaL V vector infections. PA317 cells producing
MoMuLV-based packageable genomes [either the pLNSX genome
(Miller & Rosman, 1989) or the BAG genome (Price et aL, 1987)] were
used to infect either GaLV or X-MuLV producer cells to generate
GaLV and X-MuLV pseudovirions, respectively. GaLV retroviral
vectors containing the GaLV SEATO envelope, Moloney (Mo) MuLV
core components and MoMuLV-based genomes were produced from
either PG 13 cells containing the BAG genome (Wilson & Eiden, 1991)
or from E5/10-GAS cells (Wilson, 1989) expressing the F-EVX genome
(Wilson et al., 1989).
Viral titres were determined as previously described (Wilson &
Eiden, 1991). Briefly, 3x 104 cells were exposed to filtered cell
supernatant containing viral pseudotypes and adjusted to 3 to 6 gg/ml
of polybrene (Abbott Laboratories). Titres of G418-resistant colonies
were determined by reseeding the cells 24 h after exposure to virus at
various densities in 10 cm dishes. The next day the medium was
changed to G418-containing DMEM in the following concentrations:
300 gg/ml G418 (active component) for HOS cells, 400 lag/ml G418
for bat lung fibroblasts, 450 lag/ml G418 for Rat-2 fibroblasts, and
800 gg/ml G418 for mink lung fibroblasts. After 10 to 14 days in
selective medium, G41g-resistant colonies were fixed, stained and
counted. Titres were calculated to represent the number of c.f.u./ml of
cell supernatant. Assays for cells expressing fl-galactosidase were
performed 48 to 72 h after exposure to viral pseudotypes as described
(Sanes et al., 1986). Foci of blue-staining ceils were visualized with a
microscope and counted. The number of blue foci per ml of cell
supernatant was calculated.
Cloning and sequencing of GaL V receptor cDNA homologues. A PCRbased strategy, similar to that previously described, was used to clone
regions of the cDNA for the GaLV receptor homologue (Eiden et al.,
1993). Polyadenylated RNA was isolated from either MMK or MDTF
cells (Okayama et al., 1987). Random hexanucleotides were used to
prime cDNA synthesis from mRNA using avian myeloblastosis virus
reverse transcriptase (Promega). Using synthetic nested oligonucleotide
primers corresponding to the nucleotide sequence of the human GaLV
receptor (O'Hara et al., 1990) regions of the receptor cDNA were
selectively amplified from MMK cells by PCR. Four overlapping
segments were generated: the 5' segment was made with primers GR1
(nt 370 to 396) and GR5 (nt 1091 to 1071), the second fragment with
primers GR10 (nt 1008 to 1041) and GR11 (nt 1720 to 1691), the third
with GR13 (nt 1538 to 1570) and hGR4A (nt 2383 to 2336) and the 3'
fragment with primers GR3 (nt 1734 to 1745) and GR14 (nt 2414 to
2386). One region of the MDTF GaLV receptor cDNA was amplified
by using synthetic oligonucleotide primers based on the M. m. musculus
nucleotide sequence: muGR15 (nt 1698 to 1732) and muGR4 (nt 2478
to 2453). The PCR-amplified products were ligated into the TA cloning
vector (Invitrogen) and sequenced by direct dideoxynucleotide sequencing (Sanger et al., 1977; Mierendorf & Pfeffer, 1987). Several PCR
clones were sequenced to ensure that changes were authentic and not
PCR-induced. Comparison and alignment of the deduced amino acid
sequences of different GaLV receptor cDNAs was performed using
Geneworks software (Intelligenetics) which scores mismatches and
gaps rather than matches alone.
Results
A l l m e m b e r s o f the G a L V f a m i l y u s e the s a m e cellular
r e c e p t o r to g a i n e n t r y into cells
W e u s e d a s u p e r i n f e c t i o n i n t e r f e r e n c e a s s a y to d e t e r m i n e
w h e t h e r all m e m b e r s o f t h e G a L V f a m i l y o f v i r u s e s c a n
use the s a m e c e l l u l a r r e c e p t o r to e n t e r cells. S u p e r i n f e c t i o n i n t e r f e r e n c e assays are b a s e d o n t h e a s s u m p t i o n
t h a t i n f e c t i o n o f a cell b y a v i r u s r e n d e r s t h a t v i r a l
r e c e p t o r i n a c c e s s i b l e to a c h a l l e n g e v i r u s t h a t r e q u i r e s
the s a m e r e c e p t o r . H o w e v e r , v i r u s e s t h a t use a d i f f e r e n t
r e c e p t o r c a n s u p e r i n f e c t t h e i n f e c t e d cell. T h e r a t i o o f the
v i r a l titre o n i n f e c t e d cells to the c o r r e s p o n d i n g titre o n
u n i n f e c t e d cells i n d i c a t e s t h e e x t e n t o f r e c e p t o r int e r f e r e n c e . A m p h o t r o p i c M u L V ( A - M u L V ) w a s inc l u d e d as a c o n t r o l in t h e a s s a y b e c a u s e it d o e s n o t
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Murine GaLV receptor homologues
T a b l e 1.
1903
Receptor interference patterns of S S A V and four GaLV isolates
Ratio of virus titre on infected/uninfected cell typest
Pseudotype of
superinfecting
virus*
Mink/
4070A
A-MuLV
X-MuLV(B)
X-MuLV(C)
GaLV SEATO
GaLV SF
GaLV Br
GaLV H
SSAV
0.004
ND
2"3
2.5
NO
ND
ND
2'5
Mink/
X-MuLV(B)
Mink/
X-MuLV(C)
ND~
< if001
< 0"001
3.6
ND
NO
ND
ND
Mink/
SEATO
NO
ND
0"007
0-2
ND
ND
ND
ND
HOS/
SEATO
Bat/
SEATO
Rat-2/
SEATO
0.2
0"35
NO
0.043
0"005
0"002
ND
0"003
0.67
ND
ND
0"06
< 0"00l
< 0"00l
ND
< 0"00l
1-0
ND
ND
< 0.001
< 0"001
0"002
< 0'001
0'014
0.2
0-71
0'46
< 0.001
0"008
0"032
ND
0"001
* Viral pseudotypes were generated and titrated as described. All of the pseudotyped viruses
contain a MoMLV-based recombinant genome.
t Values represent ratios of the pseudovirion titre on infected cells to the pseudovirion titre on
uninfected cells. Values shown in bold indicate cross-interference.
ND, Not determined.
Susceptibility of N I H 3T3/GRT-5 cells to
infection by members of GaLVfamily
T a b l e 2.
(b)
(a)
. . . . . . .
•
Relative virus titre on different
cell lines*
Virus
pseudotype
MoMLV
GaLV SEATO
GaLV SF
GaLV Br
GaLV H
SSAV
X-MuLV(B)
X-MuLV(C)
Rat-2
++++
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
NIH 3T3
NIH 3T3/
GRT-5
+++
-
+
+
+
+
+
+
+
+++
+ +
+ +
+ +
+ +
+ +
-
(c)
(d)
* Titres were determined by counting the number of blue foci 48 to
72 h after exposing 3 × 104 target cells to 1.0 ml of filtered infected cell
supernatant such that + + + + corresponds to > 103 blue foci/1.0 ml
of supernatant; + + + , 102 to 103 blue foci/ml; + +, 10 to 102 blue
foci/ml; + , 1 to 10 blue foci/ml; - , < 1 blue focus/ml.
T a b l e 3. Susceptibility of ceil lines from two feral Asian
mice to G A L V infection
Cell line
Virus
pseudotype
A-MuLV
GaLV SEATO
X-MuLV(B)
X-MuLV(C)
Rat-2
1.0"
1.0
1.0
I-0
NIH 3T3
MDTF
MMK
1-18
0
0-12
0-34
0.97
1.02
0-16
0-04
1.0
0
0
0
* Cells exposed to virus pseudotypes were assessed for viral infection
by histochemical staining for fl-galactosidase activity. Titres were
determined by counting the number of blue-staining foci and then
normalized to the titres observed on Rat-2 fibroblasts exposed to the
same viral pseudotype.
Fig. 1. Viral interference properties on M. m. molossinus kidney
(MMK) cells. (a) MMK cells exposed to GaLV SEATO/BAG
pseudotypes. (b) MMK cells productively infected with GaLV SEATO
wild-type virus and then exposed to GaLV SEATO/BAG pseudotypes.
(c) MMK cells exposed to X-MuLV(C)/BAG pseudotypes. (d) MMK
cells productively infected with GaLV SEATO wild-type virus and then
exposed to X-MuLV(C)/BAG pseudotypes. Forty-eight to 72 h postexposure to pseudotype virus, all cells were fixed and histochemically
stained for the presence offl-galactosidase. Bar marker in (d) represents
250 ~tm.
L a k e C a s i t a s f e r a l m o u s e in C a l i f o r n i a . T h e L a k e C a s i t a s
mice have been shown to be closely related to certain
strains of Asian mice (Inaguma
et al.,
productively infected with A-MuLV
1991). M i n k cells
were resistant to
interfere with infection by GaLVs (Sommerfelt & Weiss,
superinfection by A-MuLV
1990). X - M u L V ( B ) w a s o r i g i n a l l y i s o l a t e d f r o m B A L B / c
m u r i n e cells a f t e r i n d u c t i o n o f v i r u s e x p r e s s i o n w i t h
either of the X-MuLVs or any of the GaLV pseudotypes
iododeoxyuridine.
MuLVs
X-MuLV(C)
was
isolated
from
a
(Table
1). M i n k
pseudotypes,
but
not
to
cells i n f e c t e d w i t h e i t h e r o f t h e X -
were resistant to i n f e c t i o n by b o t h o f the X-
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1904
C. A. Wilson and others
MISTVATITS
H-ATL--ITS
TLAAVTASAP
TLAA-TAASS
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299
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Fig. 2. Comparison of the deduced amino acid sequences of the cDNAs for M . m. musculus (top) and M . m. molossinus (middle) GaLV
receptors. Residues that are identical are shaded. Dashes represent gaps introduced for alignment. The third line contains a consensus
sequence derived from the comparison of the two proteins. Potential transmembrane domains in the M . m. musculus receptor are
underlined and were determined by their Kyte-Doolittle hydrophobicity profile.
MuLVs but A-MuLV and all of the GaLV pseudotypes
were able to infect these cells. GaLV SEATO-infected
mink, human, bat or rat cells were resistant to superinfection by all of the GaLV pseudotypes but were
susceptible to infection by either A-MuLV or the XMuLVs (Table 1). These results demonstrate that all of
the GaLVs belong to the same interference group and
that this group is separate from that including A-MuLV
or any of the X-MuLVs tested in this assay.
Classification of viruses into the same interference
group suggests that these viruses use a common receptor
in a particular cell. To demonstrate that all members of
the G a L V family of viruses could use the human G a L V
SEATO receptor to infect cells, we infected N I H
3 T 3 / G R T - 5 cells with the GaLV pseudovirions. N I H
3 T 3 / G R T - 5 cells are N I H 3T3 cells which express the
human form of the receptor. As shown in Table 2, we
exposed Rat-2 fibroblasts, N I H 3T3 cells and N I H
3 T 3 / G R T - 5 cells to either MoMuLV, GaLV, or XMuLV pseudotypes. As expected, M o M u L V pseudovirions, derived from an ecotropic murine leukaemia
virus, were able to infect all three cell types. X-MuLV
pseudovirions were unable to infect either N I H 3T3 or
N I H 3 T 3 / G R T - 5 cells. None of the GaLV pseudovirions
were able to infect N I H 3T3 cells, whereas all of the
GaLVs infected N I H 3 T 3 / G R T - 5 cells and control Rat2 fibroblasts. Therefore, all of the GaLV isolates were
able to use the human receptor for cell entry.
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1905
M u r i n e G a L V receptor homologues
Analysis o f the susceptibility o f f e r a l murine cell lines to
G a L V infection
We next examined murine cell lines derived from two
Asian feral mice for their susceptibility to infection by
GaLV SEATO, A-MuLV or X-MuLV pseudovirions.
As shown in Table 3, M D T F cells, a tail fibroblast cell
line derived from an Indian feral mouse, M . dunni, like
N I H 3T3 cells, are resistant to infection by GaLV
SEATO but are susceptible to infection by X-MuLV(B)
and X-MuLV(C) as well as A-MuLV pseudovirions.
Thus M D T F cells serve as a host cell for A-MuLV and
X-MuLV but not GaLV infection. Further, we have
previously shown that the block to GaLV infection in
M D T F cells is receptor-mediated (Eglitis et al., 1993).
M M K cells, a kidney cell line derived from a Japanese
feral mouse, M . m. molossinus, are susceptible to
infection by GaLV SEATO as well as X-MuLV(B), XMuLV(C) and A-MuLV pseudovirions (Table 3).
Efficient infection by a GaLV makes M M K cells unusual,
since other murine cells are resistant to GaLV infection.
We hypothesized that a novel form of the GaLV receptor
may be used in M M K cells by both GaLV and XMuLVs. To test this hypothesis, we exposed M M K cells
productively infected with GaLV SEATO to either
GaLV SEATO or X-MuLV(C) pseudovirions (Fig. 1).
The ratio of the GaLV SEATO pseudovirion titre on
GaLV-infected cells to the titre on uninfected M M K cells
was 0.0007, whereas the ratio of the X-MuLV(C) titre on
each was 0.262, demonstrating that infection by GaLV
but not X-MuLV(C) pseudovirions was inhibited in
GaLV SEATO-infected M M K cells. Therefore, these
viruses use distinct receptors to enter M M K cells.
Cloning o f the c D N A s f o r the G a L V receptor f r o m
M M K cells and M D T F cells; comparison with M . m.
musculus-derived G a L V receptor homologue
A PCR-based strategy was used to amplify, clone and
sequence the entire c D N A for the G a L V receptor from
M M K cells. The deduced amino acid sequence of the
M M K GaLV receptor was compared to that of the
GaLV receptor homologue previously isolated from the
thymus of M . m. musculus (Johann et al., 1992) (Fig. 2).
Cells derived from M . m. musculus are resistant to GaLV
infection, hence this murine receptor homologue is nonfunctional. The M M K GaLV receptor shares an overall
88% amino acid identity with the M . m. musculusderived GaLV receptor. Differences in amino acid
residues are found throughout the protein, however
several regions are quite divergent between these two
receptors. Most of the differences are found in the
putative cytoplasmic domains: the N and C termini of
the protein contain several amino acid substitutions, the
second cytoplasmic loop contains seven amino acid
Susceptibility
to GaLV
infection
Receptor origin
Amino acid sequence
(fourth extracellular domain)
Homo sapiens
D T G D VSSK VAT
Yes
M. m. molossinus
I T G D VSSK M AI
Yes
M. m. musculus
-KQEASTKAAT
No
M. d u n n i
-KQDASTKAAT
No
Fig. 3. Alignment of the deduced protein sequences from the fourth
extracellular domain of the Homo sapiens GaLV receptor (from the
human HL-60 cell line) (O'Hara et al., 1990) the M. m. molossinus
GaLV receptor (from MMK cells) the M. m. musculus GaLV receptor
(from a thymus cDNA library) (Johann et al., 1992)and the M. dunni
GaLV receptor (from MDTF cells), correspondingto amino acids 550
to 560 of the human receptor and amino acids 553 to 562 of the M. m.
museulus receptor.
substitutions out of the 23 amino acids that make this
loop and several clusters o f amino acid substitutions are
found in the third cytoplasmic loop. In this region, the
M M K GaLV receptor shares only 22% amino acid
identity with the M . m. musculus GaLV receptor.
Comparison of the putative extracellular domains of
these two receptors reveals a region encompassing amino
acid residues 553 to 562 (based on the M . m. museulus
numbering) in which the M . m. musculus receptor has
eight amino acid residues out of 11 which differ from the
M . m. molossinus GaLV receptor sequence. This region
of the human GaLV receptor has been demonstrated to
be critical for GaLV infection (Johann et al., 1993;
Tailor et al., 1993). In an effort to define the residues in
this region that may be implicated in the functional
differences between these two receptors, we isolated and
sequenced this portion of the GaLV receptor c D N A
from a second feral mouse cell line, M D T F , which unlike
M M K cells is resistant to GaLV infection. Comparison
of this region of the M D T F receptor homologue to the
M M K receptor reveals several amino acid residues which
are conserved: Asp-553, Ser-555, Lys-557 and Ala-559
(Fig. 3). Conservation of these residues between the
M M K receptor which functions as a GaLV receptor and
the M D T F receptor which does not serve as a GaLV
receptor would suggest that these residues do not
contribute directly to the function of this protein as a
GaLV receptor.
Discussion
We have demonstrated that five different isolates of
GaLV (GaLV SEATO, GaLV SF, GaLV H, GaLV Br
and SSAV) can use the same receptor to infect cells.
Superinfection interference assays demonstrated that in
human, mink, bat and rat cells, all members of the GaLV
family cross-interfere. Furthermore, all five GaLVs could
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1906
C. A. Wilson and others
infect NIH 3T3 cells expressing the human form of the
GaLV receptor. Takeuchi et al. have demonstrated that
feline leukaemia virus subgroup B (FeLV-B) can also use
this receptor for entry into cells (Takeuchi et al., 1992).
The N-terminal 88 to 200 amino acids of various MuLV
surface (SU) envelope proteins have been shown to affect
host range and receptor interference properties (Battini
et al., 1992; Morgan et al., 1993; O t t & Rein, 1992).
Similarly, the N-terminal 87 to 92 amino acids of the
FeLV-A and -C SU proteins were shown to influence
host range (Brojatsch et al., 1992). Comparison of the
amino acid sequences of the SU envelope glycoproteins
from the various GaLV isolates and FeLV-B will reveal
regions of conserved sequences which might be important
in receptor binding.
Some xenotropic MuLVs from Asian feral mice have
been reported to interfere with GaLV superinfection
and/or to be antigenically related to GaLV or SSAV
(Lieber et al., 1975; Benveniste & Todaro, 1973). We
examined two xenotropic MuLVs, one derived from an
inbred mouse [X-MuLV(B)] and one derived from a feral
mouse in California [X-MuLV(C)] for receptor interference with GaLV infection. X-MuLV(C) was derived
from mice which have been shown to be genetically
similar to M. rn. rnolossinus (Gardner et al., 1991;
Inaguma et al., 1991). Neither of these xenotropic
MuLVs appear to use the GaLV receptor to infect
human, bat, rat, mink or M. m. molossinus cells. The
murine homologue of the GaLV receptor maps to mouse
chromosome 2 (Adamson et al., 1991) whereas susceptibility to X-MuLV infection maps to chromosome 1
of wild mice (Kozak, 1985), suggesting that these viruses
use distinct receptors. However, the GaLV-related Asian
xenotropic MuLVs isolated from M. cervicolor and M.
caroli may use a different receptor than the X-MuLVs
tested here. An ecotropic murine leukaemia virus isolated
from M. cervicolor, which does not seem to be GaLVrelated on the basis of serological cross-reactivity and
nucleic acid hybridization, has been shown to use a
receptor which maps to mouse chromosome 2 (named
the Rec-2 receptor) (Rapp & Marshall, 1980) and may in
fact be the GaLV receptor homologue.
X-MuLVs and GaLVs have for the most part an
identical host range. Most murine cells are resistant to
infection by both types of viruses whereas a broad
variety of mammalian cells are infected by these viruses,
notably human, bovine, buffalo and certain hamster cells
(Cloyd et al., 1985; Weiss & Long, 1977; Teich et al.,
1975; C. A. Wilson, unpublished data). The tail fibroblast cell line derived from the Indian feral mouse M.
dunni provides an example of a cell susceptible to XMuLV infection but resistant to GaLV infection. The
M M K cell line is the only murine cell known to be
susceptible to efficient infection by both GaLV and X-
MuLV. M. m. molossinus is thought to have arisen from
a cross between Northern Chinese M. m. musculus and
the Southeastern Asian mouse, M. rn. castaneus (Inaguma et al., 1991; Yonekawa et al., 1986). M. m.
molossinus have been shown by analysis o f Fv-4 loci to be
closely related to the California wild mice from Lake
Casitas and genetically distinct from other feral mouse
populations which are found in Europe (M. m. musculus)
and America (M. m. domesticus) (Gardner et al., 1991).
The amino acid residues which compose the putative
fourth extracellular loop have been shown to be critical
for GaLV, SSAV and FeLV-B infection (Johann et al.,
1993; Tailor et al., 1993). Analysis of mutagenesis of this
domain had suggested certain requirements for infectivity. For example, Tailor and co-workers demonstrated
that the human receptor functions as a GaLV receptor
with either glutamic acid or aspartic acid in amino acid
position 550. Substitution of a lysine at amino acid
residue 550 produced a receptor incapable of mediating
GaLV infection (Tailor et al., 1993). These results might
suggest that a negatively charged residue is required in
this position. However, the M. m. molossinus receptor
has a neutral amino acid, an isoleucine, in this position.
Substitution of the amino acid residues found in this
domain of the GaLV receptor from M. dunni for the
corresponding amino acid residues of the human GaLV
receptor results in a loss of GaLV receptor function
(Johann et al., 1993), suggesting that the fourth extracellular domain of the M. dunni receptor contributes to
loss of GaLV receptor function in the MDTF form.
We sequenced the cDNA derived from RNA encoding
the GaLV receptor isolated from the murine cell line
MMK (from the Asian feral mouse M. m. molossinus)
which is susceptible to infection by GaLV. Comparison
of the putative fourth extracellular domain of this
MMK-derived murine receptor cDNA to that of the
functional human GaLV receptor showed a striking
degree of conservation of this domain. Comparison of
this MMK GaLV receptor cDNA with other homologous cDNAs isolated from GaLV-resistant murine
cells (such as MDTF and NIH 3T3) has shown that this
region is quite divergent among the functional and nonfunctional forms of the murine GaLV receptors. However, the fourth extracellular region of two nonfunctional murine GaLV receptors (those of M. dunni
and M. m. musculus) are highly related to each other.
Other amino acid residues which differ between functional and non-functional forms of the GaLV receptor
are found in transmembrane and cytoplasmic regions.
These regions may mediate the post-binding steps of
GaLV infection (i.e. internalization).
We thank Dr BryanO'Hara for the NIH 3T3/GRT-5 cell line and
pOJ9 cDNA, Dr Claristine Kozak for the X-MuLVs, Dr Michael
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Murine G a L V receptor homologues
Brownstein for providing oligonucleotides, and Jennie Warsowe for
expert technical assistance. The authors gratefully acknowledge the
critical reading of this manuscript by Drs Richard Schneiderman and
Larry Mahan.
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