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Journal of Virology, July 2000, p. 5754-5761, Vol. 74, No. 13
Program in Molecular and Cellular
Biology1 and Departments of Comparative
Medicine2 and
Microbiology,3 University of Washington,
and Division of Human Biology, Fred Hutchinson Cancer Research
Center,4 Seattle, Washington
Received 19 January 2000/Accepted 31 March 2000
The envelope protein is a primary pathogenic determinant for
T-cell-tropic feline leukemia virus (FeLV) variants, the best studied
of which is the immunodeficiency-inducing virus, 61C. We have
previously demonstrated that T-cell-tropic, cytopathic, and
syncytium-inducing viruses evolve in cats infected with a relatively
avirulent, transmissible form of FeLV, 61E. The envelope gene of an 81T
variant, which encoded scattered single-amino-acid changes throughout
the envelope as well as a 4-amino-acid insertion in the C-terminal half
of the surface unit (SU) of envelope, was sufficient to confer the
T-cell-tropic, cytopathic phenotype (J. L. Rohn, M. S. Moser,
S. R. Gwynn, D. N. Baldwin, and J. Overbaugh, J. Virol.
72:2686-2696, 1998). In the present study, we examined the role of the
4-amino-acid insertion in determining viral replication and tropism of
FeLV-81T. The 4-amino-acid insertion was found to be functionally
equivalent to a 6-amino-acid insertion at an identical location in the
61C variant. However, viruses expressing a chimeric 61E/81T SU,
containing the insertion together with the N terminus of 61E SU, were
found to be replication defective and were impaired in the processing
of the envelope precursor into the functional SU and transmembrane (TM)
proteins. In approximately 10% of cultured feline T cells (3201)
transfected with the 61E/81T envelope chimeras and maintained over
time, replication-competent tissue culture-adapted variants were
isolated. Compensatory mutations in the SU of the tissue
culture-adapted viruses were identified at positions 7 and 375, and
each was shown to restore envelope protein processing when combined
with the C-terminal 81T insertion. Unexpectedly, these viruses
displayed different phenotypes in feline T cells: the virus with a
change from glutamine to proline at position 7 acquired a
T-cell-tropic, cytopathic phenotype, whereas the virus with a change
from valine to leucine at position 375 had slower replication kinetics
and caused no cytopathic effects. Given the differences in the
replication properties of these viruses, it is noteworthy that the
insertion as well as the two single-amino-acid changes all occur
outside of predicted FeLV receptor-binding domains.
Retroviruses are notorious for their
high degree of genetic variation. As a consequence of this variation,
there is considerable flexibility for the virus population to adapt to
different selective pressures. For example, particular virus variants
may be better able to spread from host to host whereas others are more
suited to persist within a chronically infected host. For retroviruses that cause immunodeficiency, pathogenesis is linked to the emergence of
T-cell-tropic, cytopathic (T-tropic) viruses, and it is these host-adapted viruses that appear to cause immunosuppression (18, 22). This pattern of evolution and pathogenesis has been observed for lentiviruses such as human immunodeficiency virus and simian immunodeficiency virus as well as for simple oncoretroviruses such as
feline leukemia virus (FeLV). Although FeLV infections are frequently
associated with neoplastic diseases, in some cases FeLV infection leads
to the emergence of T-tropic variants that are highly immunosuppressive
(33).
The transmissible form of FeLV is nonacutely pathogenic, whereas the
T-tropic viruses are highly pathogenic and induce immunodeficiency (26, 27). The cytopathic and pathogenic properties of these variants are conferred by the extracellular surface unit (SU) of the
envelope glycoprotein (13). The SU is generated through proteolytic cleavage of an envelope precursor protein that also includes a signal peptide and the transmembrane (TM) domain. The processed form of the TM protein anchors the SU to the cell membrane. The SU protein determines cell tropism through specific binding with
the cognate cellular receptor. Thus, changes in SU may affect pathogenesis, at least in part by altering the host cell specificity of
the virus.
We have previously demonstrated that viruses with altered cell tropism
and replication properties evolve within FeLV-infected cats (7,
32). In a cat infected with virus derived from a clone of FeLV
that is a prototype transmissible virus, 61E, we identified envelope
variants that replicated to higher levels in feline T lymphocytes than
did their progenitor virus. These envelope variants (called 81T), when
expressed in the context of the parental 61E virus, were highly
cytopathic for feline T cells in culture and caused syncytium formation
(32). Interference studies suggested that the 81T envelope
may have acquired a novel receptor specificity relative to the
progenitor viral envelope that could account for both its T-cell
tropism and its cytopathic properties (32).
The best-studied T-tropic FeLV variant, 61C, was isolated from cats
infected with an uncloned natural isolate called FeLV-FAIDS (26). The transmissible form of FeLV, 61E, was obtained
simultaneously from this isolate (26). Analyses of a panel
of 61E and 61C chimeras suggested that an insertion in the C-terminal
half of SU was a key pathogenic determinant and that other
single-amino-acid changes could have an enhancing effect on
pathogenesis (13). The 81T variants had acquired an
insertion at the same sequence location as 61C with respect to the
61E-progenitor virus, but the insertion in 81T was different in size
and sequence from the insertion in 61C. Other scattered
single-amino-acid changes were also observed in the 81T envelope SU,
some of which were also found in 61C. Because viruses expressing 61C or
81T envelope proteins were both T-cell tropic and cytopathic but only
the 81T virus was syncytium inducing, we asked what sequence changes
conferred this unique phenotype to the 81T envelope. We found that the
insertion itself was not sufficient to create a T-tropic, cytopathic,
syncytium-inducing virus. In fact, the insertion impaired envelope
protein processing in 61E/81T envelope chimeras. Over time, tissue
culture-adapted (tca) variants evolved and were selected in some
cultures transfected with the chimeric viruses. This allowed us to
identify compensatory single-amino-acid changes in both the N terminus
and the very C terminus of SU that restored envelope processing.
Interestingly, a virus that had both the amino acid change at the N
terminus and the insertion in the C-terminal half of SU was T tropic,
cytopathic, and syncytium inducing. In contrast, a virus with the
insertion paired with the C-terminal mutation exhibited replication
properties more typical of the transmissible 61E virus. These data
suggest that multiple domains in the envelope may play a role in
determining the tropism and cytopathic properties of FeLV variants.
Cell culture.
AH927 feline embryonic fibroblasts were
maintained in minimal essential medium (MEM) supplemented with 10%
fetal bovine serum. Human 293T cells were maintained in Dulbecco's MEM
(DMEM) supplemented with 10% fetal bovine serum. The feline T-cell
line 3201 was maintained in 50% Leibovitz's L15-50% RPMI 1640 supplemented with 15% fetal bovine serum. All complete media (e.g.,
cMEM and cDMEM) contained 100 U of penicillin per ml, 100 µg of
streptomycin per ml, 0.25 µg of amphotericin B per ml, and 2 mM
L-glutamine.
Construction of chimeric and mutant proviral clones.
The 61E
and 61C proviruses were derived from an FeLV-FAIDS-infected cat; the
EECC prototype FeLV-FAIDS clone was constructed from the 5' half of 61E
(including the 5' long terminal repeat [5'LTR] gag and
pol) and the 3' half (envelope 3'LTR) of a
replication-defective FeLV-FAIDS clone, 61C (26). The
EET109E [previously referred to as EET(TE)-109 in
reference 32] virus containing all of an 81T
envelope gene (81T clone 109), including the SU and TM domains, in a
61E background was described previously (32). Like the 81T-109 envelope clone, the subclones encoding the 81T-3 and 81T-6 envelope were generated from PCR-amplified envelope gene fragments obtained from the tumor of cat 40681; these clones were described previously (31). To construct the EE(ET)E chimeras, a 0.9-kb gel-purified MamI-RsrII fragment encompassing the
3' half of the envelope gene of 81T-3 and 81T-6, including coding
sequences for the C-terminal half of SU and all of TM, was cloned into
a similarly digested 3' subclone of 61E (called 3'EE
[28]). Clones with the correct chimeric structure were
identified by restriction enzyme digestion and nucleotide sequence
analysis. The full-length provirus was constructed from this chimeric
3' subclone by introducing a 6.5-kb EcoRI-XhoI
fragment carrying the 5'LTR gag and pol of 61E
into the 3' subclone as described previously (27). A similar strategy was employed for constructing the EE(CT)C chimeras, except that the 3'CC (26) subclone rather than the 3'EE subclone
was used.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Feline Leukemia Virus Envelope Sequences That
Affect T-Cell Tropism and Syncytium Formation Are Not Part of Known
Receptor-Binding Domains
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
L)E was also constructed using the
two-step cloning strategy used for construction of the
EE(ET6)E virus. In this case, the
MamI-RsrII fragment was gel isolated from a cell
culture-derived envelope clone that encoded the desired V 
L
mutation. The mutant envelope clone was obtained by amplification of
envelope gene sequences from genomic DNA from 3201 cells infected with
the tca virus EE(ET6)Etca using methods
described previously (31).
C single-base mutation predicted to encode a Q P change at
position 7 in the mature SU was engineered into EE(ET3)E by
overlap PCR to generate EE(EQPT3)E. The
single-base change that was introduced into this mutant corresponds to
a change at position 6099 (GenBank, FCVF6A [12]). The
overlap PCR method used was similar to a method described previously
for engineering mutations in the FeLV-B envelope (6).
Briefly, two overlapping fragments were generated with the desired
mutation. The 5' fragment encoded the desired change as a result of a T
G mutation engineered into the 3' primer used to generate that
product. The 3' fragment encoded the corresponding desired mutation at
its 5' end as a result of an A C change in the 5' primer used for
that PCR. The overlapping products of these reactions were then
combined, along with appropriate primers internal to the extreme 5' and 3' sequences, in a second round of PCR. A 1.3-kb fragment from this
overlap PCR product was isolated and cloned directly into the
EE(ET3)E parental virus using unique XhoI (near
the end of the pol gene) and MamI (in the middle
of the envelope) restriction sites. The presence of the desired
mutation was verified by nucleotide sequence analysis. Multiple clones
were analyzed to identify a clone for these studies that did not encode
any other predicted amino acid changes as a result of errors during PCR amplification.
For analyses of tca envelope variants, envelope fragments encompassing
sequences for the 5' envelope leader through the U3 region of the LTR
were cloned from 3201 cell DNA using primers FeLV-Pol5 and FeLV-U32B
and methods described previously (31).
Transfection and infection studies. To examine whether the EE(ET)E and EE(CT)C chimeras could generate replication-competent virus, the four proviral clones were transfected into the feline T-cell line 3201. The 61E, EECC, and EET109E clones were transfected in parallel, in all cases using electroporation. The production and spread of virus in the culture was monitored by an enzyme-linked immunosorbent assay that detects p27gag in the supernatant (ViraCHECK; Synbiotics). Cell-free virus was harvested when cells were chronically infected, as judged by detection of high levels of p27gag. Cell-free viral supernatants from these cells, diluted to each have the same level of reverse transcriptase (RT) activity, were used to infect naive 3201 T cells. To compare replication kinetics, the cells were infected with an equal dose of each virus and reverse transcriptase activity, cytopathic effects, and syncytium formation were monitored as described previously (32).
For experiments involving the mutant viruses EE(ET6,VL)E
and EE(EQPT3)E, virus was generated by
introducing the proviral clones into 293T cells by calcium phosphate
transfection. The transfection was done in duplicate to permit
infection studies with two independent virus supernatants. Cell
supernatants were collected and filtered after 48 h. Virus levels
were normalized to a control virus of known infectious titer using
p27gag enzyme-linked immunosorbent assay, and an
amount of virus equivalent to a multiplicity of infection of ~0.05
was used to infect 3201 cells. Infections were performed in duplicate
using supernatants from the transfections, and total viable-cell
numbers were averaged. Cells were monitored as described previously
(32), except that viable cells were diluted to a density of
5 × 105 cells/ml in a final volume of 4 ml. By day 12 postinfection, cells experiencing cytopathic effects were no longer
passaged but viable cell counts continued to be recorded.
The FeLV viral chimeras discussed above were also analyzed using
single-cycle viral infection assays, similar to those described previously (9). Briefly, virus particles were generated by cotransfecting the FeLV proviral clones with a murine retroviral vector
encoding 
-galactosidase, pRT43.2Tnls-gal-1 (36), into 293T cells. Cell-free viral supernatants were collected after 48 h
and filtered through 0.2-µm-pore-size filters. Serial dilutions of
viral supernatants were added to cMEM in total volumes of 1 ml each.
Virus was applied to AH927 feline fibroblasts, which were plated in
24-well plates at 2 × 104 cells per well in cMEM the
day before infection. At 48 h later, infected cells were detected
by adding
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal),
a substrate for -galactosidase. Blue nuclei were counted, and, based
on the various serial dilutions analyzed, the -galactosidase FFU per
milliliter of viral supernatant were calculated.
RT assays.
For analysis of RT activity, cell-free viral
supernatants were harvested at 2- to 3-day intervals during the 4-week
infection study and stored at 
70°C. Aliquots (10 µl) of
supernatant, and serial dilutions thereof, were assayed essentially as
described previously (15). Briefly, we measured the ability
of lysed virion preparations to catalyze the incorporation of
radiolabeled nucleotide [
-32P]dTTP (NEN), using the
polyribonucleotide template poly(A)p(dT)10 sodium salt
(Sigma) to which an oligodeoxyribonucleotide primer was annealed. The
intensity of radioactivity was quantitated by PhosphorImager analysis,
and the assigned PhosphorImager units (PIU) were adjusted by
subtracting the background radiation on the filter. PIUs for cell-free
supernatants from the duplicate 3201 infections were averaged. The same
procedure was used to assess the RT activity of the
FeLV/-galactosidase viral vectors that were generated in 293T cells
and used for subsequent single-cycle infection studies.
Metabolic labeling and RIPA of cellular lysates. Radioimmunoprecipitation analysis (RIPA) was performed with minor modification of a method described previously (8, 30). Cells were pulse-labeled for 2 h in methionine- and cysteine-deficient DMEM supplemented with 100 µCi of [35S]methionine-[35S]cysteine per ml (Trans-Label; ICN). After being labeled, the cells were chased for 3 h in cDMEM. The cells were then washed twice in cold phosphate-buffered saline and lysed in cold lysis buffer (25 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride [37]). Lysates were clarified by centrifugation at 15,000 × g at 4°C for 15 min followed by preclearing with protein A-Sepharose beads (Sigma) overnight with rocking at 4°C. Incorporated radioactivity was determined by precipitation with trichloroacetic acid. An equal number of trichloroacetic acid-precipitable counts were added to each RIPA reaction mixture.
Anti-SU monoclonal antibody C11D8 (16) and anti-TM monoclonal antibody PF6J-2A were obtained from Custom Monoclonals, Sacramento, Calif. Antibody was premixed with protein A-Sepharose beads for 1 h at 4°C. The beads were then washed three times in wash buffer (50 mM Tris [pH 7.2], 150 mM NaCl, 0.1% SDS, 0.1% Triton X-100). Cell lysates were added to antibody-protein A beads and incubated with rocking for 3 h at 4°C. The RIPA reaction mixtures were then washed five times with 1 ml of cold wash buffer on ice and resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. The eluted proteins were analyzed by SDS-PAGE (19) and fluorography.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Determinants of T-cell tropism, syncytium formation, and cytopathic
effects.
The 81T envelope gene variants were cloned from a thymic
tumor of a 61E-infected cat (40681). Although more than 90% of the envelope clones derived from the tumor encoded a predicted 4-amino-acid insertion, none of these clones were identical in sequence (31, 32). For example, we detected viruses that differed at one amino acid position within the 4-amino-acid insertion and viruses that differed at the very N terminus of the mature envelope SU. We chose
envelope clones encoding the two different insertions that were
otherwise representative of the consensus sequence of the 81T-tumor
derived clones (81T-3 [GESL] and 81T-6 [GESQ]) (Fig. 1) (31) to generate the
chimeric proviral constructs for this study. For reference, a schematic
of the 81T envelope variant (EET109E) that was recently
shown to confer a cytopathic, syncytium-inducing phenotype when
expressed in a 61E proviral context is shown (32). The FAIDS
variants EECC and the progenitor virus for the 81T variants, 61E, are
also included in Fig. 1.
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Y and Q R [Fig. 1]), these do not affect the cytopathic
phenotype of the viruses, further supporting a role for the insertion
in determining viral phenotype. These chimeras also induced syncytium
formation (data not shown), which is characteristic of 81T but not 61C
SU. This suggests that the C-terminal portion of 81T SU includes
determinants for syncytium formation. However, this does not exclude
the possibility that N-terminal sequences in 61C SU serve as cytopathic determinants.
Chimeras EE(ET3)E and EE(ET6)E (Fig. 1) were
constructed to determine whether the C-terminal portion of 81T SU is
sufficient to confer the cytopathic, T-tropic phenotype in the context
of a 61E virus. These chimeras were apparently replication defective, based on our inability to detect the production of viral
p27gag antigen in the supernatant of 3201 T
cells that had been transfected with these clones and subsequently
cultured for several weeks to allow virus spread. However, in one of
nine transfection experiments for each construct, infectious virus was
recovered after several weeks of cell passage. We examined the genotype
of the replicating virus in these cultures by amplifying and cloning
multiple envelope genes from the DNA of transfected cells and analyzing
the nucleotide sequence of these clones. We confirmed that the
predominant proviral genotype in these cells was derived from the
original transfected chimeras, since all sequences analyzed encoded the
predicted 81T insertion (data not shown).
To examine the replication properties of these tca variants,
EE(ET3)Etca and
EE(ET6)Etca, the virus recovered from the
transfected cells was used to infect naive 3201 T cells. The tca virus
EE(ET6)Etca replicated with kinetics like that
of 61E; replication of this virus did not affect cell viability or lead
to syncytium formation (Fig. 2).
Intriguingly, the other tca variant,
EE(ET3)Etca, was rapidly replicating,
cytopathic, and T tropic; this virus was also syncytium inducing (Fig.
2). Thus, tissue culture adaptation had led to the emergence of
81T-derived viruses with different cytopathic properties.
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Defining the determinants for adaptation in tissue culture.
To
examine the molecular basis for the biological differences in the tca
viruses, we examined the nucleotide sequence of multiple envelope gene
clones derived from each culture, including the region encoding both
the SU and the TM domains of the envelope. Interestingly, nucleotide
sequence analysis of the envelope gene clones showed that the adapted
variants had evolved several amino acid changes, including one that was
invariably detected in all clones examined.
EE(ET3)Etca evolved a predicted Q P change at position 7 of the mature envelope SU, near the N terminus in four of
four clones examined. This mutation is adjacent to a site where other
cytopathic clones such as 61C encode a proline change relative to the
noncytopathic clone, 61E (Fig. 1). EE(ET6)Etca evolved a predicted V L change at position 375 in the mature SU,
near the C terminus, and this mutation was found in 11 of 11 envelope
clones examined (data not shown). We hypothesized that these changes
evolved because they structurally compensate for changes in
conformation caused by the insertion.
PT3)E]
or a C-terminal change [EE(ET6,VL)E] (Fig. 1).
Unlike the original chimeras from which they evolved in culture, the
EE(EQPT3)E and
EE(ET6,VL)E mutants replicated in 3201 T cells (Fig. 3). Moreover, each virus exhibited the
characteristics of its uncloned tca counterpart,
EE(ET3)Etca and
EE(ET6)Etca, respectively. The
EE(EQPT3)E chimera encoding a Q P change
at amino acid 7 was cytopathic and syncytium inducing like
EE(ET3)Etca, whereas
EE(ET6,VL)E, which encodes a V L change at position 375, was noncytopathic like EE(ET6)Etca.
Infection of 3201 T cells with EE(ET6,VL)E did not
induce syncytium formation, similar to what has previously been
observed with 61E.
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L)E, while exhibiting similar
replication kinetics to 61E, had RT activity that increased more slowly
but to a higher peak than that of 61E. In contrast,
EE(EQPT3)E replicated more rapidly and to
higher levels than either EET109E, 61E, or clone
EE(ET6,VL)E.
Analyses of infectivity using a single-cycle replication
assay.
We also examined the infectivity of the viruses in this
study for feline fibroblast cells using a single-cycle infection assay. For this purpose, FeLV particles containing murine retroviral genomes
encoding -galactosidase were used and infection was monitored by
measuring the expression of the -galactosidase protein. Thus, this
assay requires that envelope be able to bind its cognate receptor and
initiate entry. To provide a quantitative measure of virus that is
independent of infectivity, we examined the RT activity of the viruses
(Table 1). Both the
EE(EQPT3)E and EE(ET6,VL)E
viruses were infectious for AH927 cells in this assay. In this regard,
they both resemble 61E. When normalized to RT, the relative infectivity
of EE(ET6,VL)E was approximately sevenfold lower than
that of EE(EQPT3)E, suggesting that the
ratio of infectious to noninfectious virus may be different for these
tca variants. Neither of these two mutant clones were able to infect
AH927 cells at levels comparable to the prototype FeLV, 61E, but both
were able to infect at levels similar to EET109E. Thus, the
changes in the tca viruses appear to affect both T-cell tropism and
infection in non-T-cell lines. Interestingly, both of the tca variants
were more infectious for AH927 cells than either EECC or the 61C/81T
envelope chimeras were, suggesting that the N-terminal 61C-derived
sequences may negatively affect replication in feline fibroblast cells.
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-galactosidase viral genomic marker upon exposure to the apparently replication-defective chimeras, EE(ET3)E and
EE(ET6)E (Table 1). This observation would suggest that
low levels of reverse transcription may occur as a result of a very low
level of infectious virus expressed from these proviral genomes. The
process of reverse transcription would allow the generation of
mutations that could have led to the production of the tca variants.
Certainly, any replication-competent mutants, when they occurred, would
be highly selected. However, because the levels of infectivity that we
observed with the EE(ET3)E and EE(ET6)E
chimeras were so low, it is difficult to extrapolate from these data to
argue that such rare infectious variants were the progenitor viruses
for the tca variants.
Envelope processing is affected by the insertion and restored by
the N-terminal and C-terminal changes.
The observation that
EE(ET3)E and EE(ET6)E viral chimeras were
replication defective suggested that the C-terminal insertions in 81T
SU may affect envelope structure or stability in the context of a 61E
SU. To examine this possibility, we performed RIPA of cells transfected
with these proviral clones. As shown in Fig. 4, the 61E and EET109E gp85
envelope precursor proteins were expressed at similar levels and were
processed to SU (gp70) and TM (p15E) (lanes 1 and 2). While the
EE(ET3)E and EE(ET6)E chimeras also expressed
high levels of gp85 in transfected cells, these envelope precursors
were not cleaved to detectable levels (lanes 4 and 6). Indeed, the
envelope-processing profile of these chimeras was almost identical to
that of the 61B envelope, which we have previously shown to be
defective for envelope processing (8) (lane 3). These
results indicate that the replication defect in the
EE(ET3)E and EE(ET6)E chimeras is due to a
defect in envelope processing.
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PT3)E and EE(ET6,VL)E shows that the single-amino-acid changes evolved in the tca viruses restore proper processing of the envelope protein. This can be most
clearly detected with the antibody against the envelope TM protein,
although a faint smear characteristic of heavily glycosylated gp70 can
also be detected with the anti-SU antibody (Fig. 4, lanes 5 and 7). Our
data suggest that other amino acid changes in the 81T and 61C envelopes
compensate for structural changes induced by the insertions in these
viruses. In support of this hypothesis, we found that
EE(CT3)E and EE(CT6)E, which contain the 81T-3
and 81T-6 insertions, respectively, in the context of a 61C envelope, exhibit proper envelope processing (lanes 8 and 9), and these viruses
are replication competent.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A small, 6-amino-acid insertion in the C-terminal half of the FeLV-FAIDS variant envelope protein was previously shown to be a critical determinant of its highly pathogenic, immunosuppressive phenotype (13, 26). Although this insertion appeared to be the result of an imperfect duplication of adjacent sequences, the precise progenitor of the FeLV-FAIDS variants is unknown (26). In contrast, the progenitor for the 81T variants is known because these envelope sequences were obtained from a cat infected with a virus derived from a molecularly cloned, transmissible form of FeLV, 61E (31). These 81T variants had acquired an insertion of unknown origin in the same position (position 352 in SU [31]) as in the 61C virus. Here we show that viruses encoding the C-terminal portion of SU of 81T, including the 4-amino-acid insertion, and the N-terminal portion of 61C SU are T-cell tropic and cytopathic. Of note, tumor-derived 81T variants encoding two different 4-amino-acid insertions (GESL and GESQ) were equally cytopathic when combined with the C terminus of 61C SU, suggesting that the single-amino-acid difference in the insertion does not affect T-cell tropism and cytopathicity. Moreover, our data suggest that the 4-amino-acid insertion(s) in 81T and the 6-amino-acid insertion in 61C are functionally interchangeable despite their difference in size and sequence. A chimera encoding a segment of the 81T envelope containing the insertion in a background of the 61C envelope [e.g., EE(CT3)C] was also syncytium inducing, which is a unique property of the 81T virus. The observation that 4- or 6-amino-acid insertions with very different sequences can function as determinants of T-cell tropism suggests that the inserted sequences may not be altering replication and cell tropism by a direct interaction with the cell surface receptor. Rather, these data are more consistent with a model in which the insertion has a conformational effect that affects receptor-binding determinants elsewhere on the envelope protein.
The domains of the FeLV envelope that are important for receptor specificity have been defined through analyses of viral envelope chimeras. To date, these studies have focused on subgroup B FeLVs because the receptor for this FeLV was the first to be identified (24, 35). These studies suggest that sequences in the N terminus of FeLV SU are important for receptor recognition (6, 34). This region of the envelope encompasses the variable region A and B (VRA and VRB) domains, which have also been described as receptor-binding domains for the related murine leukemia viruses (MuLVs) (2-5, 10, 11, 17, 21, 23, 25, 29). It is noteworthy that the insertion at position 352 that plays a key role in defining the replication properties of the T-tropic FeLV variants is C terminal to the described receptor recognition domains for FeLV-B and MuLV. These findings suggest that segments of envelope outside of VRA and VRB may also play an important role in determining cell tropism and, by extension, may play a role in determining receptor specificity.
The insertion and scattered nearby mutations in the SU of the 81T envelope impairs envelope protein processing within a background of the parental 61E envelope protein. The emergence of replication-competent tca viruses in cells expressing proviruses encoding chimeric 61E/81T SU proteins allowed us to identify two independent compensatory mutations that restored envelope protein processing, one at the very N terminus of SU and another near the C terminus of SU. Viruses encoding these changes were infectious in feline fibroblast cells, as judged by single-cycle infection assays. Interestingly, 61E/81T chimeric viruses encoding these amino acid changes displayed very distinct phenotypes in feline T cells. A virus with the C-terminal SU change coupled with the 4-amino-acid insertion replicated with similar kinetics to that of the avirulent 61E virus. Thus, the replication properties of this mutant virus suggest that the insertion alone cannot confer the T-tropic, cytopathic phenotype of 81T. We cannot determine from these studies whether differences in replication and T-cell tropism may directly impact the cytopathic properties of the virus. Interestingly, the N-terminal change, when coupled with the insertion, rendered the virus highly cytopathic and syncytium inducing in feline T cells. In addition, this virus was as infectious in feline fibroblast cells as a virus encoding the complete 81T SU (e.g., EET109E). We have previously provided evidence that the 81T variants are dually tropic; they exhibited interference properties that suggest that they use a receptor specific for the T-tropic 61C virus as well the more ubiquitously expressed FeLV-61E receptor, perhaps at reduced efficiency (32). The studies described here suggest that the N-terminal change, which makes the virus both T-cell tropic and able to infect fibroblast cells, may be a key determinant for the proposed dual-receptor specificity of the virus.
The N-terminal proline change that evolved in the tca virus clusters with similar mutations in the original 81T viruses isolated from the cat thymic tumor. Thus, mutations in this region of SU evolved independently in viruses selected for replication in an infected cat and in viruses selected for replication in feline T cells in culture. Moreover, 61C and other FeLV-FAIDS-derived variants also have amino acid differences relative to 61E in this very N-terminal portion of envelope SU. Proline is a particularly common amino acid, occurring at position 6 in the mature SU of several FeLV-FAIDS variants (27) and in some of the 81T variants (31, 32). FeLV-61E encodes a histidine at position 6. However, some of the 81T variants, including the 81T-109 virus previously shown to be T tropic, encode an aspartic acid at this position, suggesting that amino acid changes other than proline may also alter replication and processing in the context of certain envelope sequence. Interestingly, a histidine residue at position 8 of the MuLV SU affects the efficiency of receptor binding and/or postbinding events (1, 20, 38). Together, these data suggest that the very N terminus of the FeLV and MuLV envelopes may play a critical role in defining the replication properties of the virus.
Our studies suggest that sequence determinants of FeLV T-cell tropism, which include the N-terminal change and a C-terminal insertion, lie outside previously defined VRA and VRB receptor domains. Together, these data indicate that conformational changes outside of envelope domains that may not specifically contact the receptor could significantly influence receptor specificity. There have been several studies of envelope receptor interactions lacking N-terminal or C-terminal sequences outside of VRA and VRB (5, 10, 11, 17). Our studies suggest that such analyses, while important for identifying residues directly involved in binding, may not provide a broader view of the envelope-receptor interactions in the context of an infecting virus. Indeed, the crystal structure that has been solved for a fragment of MuLV SU does not include the C-terminal half of SU (14). Thus, it will be of interest to determine how an insertion in that portion of SU could influence envelope structure and subsequent viral tropism. The FeLV variants described here, and particularly the tissue culture-derived viruses exhibiting distinct replication properties, will allow us to further examine the role of sequences outside of VRA and VRB in cell tropism and receptor interactions.
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ACKNOWLEDGMENTS |
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We thank Maribeth Eiden for providing pRT43.2Tnls-gal-1 and
Maria Anderson for technical assistance and helpful discussions.
This work was supported by NIH grant CA 51080. F.C.H. is supported in part by NIH training grant 5 T32 RR07019. A.S.L. is supported in part by the Poncin Scholarship Fund.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Human Biology, Fred Hutchinson Cancer Center, 1100 Fairview Ave. N., C3-168, Seattle, WA 98109-1024. Phone: (206) 667-3524. Fax: (206) 667-1535. E-mail: joverbau{at}fhcrc.org.
This paper is dedicated to the memory of our friend and colleague,
Samuel Rudolph Gwynn.
Deceased.
§ Present address: Leadd BV, 2300 AA Leiden, The Netherlands.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, July 2000, p. 5754-5761, Vol. 74, No. 13
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