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Journal of Virology, December 2001, p. 11426-11436, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11426-11436.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Conserved, N-Linked Carbohydrates of Human Immunodeficiency
Virus Type 1 gp41 Are Largely Dispensable for Viral
Replication
Welkin E.
Johnson,
Jennifer
M.
Sauvron, and
Ronald C.
Desrosiers*
Department of Microbiology and Molecular
Genetics, New England Regional Primate Research Center, Harvard
Medical School, Southborough, Massachusetts 01772-9102
Received 16 April 2001/Accepted 20 August 2001
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ABSTRACT |
The transmembrane subunit (TM) of human immunodeficiency virus type
1 (HIV-1) envelope protein contains four well-conserved sites for
the attachment of N-linked carbohydrates. To study the contribution of
these N-glycans to the function of TM, we systematically mutated the
sites individually and in all combinations and measured the effects of
each on viral replication in culture. The mutants were derived from
SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope
sequence that originated from a primary HIV-1 isolate. The attachment
site mutants were generated by replacing the asparagine codon of each
N-X-S/T motif with a glutamine codon. The mobilities of the variant
transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis suggested that all four sites are utilized for
carbohydrate attachment. Transfection of various cell lines with the
resulting panel of mutant viral constructs revealed that the N-glycan
attachment sites are largely dispensable for viral replication.
Fourteen of the 15 mutants were replication competent,
although the kinetics of replication varied depending on the mutant and
the cell type. The four single mutants (g1, g2, g3, and g4) and all six
double mutants (g12, g13, g14, g23, g24, and g34) replicated in both
human and rhesus monkey T-cell lines, as well as in primary rhesus
peripheral blood mononuclear cells. Three of the four triple
mutants (g124, g134, and g234) replicated in all cell types tested. The
triple mutant g123 replicated poorly in
immortalized rhesus monkey T cells (221 cells) and did not replicate
detectably in CEMx174 cells. However, at 3 weeks posttransfection of
221 cells, a variant of g123 emerged with a new N-glycan
attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental
virus. The quadruple mutant (g1234) did not replicate in any cell line
tested, and the g1234 envelope protein was nonfunctional in a
quantitative cell-cell fusion assay. The synthesis and processing of
the quadruple mutant envelope protein appeared similar in transient
assays to those of the parental SHIV-KB9 envelope. Given their high
degree of conservation, the four N-linked carbohydrate attachment sites
on the external domain of gp41 are surprisingly dispensable for
viral replication. The viral variants described in this report should
prove useful for investigation of the contribution of carbohydrate
moieties on gp41 to recognition by antibodies, shielding from
antibody-mediated neutralization, and structure-function relationships.
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INTRODUCTION |
Proteins containing amino acid
motifs of the type N-X-S/T are subject to cotranslational glycosylation
as they emerge from membrane-bound ribosomes (24). The
envelope precursor of human immunodeficiency virus (HIV) and simian
immunodeficiency virus (SIV), gp160, contains about 28 sites for
N-linked carbohydrate attachment. After translation and
oligomerization, gp160 is cleaved by a cellular protease to generate
the surface (SU) and transmembrane (TM) proteins. Approximately 24 sites for N-linked glycosylation are found in the HIV type 1 (HIV-1) SU
subunit (gp120), and glycosylation accounts for about 50% of this
protein's mass (18). The HIV-1 TM protein, gp41,
typically contains three or four sites for N-glycan attachment, located
within a short stretch (20 to 30 residues) of the C-terminal half of
the ectodomain (Fig. 1). There is a growing body of evidence demonstrating that N-linked glycosylation can
serve to modulate the exposure of the HIV and SIV gp120 proteins to
immune surveillance in patients or experimentally infected animals
(2, 3, 6, 25, 29-32). For example, Reitter et al.
demonstrated that SIVmac239 variants lacking specific N-linked carbohydrate attachment sites in gp120 were more sensitive to antibody-mediated neutralization and better elicitors of neutralizing antibody responses in rhesus monkeys (29). However,
effects of glycosylation on the antigenicity and immunogenicity of the gp41 subunit have not been reported.
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FIG. 1.
The TM proteins of HIV-1 envelopes contain three to four
conserved sites for N-linked glycosylation. (A) Alignment of 10 representative HIV-1 amino acid sequences. The alignment extends from
the conserved cysteine residues (boldface C) to the first two amino
acids of the predicted membrane-spanning domain (grey box). Boldface
capital letters at the beginning of each line indicate clades (E, B,
and D) of group M; also included are two isolates of group O (grpO).
Subscripts indicate the accession numbers for each sequence. Canonical
carbohydrate attachment sites are indicated by boldface, underlined
letters. (B) Amino acid sequence of the glycosylated region of the
virus used in this study, SHIV-KB9.
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The extracellular domain of gp41 consists of an amino-terminal fusion
domain, N- and C-terminal heptad repeats, a short disulfide loop, and a
tryptophan-rich domain (Fig. 1) (11). Sequential binding
of gp120 to receptor (CD4) and coreceptor triggers conformational changes that promote insertion of the hydrophobic N terminus of gp41
into the target cell membrane. The conformational changes cause the
gp41 oligomer to fold into a highly stable coiled-coil structure
(4, 19, 35). Formation of the coiled-coil structure probably serves to bring the viral and cellular membranes into close
proximity, allowing mixing of the lipid bilayers and release of the
viral nucleoprotein complex into the target cell cytoplasm (5). gp41 is also required for oligomerization of the
SU/TM complex and anchoring of the envelope complex in the viral
membrane (11).
Several reports have described the effects of individual substitutions
of the four conserved, N-linked glycosylation sites on the function of
gp41. However, the results are difficult to compare because the studies
differed in the choice of mutagenesis strategy, expression system, and
cell line(s) used (7, 8, 10, 17, 26). While some reports
claimed that specific attachment sites appear to be required for proper
gp41 expression (7, 10), others reported that mutations at
the same sites had only modest effects on protein function
(8). Only two studies reported the effects of such
mutations in the context of viral replication (8, 17), and
to our knowledge, there are no reports on the replication of viruses
lacking multiple N-linked glycans of gp41. In this study we have
mutated each of the four sites singly and in all possible combinations
and have tested the effects of the substitutions on viral replication
in immortalized T-cell lines and activated peripheral blood mononuclear
cells (PBMC). The mutations were constructed and analyzed in the
context of the chimeric virus SHIV-KB9, providing a useful tool for
studying the impact of the HIV-1 gp41 N-glycan cluster on replication
and pathogenesis in vivo.
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MATERIALS AND METHODS |
Site-directed mutagenesis and subcloning.
Plasmids
containing the 5' and 3' halves of the SHIV-89.6P molecular clone
SHIV-KB9, and the SHIV-KB9 envelope expression vector, were kindly
provided by B. Etemad-Moghadam and J. Sodroski (13).
SHIV-KB9 proviral nucleotides are numbered as found in the GenBank
database, accession no. U89134.
Individual N-linked glycosylation sites were mutagenized by the method
of PCR followed by splicing by overlap extension (SOE)
(
34), using as a template a plasmid containing the 3' half
of
the SHIV-KB9 provirus (from the
SphI site at position
5929 through
the 3' long terminal repeat [LTR] and including all of
env). For
SOE, two overlapping PCR products are generated,
with the desired
substitution incorporated into the region of overlap.
Then, in
a second round of PCR, the two overlapping products are mixed
together and fused by annealing and extension of the overlapping
ends.
Outside primers are included in the second round to amplify
the fusion
product. The result is a single PCR product containing
the mutagenized
site(s). Inclusion of relevant restriction sites
in the outside primers
facilitates subsequent
cloning.
Overlapping first-round products, containing the four single-site
mutations or the double substitution in sites 1 and 2, were
generated
by PCR. The overlapping fragments were then mixed and
matched to
construct full-length, SOE-fused fragments containing
each of the
single (g1, g2, g3, and g4) substitutions and one
double (g12)
substitution. DNA fragments generated by SOE were
then digested with
the restriction endonucleases
KpnI and
BamHI,
and
the resulting fragment was swapped into the SHIV-KB9 3'-half
plasmid
(
13). The remaining double, triple, and quadruple mutants
were also constructed by SOE, using the finished single and double
mutant constructs as templates for the first-round PCR. For transient
expression and fusion assays,
KpnI-
BamHI
fragments corresponding
to each mutant were also subcloned into a
SHIV-KB9 envelope expression
construct (
9).
The outside primers used for amplification were WEJ-1 (positions 6457 to 6477; 5' ATG GGG TAC CTG TGT GGA GAG 3') and R13
(9092 to 9071; 5'
CCA AGG ATC CGT TCA CTG ATG G 3'). The following
paired, overlapping
primers were used for mutagenesis (the target
site and position are
indicated in parentheses, and lowercase
letters specify the substituted
nucleotides): WEJ-5 (g1, 8149
to 8201), 5'CTT CTG TGC CTT GGc AaG TTA
GTT GGA GTA ATA AAT CTG
TGG ATG ATA TTT GG3'; WEJ-6 (g1, 8185 to 8136),
5' GAT TTA TTA
CTC CAA CTA ACt TgC CAAGGC ACA GAA GTG GTG CAA ATG AG3';
WEJ-7
(g2, 8166 to 8201), 5' GTT AGT TGG AGT cAa AAA TCT GTG GAT GAT
ATT TGG3'; WEJ-8 (g2, 8201 to 8152), 5' CCA AAT ATC ATC CAC AGA
TTT tTg
ACT CCA ACT AAC ATT CCA AGG CAC AG 3'; WEJ-9 (g3, 8187
to 8240), 5' GTG
GAT GAT ATT TGG AAT cAa ATG ACC TGG ATG GAG TGG
GAA AGA GAA ATT GAC 3';
WEJ-10 (g3, 8222 to 8176), 5' CTC CAT
CCA GGT CAT tTg ATT CCA AAT ATC
ATC CAC AGA TTT ATT AC 3'; WEJ-11
(g4, 8224 to 8271), 5' GGG AAA GAG
AAA TTG ACc AaT ACA CAG ACT
ATA TAT ATG ACT TAC TTG 3'; WEJ-12 (g4,
8264 to 8210), 5' GTC
ATA TAT ATA GTC TGT GTA tTg GTC AAT TTC TCT TTC
CCA CTC CAT CCA
GGT C 3'; WEJ-13 (g12, 8149 to 8201), 5' CTT CTG TGC
CTT GGc AaG
TTA GTT GGA GTc AaA AAT CTG TGG ATG ATA TTT GG 3'; and
WEJ-14
(g12, 8200 to 8143), 5' CAA ATA TCA TCC ACA GAT TTt TgA CTC CAA
CTA ACt TgC CAA GGC ACA GAA GTG GTG C 3'.
Viral replication and cell culture.
221 cells (an
immortalized rhesus monkey T-cell line), CEMx174 cells, and C8166-45
cells were maintained as described previously (21, 22).
Rhesus monkey PBMC were purified by Ficoll separation of blood taken
from specific-pathogen-free animals. Lymphocytes were activated by
mixing PBMC from multiple animals in the same flask for 2 to 3 days;
PBMC were then transferred to RPMI medium containing 10% fetal bovine
serum and 10% interleukin-2.
To assay viral replication and to generate viral stocks, 5' and 3'
viral clones (
13) were digested with
SphI and
XhoI (5'
clone) or
SphI and
NotI (3'
clone), and the digested DNA was ligated
overnight at 16°C with T4
DNA ligase to generate full-length proviral
DNA. Five micrograms of
ligated DNA was then used to transfect
221 cells, CEMx174 cells, or
C8166-45 cells by the DEAE-dextran
method (
23). The medium
was changed every 2 to 3 days, and viral
replication was measured by
monitoring the appearance of p27 in
the supernatant. The p27
concentration was determined by antigen
capture assay (Coulter
Corporation, Hialeah, Fla.). For infections,
medium was cleared of
cells and debris by centrifugation, and
aliquots containing 10 ng of
p27 were used to inoculate 5 million
pelleted cells. After 24 h,
cells were pelleted, rinsed, and placed
in fresh
medium.
Env expression, immunoblots, and fusion assays.
Envelope
expression constructs were constructed by replacing the
KpnI-BamHI fragment of a SHIV-KB9 envelope
expression construct (9) with the corresponding fragment
from each of the mutant constructs. Expression was driven by the viral
LTR and was dependent on coexpression of the tat and
rev genes. For transient expression, 293T cells or COS-7
cells were transfected with 5 to 10 µg of calcium
phosphate-precipitated DNA using the Profection Mammalian Transfection
kit (Promega, Madison, Wis.).
For Western blotting, transfected 293T cells were rinsed in
phosphate-buffered saline and lysed in radioimmunoprecipitation
assay
buffer. Lysates were boiled in sample buffer, separated
by
electrophoresis, and transferred to membranes. The membranes
were then
treated sequentially with primary antibody (either anti-gp120
or
anti-gp41) and secondary, horseradish peroxidase (HRP)-conjugated
antibody and visualized using a chemiluminescent HRP detection
system
(Amersham). Primary antibodies were AD3 (anti-gp120) and
2F5
(anti-gp41) (National Institutes of Health AIDS Research and
Reference
Reagent
Program).
To assay levels of gp120 shedding by transfected cells, gp120
concentrations were determined using an HIV-1 gp120 antigen
capture
enzyme-linked immunosorbent assay (Advanced Biotechnologies,
Inc.,
Columbia, Md.). gp120 levels in cell-free supernatants and
in
whole-cell lysates were determined; the level of gp120 in supernatants
was calculated as a percentage of total gp120 (supernatant plus
cell
lysate) for each transfection. A construct expressing a secreted,
soluble form of the HxBC2 gp120 protein was included as a positive
control. The HxBC2 plasmid was a gift of S. Basmaciogullari and
J.
Sodroski.
The fusogenicity of various mutant envelopes was tested by cell-cell
fusion assay. Briefly, target cells (293T cells) were
seeded into
48-well plates, incubated overnight, and cotransfected
with plasmids
expressing Tat and either the parental or mutant
Env proteins. At 18 to
24 h posttransfection the medium was removed,
the cells were
rinsed in phosphate-buffered saline, and fresh
medium was added. At
48 h posttransfection the target cells were
overlaid with effector
cells. Effector cells expressed the CD4
receptor and an appropriate
coreceptor and contained a Tat-inducible
gene for secreted alkaline
phosphatase (SEAP). In the event of
Env-mediated fusion, the two cell
types should fuse, allowing
the Tat protein produced in the target
cells to access and transactivate
the SEAP reporter gene provided by
the effector cells. Effector
cells were either CEMx174 cells or
C8166-45 cells engineered to
contain the SEAP reporter
(
20). Fusogenicity was measured as
the accumulation of
SEAP activity in the supernatant over time.
SEAP activity was assayed
using the Phospha-Light Assay system
(Tropix, Bedford, Mass.).
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RESULTS |
Conserved N-linked glycosylation sites of HIV-1 gp41.
Sequence
alignments of the HIV-1 envelope protein revealed the presence of four
well-conserved sites for the attachment of N-linked carbohydrates
clustered within a 30-amino-acid stretch of the gp41 ectodomain (Fig.
1A). Related lentiviruses such as the SIVs typically have three or four
sites for N-glycan attachment in this same region (16).
Many of the nonprimate lentiviruses also have attachment sites in the
analogous portions of their TM proteins. Using site-directed
mutagenesis, we have replaced the asparagine residue of each of the
conserved HIV-1 gp41 carbohydrate attachment sites with a glutamine
(N-X-S/T 
Q-X-S/T). There are four such sites in the gp41 ectodomain
of SHIV-KB9; these sites were replaced singly and in combinations to
generate all 15 possible variants. Substitutions were made by altering
the first and third positions of each asparagine codon to create a
codon for glutamine (AAT CAA). Glutamine was chosen because it is
structurally similar to asparagine, differing by only a single
methylene group. Moreover, a minimum of two nucleotide changes are
necessary for the altered sequence to revert to any codon specifying
asparagine, thereby reducing the likelihood of direct reversion of the
targeted codon during viral replication experiments. Mutants are
indicated by a lowercase letter g followed by numerals designating
which of the four attachment sites have been modified in that
particular variant. For example, g123 refers to the triple mutant
lacking the first, second, and third attachment sites of the gp41
protein (Fig. 1 and Table 1).
PCR fragments containing each mutation were generated using mutagenic
primers and the method of SOE (see Materials and Methods
for details);
the template for PCR was a plasmid containing the
3' half of the
SHIV-KB9 genome (
13). Once the single mutants
were
generated, each combination mutant was also generated by
SOE, using the
single mutant clones as templates for the first
round of PCR.
KpnI-
BamHI fragments containing the substitutions
were then used to replace the corresponding fragment in the parental
plasmid. All mutant constructs were sequenced on both strands
to
confirm the absence of off-site mutations before being used
for viral
replication
experiments.
Contribution of gp41 glycosylation to viral replication.
To
assess the effects of the substitutions on viral replication, cloned
viral DNAs containing each of the mutant envelope genes were
transfected into CEMx174 cells, immortalized rhesus monkey T cells (221 cells), and, in some cases, the human T-cell line C8166-45.
Transfection of 14 of the 15 mutants gave rise to replication-competent
virus (Table 1), although the kinetics of replication varied
considerably depending on the variant and the cell line. The
replication-competent variants included all four of the single mutants
(g1, g2, g3, and g4), the six double mutants (g12, g13, g14, g23, g24,
and g34), and three of the four triple mutants (g123, g124, g134, and g234).
Results of representative transfection experiments are shown in Fig.
2. For these experiments, 221 cells (a
rhesus monkey
T-cell line) were transfected in parallel with each of
the 15
mutants and the parental virus, SHIV-KB9. Replication of 13 of
the 15 mutants produced peak yields within several days of the
parental
virus (as measured by p27 production in the supernatant)
(Fig.
2), and
a similar result was obtained with transfections
of CEMx174 cells (data
not shown). However, replication of the
triple mutant g123 was severely
reduced compared to that of the
parental virus (Fig.
2D). The most
severe defect in replication
was exhibited by the g1234 mutant, which
did not give rise to
detectable virus production in repeated
transfections (Fig.
2D
and E). To rule out the possibility of
unintended, inactivating,
off-site mutations, the entire g1234
construct was regenerated
twice, sequence confirmed, and retested by
transfection. No replication
was detected with either of the
independent constructions upon
multiple transfections with each (data
not shown).
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FIG. 2.
Replication of glycosylation mutants in immortalized
rhesus monkey T cells (221 cells) following transfection. Full-length
viral DNAs containing the indicated substitutions were used to
transfect 221 cells, and viral replication was monitored by the
appearance of the p27 capsid antigen in the tissue culture supernatant.
(A) Replication of SHIV-KB9 and the four single-site mutants. (B)
Replication of four double mutants. (C) Replication of the g124 and
g134 triple mutants. (D) Replication of g123, g234, and the quadruple
mutant g1234. (E) Replication of various N-glycan attachment mutants,
including g13 and g14. (F) Illustration depicting the glycosylation
status of the four gp41 N-linked glycosylation sites in SHIV-KB9 and
the 15 mutants used in this study.
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Viral p27 antigen was detected in the supernatant of the culture
transfected with the g123 triple mutant DNA approximately
20 days after
transfection of 221 cells (Fig.
3). This
is in sharp
contrast to the case for the other three triple mutants,
which
peaked at around day 9 posttransfection (Fig.
2). When
virus-containing
supernatant harvested on day 27 was used to infect
fresh 221 cells,
viral replication kinetics were similar to those of
the parental
virus (Fig.
3B). This result was consistent with
reversion of
one or more of the substituted N-X-S/T motifs in the g123
variant
(designated g123*) or with the acquisition of
compensatory changes.
Sequencing of viral DNA amplified from
infected 221 cells revealed
a single G
A substitution in the
gp41 ectodomain of g123, resulting
in a serine-to-asparagine change at
position 611 (Fig.
3C). The
newly acquired asparagine replaced
the serine residue of the first
conserved attachment site and resulted
in the creation of a new
N-X-S/T glycosylation motif overlapping the
first of the four
conserved attachment sites. Since the appearance of
compensatory
changes is likely to be dependent upon errors made during
viral
replication, this result indicates that the original g123 mutant
was replicating in the transfected 221 cells, albeit at or below
the
limit of detection of the p27 antigen capture assay. Transient
expression and Western blotting confirm that the new N-X-S/T motif
in
the g123* protein is utilized for N-glycan attachment (see
below).
Sequence analysis was also performed on the g12 mutant,
which showed a
slight delay in peak replication. In the case of
the g12 double mutant,
the original substitutions were retained
and there were no
further coding changes in the gp41 ectodomain
sequence.
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FIG. 3.
Compensatory change in g123. (A) Replication of g123
after transfection of 221 cells. (B) 221 cells infected with
supernatant harvested 24 days after transfection of mutant g123 (g123*)
or with supernatant containing the parental virus (SHIV-KB9). (C) Top,
sequence of the original g123 construct in the region of sites 1 and 2. Bottom, corresponding sequence of g123*, a variant of g123 which
emerged at 24 days posttransfection. Dashed-line boxes indicate the
substituted first and second sites in g123. The solid-line box
indicates the new attachment site resulting from a single G A
nucleotide substitution.
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Altogether, replication of each attachment site mutant was tested
in a minimum of three independent transfections: two transfections
of
rhesus 221 cells and one transfection of CEMx174 cells. In
addition, certain mutants were also tested for replication
in
C8166-45 cells (Table
1). Interestingly, the replication deficiency
of mutant g123 was not apparent on C8166-45 cells (Table
1).
Collectively, results from transfection experiments did not
indicate
that any individual attachment site(s) was absolutely
required
for viral replication, although mutants with
substitutions in
sites 1 and 2 were delayed (g12, g124, and g123) or
defective
(g1234) for replication in multiple experimental tests (Table
1 and Fig.
2).
The infectious titer of viral stocks was measured on
CEMx174SIV-SEAP and C8166-45SIV-SEAP indicator cells. These cell lines
have been engineered to contain a gene for SEAP under the control
of
the SIV LTR promoter, which is activated by the SIV or HIV-1
Tat
proteins (
20). SEAP expression in the supernatant requires
successful completion of the early stages of infection, including
entry, reverse transcription, integration, and expression of the
viral
Tat protein. Viral stocks were normalized to contain the
equivalent
concentrations of p27 capsid antigen; stocks were then
diluted and
titers were determined on CEMx174SIV-SEAP and C8166-45SIV-SEAP
cells
(Fig.
4). SEAP activity was measured at
72 h postinfection
to ensure that the results represented
the initial infection events,
prior to significant spread through
the culture (
20).
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FIG. 4.
Infectious titers of attachment site mutants. Stocks of
each virus were generated by transfection and normalized for p27
content. CEMx174SIV-SEAP (A) or C8166-45SIV-SEAP (B) indicator cells
were infected with normalized amounts of each variant, and Tat-induced
SEAP expression was measured on day 4 postinfection.
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The C8166-45SIV-SEAP indicator cells were highly sensitive to infection
by the parental virus as well as most of the mutant
derivatives; the
parental, g1, g12, g23, and g34 viruses showed
little or no decrease in
SEAP induction over the entire range
of dilutions (12.5 to 0.2 ng of
p27). The g2, g4, and g134 variants
showed slight decreases at
higher dilutions, and the only dramatic
effect of the gp41 mutations on
titer was evidenced by the g124
and g234 triple mutants (Fig.
4).
When CEMx174SIV-SEAP indicator
cells were used as target
cells, differences in infectious titer
were more apparent even at the
lowest dilution (12.5 ng of p27).
Although the two cell lines differed
in overall sensitivity to
infection, the rank orders of infectivities
were similar. The
parental virus and single-site mutants were among the
most infectious,
and the triple mutants were the least infectious.
However, some
variants, such as g12, did vary considerably in terms of
relative
infectivity (Fig.
4).
We also used normalized aliquots of each virus to infect pooled,
activated rhesus monkey PBMC. Although none of the attachment
site
mutants replicated as well as the parental virus in PBMC,
the single
mutants and most of the double mutants replicated with
only a slight
delay (Fig.
5 and data not shown). g12,
g34, g124,
and g134 did not replicate as well as the other mutants on
PBMC,
with peak p27 levels of less than 100 ng/ml of supernatant. The
results of both transfection and infection experiments indicate
that no
single attachment site is absolutely required for viral
replication.
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FIG. 5.
Replication of attachment site mutants in activated
rhesus monkey PBMC. Approximately 5 million cells were infected with
supernatant containing the indicated mutants after normalization for
p27 content. Replication was monitored for 2 weeks by p27 antigen
capture assay.
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Expression and processing of glycosylation-deficient envelope
proteins.
The effects of attachment site substitutions within the
N-glycan cluster of gp41 on viral replication ranged from subtle (g1, g2, g3, and g4) to extreme (g123 and g1234). To analyze potential defects in expression or processing of the envelopes as possible causes
for these differences, we cloned the mutant genes into an LTR-driven,
tat- and rev-dependent expression vector.
Expression constructs were then transfected into 293T cells, the cells
were lysed at various time points, and the mutant proteins were
visualized by Western blotting (Fig. 6).
Expression and processing of the quadruple mutant protein (g1234),
which is derived from a replication-defective variant, did not differ
noticeably from expression and processing of the parental envelope
protein or the g24 and g234 mutant proteins (Fig. 6).
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FIG. 6.
Expression and processing of attachment site mutants.
293T cells transfected with constructs expressing the parental (KB9)
and g24, g234, and g1234 mutant proteins were lysed at 48, 72, and
96 h posttransfection, proteins were separated by SDS-PAGE, and
envelope proteins were visualized by Western blotting with an
anti-gp120 antibody (AD3) and an appropriate HRP-conjugated secondary
antibody.
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To ascertain whether substitution of the N-glycan attachment sites
affected the stability of the gp120-gp41 association, we
investigated
whether removal of the gp41 N-glycans led to an increase
in shedding of
gp120 from the surface of transfected cells. 293T
cells and COS-7 cells
were transfected with the constructs expressing
the parental (SHIV-KB9)
envelope, as well as the g134, g234, and
g1234 combination mutant
proteins. Antigen capture enzyme-linked
immunosorbent assay was used to
quantitate the total gp120 protein
in the transfected-cell supernatants
and in whole-cell lysates
at 48, 72, and 96 h posttransfection. As
a positive control, cells
were also transfected with a construct
expressing a soluble, secreted
form of HIV-1 gp120. gp120 in cell-free
supernatant was then calculated
as a percentage of the total
(supernatant plus whole-cell lysate)
gp120 production for each envelope
variant (Table
2). The two
triple mutants
displayed a slight increase in gp120 shedding compared
to the parental
envelope protein (less than twofold), while the
g1234 quadruple mutant
exhibited significantly increased levels
of shedding (Table
2).
To confirm that each of the four predicted glycosylation sites of
SHIV-KB9 is utilized for carbohydrate attachment, a subset
of the
mutant envelope genes was expressed by transient
transfection
of 293T cells. Proteins in transfected-cell lysates were
separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
and analyzed by Western blotting. Migration of the altered
gp41
proteins was visualized by reaction with a gp41-specific
monoclonal
antibody followed by incubation with an appropriate
HRP-conjugated
secondary antibody (see Materials and
Methods).
On average, glycosylation of a single site is predicted to contribute
approximately 2 to 3 kDa to the apparent molecular mass
of a protein
(
15). Comparison of the migration patterns of several
of
the mutant proteins reveals that each of the four sites appears
to be
utilized for N-linked carbohydrate attachment (Fig.
7).
For example, all four triple mutant
proteins (g123, g124, g134,
and g234) migrate at the same
position, indicating that each of
the four sites (4, 3, 2, and 1, respectively) can be utilized
for N-linked glycosylation. The
fastest-migrating species is the
quadruple mutant g1234, at a
position consistent with the loss
of one additional site
compared to any of the four triple mutant
proteins. Moreover,
the difference in migration between the g24
and g124 proteins
is equivalent to the expected contribution of
a single
N-glycan.
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|
FIG. 7.
Expression of gp41 attachment site mutants. Whole-cell
lysates from 293T cells transfected with expression constructs were
separated by SDS-PAGE and visualized by Western blotting. Proteins were
visualized by probing with a monoclonal antibody to gp41 (2F5) followed
with an HRP-conjugated secondary antibody. The mobility shifts of the
double mutant (g24), each of the four triple mutants (g123, g124, g134,
and g234), and the quadruple mutant (g1234) relative to the parental
protein indicate that each of the canonical attachment sites is
utilized for N-linked glycosylation. The slower migration of g123* is
consistent with the acquisition of a new attachment site (compare lanes
3 and 4).
|
|
g123* (Fig.
7, lane 4) is the envelope protein of the variant
that arose in cells originally transfected with the g123 viral
clone (see above). The g123* gp41 sequence contains a new N-X-S/T
glycosylation motif in addition to the unaltered fourth site and
thus
has the potential to be glycosylated at two positions (Fig.
3). The
protein containing the new site migrates slower than the
original g123
mutant protein but at the same position as the g24
double mutant (which
contains two attachment sites), indicating
that the newly acquired
motif is in fact utilized for N-linked
carbohydrate attachment (Fig.
7,
lanes 2 and
4).
Fusogenicity of carbohydrate attachment site mutants.
To assay
fusogenicity, mutant envelope proteins and the HIV-1 Tat protein were
coexpressed in target cells by transient transfection. At 48 h
posttransfection, the target cells were mixed with effector cells. The
effector cells express the viral receptor and coreceptor and contain an
integrated, Tat-inducible SEAP reporter gene (20). In the
event of Env-mediated fusion between target and effector cells, the
contents of the two cell types mix and Tat protein produced by the
target cells induces expression of the SEAP reporter gene. Fusion can
then be visualized as the formation of syncytia and can also be
measured as the accumulation of SEAP activity in the culture
supernatant over time. Vectors expressing the parental, g24, g123,
g124, g234, and g1234 envelope proteins were transfected into 293T
target cells. CEMx174-SEAP and C8166-45SEAP cells, both of which
support entry and replication of HIV-1 and SIV, were used as effector
cells (20).
Formation of syncytia between the transfected target cells and
CEMx174SEAP effector cells (Fig.
8) or
C8166-45 cells (not
shown) was observed using light microscopy.
Expression of the
parental envelope protein and the g24 protein gave
rise to observable
syncytium formation by 9 h after mixing of the
two cell types,
whereas there were no apparent syncytia in assays
performed with
the g1234 quadruple mutant, even at the 30-h time point
(Fig.
8).
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|
FIG. 8.
Syncytium formation by SHIV-KB9 envelope and selected
gp41 N-linked glycosylation mutants. The fusion assay was performed as
described in the legend to Fig. 7. Representative fields were
photographed at 30 min, 9 h, and 30 h after mixing of target
and effector cells. Top row, SHIV-KB9 envelope. Middle row, g24
envelope. Bottom row, g1234 envelope.
|
|
Fusion-induced SEAP activity was also used to compare the activities of
the parental and mutant envelope proteins. As can
be seen in Fig.
9, the g123 envelope can mediate fusion
with either
effector cell type, despite being derived from a
replication-deficient
virus. Only the g1234 quadruple mutant exhibited
no detectable
fusogenic activity, even though the protein was expressed
and
processed to significant levels after transient transfection of
293T target cells (Fig.
6). Tat protein alone did not induce detectable
SEAP expression, indicating that envelope expression is required
for
mixing of the contents of the two cell types (Fig.
8). The
Tat-only
control also rules out the possibility that soluble,
secreted Tat
interferes with the assay.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 9.
Fusogenicity of attachment site mutant envelopes.
Effector cells were transfected with expression constructs and mixed
with target cells at 48 h posttransfection. In the event of
Env-mediated fusion, Tat protein from the effector cell induces SEAP
expression from the target cell. Target cells are indicated in the
upper left corner of each graph.
|
|
The envelope gene in SHIV-89.6, the progenitor of SHIV-KB9, was
originally derived from a dual-tropic HIV-1 isolate (
27).
To test the receptor specificity of the envelope proteins, the
fusion
assay was repeated using GHOST cells as the effector cells.
GHOST cells
are a panel of cell lines expressing CD4 in the context
of different
coreceptors; additionally, each GHOST cell line contains
an LTR-driven,
Tat-inducible gene for green fluorescent protein
(
33).
Successful fusion between an Env-expressing target cell
and a
GHOST effector cell is indicated by the formation of large,
green
syncytia. Using GHOST cells as effectors in the cell-cell
fusion
assay, we found that the parental SHIV-KB9 envelope, as
well as the g24
double mutant and the g123 and g234 triple mutant
envelopes, can
mediate cell-cell fusion using either CCR5 or CXCR4
as a coreceptor
(data not shown). As with the CEMx174SEAP and
C8166-45SEAP effector
cells, expression of the g1234 protein did
not result in detectable
fusion with GHOST cells bearing either
coreceptor (data not
shown).
| |
DISCUSSION |
Studies utilizing SIV/HIV chimeric viruses (SHIV) combine the
experimental advantages of a well-documented animal model for AIDS with
access to a wealth of available HIV-1 reagents, including antibodies of
defined specificity, banked sera, standardized assays, and established
cell lines. Because one of the long-term aims of this work is to assess
the potential effects of gp41 glycosylation on immunological control of
the virus in vivo, the mutations described were engineered in the
chimeric virus SHIV-KB9.
The ectodomain of the HIV-1 gp41 protein contains four canonical
recognition sequences for the attachment of N-linked carbohydrates (16). The effects of single-site substitutions on protein
expression and function have been reported (7, 8, 10, 17,
26), but only two of these studies looked at the role of
individual N-glycans in viral replication (8, 17). In
order to more thoroughly assess the role of the gp41 N-glycan cluster
in viral replication, we constructed all possible multiply substituted variants and tested the effects of these substitutions on viral replication by transfection or infection of T-cell lines and primary rhesus monkey PBMC. Surprisingly, only 2 of the 15 possible attachment site mutants were severely deficient for viral replication (g123 and
g1234). Each of the individual sites is dispensable for viral replication, although all sites appear to contribute to optimal replication efficiency. In general, the more completely glycosylated variants replicated better than those lacking multiple sites, with the
triple mutants and the quadruple mutants displaying the greatest
deficiency in viral replication. The same trend was seen when viral
infectivity was measured on CEMx174SIV-SEAP indicator cells, suggesting
that infectivity differences caused by combined substitution of
multiple sites are a contributing factor to the differences in
replication kinetics seen in viral replication assays.
The differences in replication, and the severe defects in replication
displayed by some mutants, are not the result of gross defects in
expression or processing of the envelope complex. Although cell surface
expression was not addressed directly in this study, the positive
results in fusion assays indicate that sufficient levels of the g123
envelope protein were expressed on the cell surface to promote
cell-cell fusion. These experiments did not rule out subtle differences
in the kinetics of expression and processing of the mutant proteins.
However, such differences, if they exist, are not likely to explain the
severity of the replication defect found in the g123- and
g1234-containing viruses.
In addition to promoting membrane fusion, gp41 is also necessary for
oligomerization of the envelope complex (11). It is possible that the various mutants tested here have less-than-optimal formation of functional envelope oligomers, with the most severe defect
demonstrated by the g123 and g1234 mutants. A decrease in functional
multimers could explain the general decrease in replication, if indeed
glycosylation affects oligomerization. Once envelope complexes reach
the cell surface, they also have to be incorporated into emerging
virions. A defect in virion incorporation would not be detected in the
cell-cell fusion assay. The experiments reported here do not address
the possibilities that removal of N-linked attachment sites from gp41
can affect transport of envelope protein to the cell surface,
incorporation of envelope complexes into virions, and/or gp41 oligomer
stability. It remains possible that the g1234 mutant, which was not
fusogenic in cell-cell fusion assays, may have a defect in the
stability of the gp120-gp41 heterodimer (Table 2). Whether the degree
of shedding seen with g1234 (three- to fourfold higher than that of the
wild type) is sufficient to explain the defect in replication is not known.
Previous work in this laboratory has demonstrated that multiple
N-linked carbohydrate attachment sites can be removed from the gp120
subunit of SIV, with little or no apparent effect on viral replication
(28). Moreover, work done with SIV by our laboratory
(29) and others (25), as well as with HIV-1
(1, 2, 31) and SHIV (6), has demonstrated a
role for N-linked glycans in limiting recognition of gp120 protein
sequences by antiviral antibodies. As far as we know, these types of
observations have not been extended to include the N-glycan cluster of
gp41. Interestingly, the regions of gp41 just N terminal and C terminal to the N-glycan cluster are highly antigenic, and numerous antibodies and polyclonal antisera directed to epitopes in these regions have been
identified and characterized (14). In fact, the vast majority of antibodies against gp41 can be grouped into one of two
clusters, depending on whether they recognize epitopes just N terminal
to the N-glycans (cluster I) or just C terminal to the N-glycans
(cluster II) (36). One possible explanation for the
paucity of known antibodies to the region separating the two highly
antigenic clusters is the presence of these four conserved N-glycans.
The attachment site mutants of SHIV-KB9 described in this study will
provide a useful framework for testing this possibility in vivo, using
the well-characterized rhesus monkey model for AIDS pathogenesis
(12).
| |
ACKNOWLEDGMENTS |
W.E.J. is supported by an Elizabeth Glaser Pediatric AIDS
Foundation 2-Year Scholar Award, grant PF-77385. This work was also supported by Public Health Service grants AI25328, AI35365, and RR00168.
We thank B. Etemad-Moghadam, S. Basmaciogullari, and J. Sodroski for
providing SHIV-KB9 constructs and the HxBC2 gp120 plasmid.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: New England
Regional Primate Research Center, One Pine Hill Dr., Box 9102, Southborough, MA 01772-9102. Phone: (508) 624-8002. Fax: (508)
460-0612. E-mail: ronald_desrosiers{at}hms.harvard.edu.
| |
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Abstract
Introduction
