Department of Microbiology and Molecular
Genetics, New England Regional Primate Research Center, Harvard
Medical School, Southborough, Massachusetts 01772-9102
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.
 |
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.
View larger version (38K):
[in this window]
[in a new window]
|
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.
|
|
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.
| |
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.).
| |
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).
View larger version (34K):
[in this window]
[in a new window]
|
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.
|
|
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.
View larger version (24K):
[in this window]
[in a new window]
|
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.
|
|
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).
View larger version (30K):
[in this window]
[in a new window]
|
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.
|
|
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.
View larger version (24K):
[in this window]
[in a new window]
|
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.
|
|
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).
View larger version (26K):
[in this window]
[in a new window]
|
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.
|
|
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.
View larger version (57K):
[in this window]
[in a new window]
|
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).
View larger version (147K):
[in this window]
[in a new window]
|
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).
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.
| 1.
|
Alexander, S., and J. H. Elder.
1984.
Carbohydrate dramatically influences immune reactivity of antisera to viral glycoprotein antigens.
Science
226:1328-1330[Abstract/Free Full Text].
|
| 2.
|
Back, N. K.,
L. Smit,
J. J. De Jong,
W. Keulen,
M. Schutten,
J. Goudsmit, and M. Tersmette.
1994.
An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization.
Virology
199:431-438[CrossRef][Medline].
|
| 3.
|
Chackerian, B.,
L. M. Rudensey, and J. Overbaugh.
1997.
Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies.
J. Virol.
71:7719-7727[Abstract].
|
| 4.
|
Chan, D. C.,
D. Fass,
J. M. Berger, and P. S. Kim.
1997.
Core structure of gp41 from the HIV envelope glycoprotein.
Cell
89:263-273[CrossRef][Medline].
|
| 5.
|
Chan, D. C., and P. S. Kim.
1998.
HIV entry and its inhibition.
Cell
93:681-684[CrossRef][Medline].
|
| 6.
|
Cheng-Mayer, C.,
A. Brown,
J. Harouse,
P. A. Luciw, and A. J. Mayer.
1999.
Selection for neutralization resistance of the simian/human immunodeficiency virus SHIVSF33A variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify N-linked glycosylation.
J. Virol.
73:5294-5300[Abstract/Free Full Text].
|
| 7.
|
Dash, B.,
A. McIntosh,
W. Barrett, and R. Daniels.
1994.
Deletion of a single N-linked glycosylation site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion.
J. Gen. Virol.
75:1389-1397[Abstract/Free Full Text].
|
| 8.
|
Dedera, D. A.,
R. L. Gu, and L. Ratner.
1992.
Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.
Virology
187:377-382[CrossRef][Medline].
|
| 9.
|
Etemad-Moghadam, B.,
G. B. Karlsson,
M. Halloran,
Y. Sun,
D. Schenten,
M. Fernandes,
N. L. Letvin, and J. Sodroski.
1998.
Characterization of simian-human immunodeficiency virus envelope glycoprotein epitopes recognized by neutralizing antibodies from infected monkeys.
J. Virol.
72:8437-8445[Abstract/Free Full Text].
|
| 10.
|
Fenouillet, E.,
I. Jones,
B. Powell,
D. Schmitt,
M. P. Kieny, and J. C. Gluckman.
1993.
Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.
J. Virol.
67:150-160[Abstract/Free Full Text].
|
| 11.
|
Hunter, E.
1997.
gp41., A multifunctional protein involved in HIV entry and pathogenesis, p. III-55-III-73.
In
B. Korber, B. Hahn, B. Foley, J. W. Mellors, T. Leitner, G. Myers, F. McCutchan, and C. L. Kuiken (ed.), Human retroviruses and AIDS 1997: a compilation and analysis of nucleic acid and amino acid sequences. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 12.
|
Joag, S. V.
2000.
Primate models of AIDS.
Microbes Infect.
2:223-229[CrossRef][Medline].
|
| 13.
|
Karlsson, G. B.,
M. Halloran,
J. Li,
I. W. Park,
R. Gomila,
K. A. Reimann,
M. K. Axthelm,
S. A. Iliff,
N. L. Letvin, and J. Sodroski.
1997.
Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.
J. Virol.
71:4218-4225[Abstract].
|
| 14.
|
Korber, B.,
C. Brander,
B. F. Haynes,
J. P. Moore,
R. Koup,
B. D. Walker, and D. I. Watkins (ed.).
1999.
HIV molecular immunology database.
Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 15.
|
Kornfeld, R., and S. Kornfeld.
1985.
Assembly of asparagine-linked oligosaccharides.
Annu. Rev. Biochem.
54:631-664[CrossRef][Medline].
|
| 16.
|
Kuiken, C.,
B. Foley,
B. Hahn,
B. Korber,
F. McCutchan,
P. Marx,
J. Mellors,
J. Mullins,
J. Sodroski, and S. Wolinksy (ed.).
1999.
Human retroviruses and AIDS 1999: a compilation and analysis of nucleic acid and amino acid sequences.
Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N.Mex.
|
| 17.
|
Lee, W. R.,
X. F. Yu,
W. J. Syu,
M. Essex, and T. H. Lee.
1992.
Mutational analysis of conserved N-linked glycosylation sites of human immunodeficiency virus type 1 gp41.
J. Virol.
66:1799-1803[Abstract/Free Full Text].
|
| 18.
|
Leonard, C. K.,
M. W. Spellman,
L. Riddle,
R. J. Harris,
J. N. Thomas, and T. J. Gregory.
1990.
Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.
J. Biol. Chem.
265:10373-10382[Abstract/Free Full Text].
|
| 19.
|
Malashkevich, V. N.,
D. C. Chan,
C. T. Chutkowski, and P. S. Kim.
1998.
Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides.
Proc. Natl. Acad. Sci. USA
95:9134-9139[Abstract/Free Full Text].
|
| 20.
|
Means, R. E.,
T. Greenough, and R. C. Desrosiers.
1997.
Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus.
J. Virol.
71:7895-7902[Abstract].
|
| 21.
|
Mori, K.,
D. J. Ringler,
T. Kodama, and R. C. Desrosiers.
1992.
Complex determinants of macrophage tropism in env of simian immunodeficiency virus.
J. Virol.
66:2067-2075[Abstract/Free Full Text].
|
| 22.
|
Morrison, H. G.,
F. Kirchhoff, and R. C. Desrosiers.
1993.
Evidence for the cooperation of gp120 amino acids 322 and 448 in SIVmac entry.
Virology
195:167-174[CrossRef][Medline].
|
| 23.
|
Naidu, Y. M.,
H. W. de Kestler,
Y. Li,
C. V. Butler,
D. P. Silva,
D. K. Schmidt,
C. D. Troup,
P. K. Sehgal,
P. Sonigo,
M. D. Daniel, et al.
1988.
Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.
J. Virol.
62:4691-4696[Abstract/Free Full Text].
|
| 24.
|
Opdenakker, G.,
P. M. Rudd,
C. P. Ponting, and R. A. Dwek.
1993.
Concepts and principles of glycobiology.
FASEB J.
7:1330[Abstract].
|
| 25.
|
Overbaugh, J., and L. M. Rudensey.
1992.
Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques.
J. Virol.
66:5937-5948[Abstract/Free Full Text].
|
| 26.
|
Perrin, C.,
E. Fenouillet, and I. M. Jones.
1998.
Role of gp41 glycosylation sites in the biological activity of human immunodeficiency virus type 1 envelope glycoprotein.
Virology
242:338-345[CrossRef][Medline].
|
| 27.
|
Reimann, K. A.,
J. T. Li,
G. Voss,
C. Lekutis,
K. Tenner-Racz,
P. Racz,
W. Lin,
D. C. Montefiori,
D. E. Lee-Parritz,
Y. Lu,
R. G. Collman,
J. Sodroski, and N. L. Letvin.
1996.
An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.
J. Virol.
70:3198-3206[Abstract].
|
| 28.
|
Reitter, J. N., and R. C. Desrosiers.
1998.
Identification of replication-competent strains of simian immunodeficiency virus lacking multiple attachment sites for N-linked carbohydrates in variable regions 1 and 2 of the surface envelope protein.
J. Virol.
72:5399-5407[Abstract/Free Full Text].
|
| 29.
|
Reitter, J. N.,
R. E. Means, and R. C. Desrosiers.
1998.
A role for carbohydrates in immune evasion in AIDS.
Nat. Med.
4:679-684[CrossRef][Medline].
|
| 30.
|
Schonning, K.,
A. Bolmstedt,
J. Novotny,
O. S. Lund,
S. Olofsson, and J. E. Hansen.
1998.
Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120.
AIDS Res. Hum. Retroviruses
14:1451-1456[Medline].
|
| 31.
|
Schonning, K.,
B. Jansson,
S. Olofsson, and J. E. Hansen.
1996.
Rapid selection for an N-linked oligosaccharide by monoclonal antibodies directed against the V3 loop of human immunodeficiency virus type 1.
J. Gen. Virol.
77:753-758[Abstract/Free Full Text].
|
| 32.
|
Schonning, K.,
B. Jansson,
S. Olofsson,
J. O. Nielsen, and J. S. Hansen.
1996.
Resistance to V3-directed neutralization caused by an N-linked oligosaccharide depends on the quaternary structure of the HIV-1 envelope oligomer.
Virology
218:134-140[CrossRef][Medline].
|
| 33.
|
Trkola, A.,
T. Ketas,
V. N. Kewalramani,
F. Endorf,
J. M. Binley,
H. Katinger,
J. Robinson,
D. R. Littman, and J. P. Moore.
1998.
Neutralization sensitivity of human immunodeficiency virus type 1 primary isolates to antibodies and CD4-based reagents is independent of coreceptor usage.
J. Virol.
72:1876-1885[Abstract/Free Full Text].
|
| 34.
|
Vallejo, A. N.,
R. J. Pogulis, and L. R. Pease.
1995.
Mutagenesis and synthesis of novel recombinant genes using PCR, p. 603-612.
In
C. W. Dieffenbach, and G. S. Dveksler (ed.), PCR primer: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 35.
|
Weissenhorn, W.,
A. Dessen,
S. C. Harrison,
J. J. Skehel, and D. C. Wiley.
1997.
Atomic structure of the ectodomain from HIV-1 gp41.
Nature
387:426-430[CrossRef][Medline].
|
| 36.
|
Xu, J. Y.,
M. K. Gorny,
T. Palker,
S. Karwowska, and S. Zolla-Pazner.
1991.
Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using 10 human monoclonal antibodies.
J. Virol.
65:4832-4838[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
