Effect of Point Mutations in the N Terminus of the Lentivirus
Lytic Peptide-1 Sequence of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41 on Env Stability*
Sheau-Fen
Lee
,
Chiung-Yuan
Ko,
Chin-Tien
Wang§, and
Steve S.-L.
Chen¶
From the Division of Infectious Diseases, Institute
of Biomedical Sciences, Academia Sinica, Taipei 11529, § Institute of Clinical Medicine, National Yang-Ming
University, School of Medicine, and Department of Medical Research
and Education, Taipei Veterans General Hospital,
Taipei 11217, Taiwan, Republic of China
Received for publication, February 13, 2002
 |
ABSTRACT |
To understand the role of the lentivirus lytic
peptide-1 region of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp) 41 in viral infection, we examined the
effects on virus replication of single amino acid deletions spanning
this region in an infectious provirus of the HXB2 strain. Among the
mutants analyzed, only the deletion of one of the two adjacent valine
residues located at positions 832 and 833 (termed the 
833 mutant for
simplicity) greatly reduced the steady-state, cell-associated levels of
the Env precursor and gp120, as opposed to the wild-type virus. The altered Env phenotype resulted in severely impaired virus infectivity and gp120 incorporation into this mutant virion. Analyses of additional mutants with deletions at Ile-830, Ala-836, and Ile-840 demonstrated that the 830 mutant exhibited the most significant inhibitory effect
on Env steady-state expression. These results indicate that the N
terminus of the lentivirus lytic peptide-1 region is critical for Env
steady-state expression. Among the mutant viruses encoding Env proteins
in which residues Val-832 and Val-833 were individually substituted by
nonconserved amino acids Ala, Ser, or Pro, which were expected to
disrupt the 
-helical structure in the increasingly severe manner of
Pro > Ser > Ala, only the 833P mutant exhibited
significantly reduced steady-state Env expression. Pulse labeling and
pulse-chase studies demonstrated that the 830, 833, and 833P
mutants of Env proteins degraded more rapidly in a
time-dependent manner after biosynthesis than did the
wild-type Env. The results indicate that residue 830 and 833 mutations
are likely to induce a conformational change in Env that targets the mutant protein for cellular degradation. Our study has implications about the structural determinants located at the N terminus of the
lentivirus lytic peptide-1 sequence of gp41 that affect the fate of Env
in virus-infected cells.
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INTRODUCTION |
The cytoplasmic domain of the human immunodeficiency virus, type 1 (HIV-1),1 transmembrane (TM)
protein gp41 has been implicated in several steps of the virus life
cycle. The viral replication functions that are affected by large
truncations, deletions, or mutations in this region include virus
replication, infectivity, transmission, cytopathogenicity (1-8), viral
uncoating, or penetration of the viral core into host cells (6), Env
incorporation into virions (5, 7-10), and interactions with the viral
matrix protein MA during virus assembly (11-16). In addition, the
cytoplasmic tail has a role in regulation of Env expression on the cell
surface (17, 18), basolateral targeting of virus budding in polarized cells (19, 20), and in interactions with cellular components such as
induction of apoptosis (21, 22) and binding to the medium chain µ1
and µ2 subunits of the AP-2 and AP-1 clathrin-associated adaptor
complexes (23, 24).
Although the gp41 cytoplasmic tail does not exhibit typical
membrane-binding sequences, the HIV-1 isolates show remarkable conservation of amphipathic -helical secondary structures. The segments 828-856, 770-795, and 786-812, referred to as lentivirus lytic peptides (LLP)-1, LLP-2, and LLP-3, respectively, all exhibit features of an amphipathic -helix (25-27). Peptides mimicking these
sequences show strong interactions with membranes, resulting in
perturbation of membrane permeability and increased bilayer conductance
(28-33). The -helical motifs of LLP-1 and LLP-2 have also been
implicated in virus-mediated cytopathicity by binding to calmodulin
(34-37), a critical mediator of secondary messenger calcium in
cytoplasmic signal transduction cascades (38). Binding to these
peptides thus inhibits calmodulin-dependent enzymes
in vitro and phospholipid systems in T cells.
Our previous results (39) showed that an Env mutant that lacks the
entire cytoplasmic tail is able to interfere in trans with
wild-type (wt) virus infectivity through formation of a dysfunctional hetero-oligomer with the wt Env subunit. This cytoplasmic tail truncation mutant also exhibits dominant interference with virus transmission mediated by homologous and heterologous T-tropic isolates
when the mutant env gene is targeted to human
CD4+ cells (40). We further demonstrated that the
C-terminal two-thirds segment of the gp41 cytoplasmic tail possesses
the potential to self-assemble into a high ordered multimer and that
this segment confers cellular membrane-binding ability (41, 42). The
correlation of membrane-binding ability and the multimerization
potential of subdomains of the cytoplasmic tail may have implications
for understanding the role of the cytoplasmic tail in the virus life cycle.
The gp41 cytoplasmic tail may play another role besides those of virus
infectivity and transmission. Truncation of the last 147 amino acids
from the C terminus of gp41, i.e. mutant TM709, significantly decreases Env stability, as judged by the greatly reduced
Env steady-state level of this mutant compared with the wt virus (7).
However, mutants bearing shorter truncations in the C-terminal
cytoplasmic tail than in the TM709 mutant showed no effect on Env
stability (7). In the context of the entire viral genome, Dubay
et al. (5) demonstrated that removal of the last 19 amino
acids, i.e. residues Arg-838 to Leu-856, reduces the
stability of Env but that further deletions toward the TM region do not
affect Env stability. This 19-amino acid deletion mutant shows no
effect on Env stability when Env is expressed in the absence of other
viral proteins (5). Gabuzda et al. (6) reported that in
COS-1 cells, Env mutants with deletions from 814 to 856 and from 840 to
856 decrease the steady-state levels of mutant Env proteins compared
with the steady-state level expressed by wt Env. In contrast, deletions
of residues in the C-terminal to residue 846 do not affect steady-state
Env expression (6). The observations of Dubay et al. (5) and
Gabuzda et al. (6) suggest that the integrity of the
cytoplasmic tail N-terminal to residue 846 is crucial, in a cell
type-dependent manner, for Env stability. However, the
amino acid residues and/or or structural determinants located in the
cytoplasmic tail critical for Env stability remain to be determined.
To define the role of the LLP-1 sequence in virus replication, in this
study we introduced single point deletions and substitutions in the
LLP-1 region and examined the effects of these mutations on virus
replication. We found that a single deletion of one of the two adjacent
valine residues located at 832 and 833 of Env (termed the 833 mutant
for simplicity) greatly reduced Env steady-state expression compared
with that of the wt virus. Studies of additional deletion mutants at
Ile-830, Ala-836, and Ile-840 also showed that the N terminus of the
LLP-1 motif is important in Env steady-state expression. Further
analysis of amino acid specificity for Val-832 and Val-833 illustrated
that Env steady-state expression could be altered by a single
substitution of a Pro residue for Val-833. We also found that the
830, 833, and 833P mutant Env proteins were less stable than the
wt Env after biosynthesis, presumably due to their transport to an
intracellular site for degradation. This is the first demonstration to
provide a basis for understanding the viral molecular determinants that
modulate HIV-1 Env stability.
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EXPERIMENTAL PROCEDURES |
Cells and Hybridoma--
293 and HeLa cells were cultured in
Dulbecco's modified Eagle's medium containing 10% heat-inactivated
fetal bovine serum. CEM-SS and PM1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Hybridoma Chessie 13, Chessie 8, 902, and 183 were maintained in RPMI 1640 supplemented with
10% fetal bovine serum and injected intraperitoneally into BALB/c mice
to produce ascitic fluids. Mouse monoclonal antibody (mAb) directed against an N-terminal peptide of 
-actin was purchased from Sigma. Horseradish peroxidase (HRP)-linked sheep anti-mouse Ig and
streptavidin-biotinylated HRP complex were obtained from Amersham
Biosciences. Peroxidase-labeled affinity-purified rabbit anti-sheep IgG
was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
Mutagenesis and Construction of Plasmids--
Single point
deletions and substitutions as shown in Fig. 1 were introduced into the
gp41 cytoplasmic tail-coding sequence of the HXB2R3 provirus (15) by
oligonucleotide-directed, site-specific mutagenesis using a PCR overlap
extension (43, 44). The paired primers used in PCR encoding each point
deletion in the gp41 C-terminal cytoplasmic tail were as
follows: 818, 5'-GCTTGCTCAATGCCGCCATAGCAGTAGCTGAGGG-3' (sense) and
5'-GCTACTGCTATGGCGGCATTGAGCAAGCTAACAGC-3' (antisense); 830,
5'-GAGGGGACAGATAGGGTTGAAGTAGTACAAGGA-3' (sense) and
5'-AGCTCCTTGTACTACTTCAACCCTATCTGTCCC-3' (antisense); 833,
5'-GGGTTATAGAAGTACAAGGAGCTTG-3' (sense) and 5'-GCTCCTTGTACTTCTATAACCCTATCTG-3' (antisense); 836,
5'-ATAGAAGTAGTACAAGGATGTAGAGCTATTCGC-3' (sense) and
5'-GTGGCGAATAGCTCTACATCCTTGTACTACTTC-3' (antisense); 840,
5'-CAAGGAGCTTGTAGAGCTCGCCACATACCTAGA-3' (sense) and
5'-TCTTCTAGGTATGTGGCGAGCTCTACAAGCTCC-3' (antisense); 843,
5'-GCTATTCGCCACCCTAGAAGAATAAGAC-3' (sense) and
5'-CTTATTCTTCTAGGGTGGCGAATAGCTCTAC-3' (antisense); 847,
5'-CATACCTAGAAGAAGACAGGGCTTGGAA-3' (sense) and
5'-CAAGCCCTGTCTTCTTCTAGGTATGTGGC-3' (antisense); 854, 5'-CAGGGCTTGGAAAGGTTGCTATAAGATGGGTGGC-3' (sense) and
5'-CCCATCTTATAGCAACCTTTCCAAGCCCTGTC-3' (antisense); 832A,
5'-GGGTTATAGAAGCAGTACAAGGAGCTTG-3' (sense) and
5'-GCTCCTTGTACTGCTTCTATAACCCTATCTG-3' (antisense); 832S,
5'-GGGTTATAGAATCGGTACAAGGAGCTTG-3' (sense) and
5'-GCTCCTTGTACCGATTCTATAACCCTATCTG-3' (antisense); 832P,
5'-GGGTTATAGAACCAGTACAAGGAGCTTG-3' (sense) and
5'-GCTCCTTGTACTGGTTCTATAACCCTATCTG-3 (antisense); 833A,
5'-AGGGTTATAGAAGTAGCACAAGGAGCTTGTAGA-3' (sense) and
5'-AGCTCTACAAGCTCCTTGTGCTACTTCTATAAC-3' (antisense); 833S, 5'-AGGGTTATAGAAGTATCGCAAGGAGCTTGTAGA-3' (sense) and
5'-AGCTCTACAAGCTCCTTGCGATACTTCTATAAC-3' (antisense); and 833P,
5'-AGGGTTATAGAAGTACCACAAGGAGCTTGTAGA-3' (sense) and
5'-AGCTCTACAAGCTCCTTGTGGTACTTCTATAAC-3' (antisense).
In the first round of PCR, two amplification reactions were performed,
both using the HXB2gpt infectious clone (45) as the template. One amplification used oligonucleotide 8423f
5'-GAAGAAGAAGGTGGAGAGAGA-3' (sense; nucleotides positioned at
8423-8443 of the HXB2 strain of the provirus) and the antisense
oligonucleotide as specified for each mutation as the primers. The
other amplification used 8933r 5'-GCTACTTGTGATTGCTCC-3' (antisense;
nucleotides positioned at 8933 to 8916) and the sense oligonucleotide
as specified for each mutation as the primers. The two PCR products
obtained were then used as the templates, and oligonucleotides 8423f
and 8933r were used as the primers in the second round of PCR. All PCRs were performed using Vent DNA polymerase (New England Biolabs, Beverly,
MA) according to a PCR amplification program described previously (42).
The PCR-amplified DNA fragments were ligated via the BamHI
and XhoI sites into pGEM-7Zf (Promega Corp.; Madison, WI).
After confirmation of the insert sequences in the pGEM-7Zf constructs,
the BamHI-XhoI fragments were isolated and then
inserted into the HXB2R3-TM844 provirus (7) at the corresponding sites. To obtain a wt HXB2R3 provirus, the BamHI-XhoI
sequence in HXB2R3-TM844 was replaced by the homologous sequence
derived from HXB2gpt. To generate HIV-1 long terminal
repeat (LTR)-controlled mutant Env expression plasmids, the
KpnI-XhoI fragments isolated from mutant
proviruses were substituted for the homologous sequence in a version of
wt pSVE7-puro (40) in which the XhoI site located in the 5'-LTR was deleted. All mutant pGEM-7Zf, HXB2R3, and
pSVE7-puro constructs were autosequenced using 8423f as the
primer to confirm the sequences of the mutated
BamHI-XhoI fragments.
pCDNA3 (Invitrogen; Carlsbad, CA), a cytomegalovirus
enhancer/promoter-driven plasmid that also encodes T7 and Sp6 promoters flanking the polycloning sites, was used to construct pCDNA3/wt env encoding the entire HIV-1 env gene for
in vitro transcription/translation. The BamHI
site in pCDNA3 was first abolished by BamHI digestion followed by overhang filling using T4 DNA polymerase in the presence of
dNTPs and by ligation using T4 DNA ligase. The full-length wt
env gene was generated by PCR using wt pSVE7-puro
as the template, and
5'-CGGAATTCGCCGCCACCATGAGAGTGAAGGAGAAATATCAG-3' and
5'-GCTCTAGATTATAGCAAAATCCTTTCCA-3' as the forward and reverse
primers, respectively. The amplification was performed in Vent
polymerase buffer with a final concentration of 4 mM
MgSO4 and 5% dimethyl sulfoxide according to a PCR program described previously (41). The 2.6-kb EcoRI-XbaI
fragment was then inserted into the corresponding sites in the
BamHI-deleted pCDNA3 plasmid. The resultant
pCDNA3/wt env plasmid encoded a Kozak sequence 5' to the
ATG initiation codon of the Env protein, which was followed by the
entire env coding sequence with a stop codon 5' to the
XbaI recognition sequence. The entire env gene was confirmed by DNA autosequencing. Mutant pSVE7-puro
plasmids encoding the 830, 833, or 833P proteins were
individually used as templates in PCR to generate the 0.33-kb mutated
BamHI-XbaI fragments. These fragments were then
used to replace the homologous sequence in pCDNA3/wt env
to yield various mutant pCDNA3/env plasmids. The mutant
pCDNA3/env clones were also confirmed by DNA sequencing.
Plasmid DNA Transfection--
To generate virus stocks or
examine viral protein expression, 293 cells grown in 100-mm Petri
dishes were transfected with 10 µg each of the wt or mutant
proviruses by a standard calcium phosphate coprecipitation method (39,
46). To prepare vesicular stomatitis virus (VSV) glycoprotein
G-pseudotyped virus stocks, 293 cells were cotransfected with 7.5 µg
of the human cytomegalovirus virus (HCMV) promoter-directed VSV G
protein expression plasmid pHCMV-VSV G (47) together with 7.5 µg each
of the wt or mutant proviruses. For examination of viral proteins
encoded by proviruses, HeLa cells grown in 100-mm Petri dishes were
transfected with 10 µg each of the wt or mutant proviruses by the
LipofectAMINE transfection method (Invitrogen). For examination of Env
and Rev expressions, HeLa cells grown in 60-mm Petri dishes were
cotransfected with 4 µg of wt or mutant pSVE7-puro
plasmids together with 0.5 µg of an HIV-1 LTR-driven Tat expression
plasmid pIIIextat.
Virus Infection and Reverse Transcriptase (RT) Activity--
Two
days after the 293 transfection, cell-free viruses were prepared and
normalized for RT activity as described previously (40, 48). Virus
infectivity was assayed with CEM-SS or PM1 cells using aliquots of
viruses containing RT activity as specified in each experiment. RT
activity of viruses obtained from supernatants of infected cultures was
then monitored postinfection. For VSV-G pseudotype infection, CEM-SS
cells were infected with pseudotyped viruses containing RT activity as
indicated in each experiment.
Western Blot Analysis--
Two days after transfection with
proviruses or 3 days after infection with VSV G-pseudotyped recombinant
viruses, viruses were isolated from culture supernatants over a 20%
sucrose cushion prepared in phosphate-buffered saline (PBS) as
described previously (39, 46). Cells and viral pellets were lysed with
PBS containing 1% Nonidet P-40, 1% sodium deoxycholate, and the
complete protease inhibitor mixture (Roche Molecular Biochemicals).
Equal volumes of cell and virion lysates were resolved by SDS-PAGE
followed by Western blot analysis using appropriate first antibodies.
Mouse mAbs 902, Chessie 13, Chessie 8, and 183 were used to
simultaneously detect Env and Gag products. For examination of gp160,
gp120, and gp41 in pSVE7-puro transfections, mAbs 902, Chessie 13, and Chessie 8 were used. When only gp160 and gp120 were
targeted, mAbs 902 and Chessie 13 were used as indicated in Fig.
3A. Alternatively, sheep anti-gp120 was used to detect gp160
and gp120 as indicated in Fig. 4B. A mAb directed against a
peptide corresponding to the C terminus of the HIV-1 Rev (kindly
provided by Dr. J. Karn) was used to detect HIV-1 Rev expression.
HRP-conjugated anti-mouse or anti-sheep IgG was used as a second
antibody to detect the immune complexes.
Metabolic Labeling, Pulse Chasing, and
Immunoprecipitation--
Env-expressing HeLa cells were washed twice
with PBS 2 days after transfection and supplemented with 1 ml of
methionine-free Dulbecco's modified Eagle's medium containing 2%
dialyzed fetal calf serum. After incubation at 37 °C for 30 min, 100 µCi of Easy Tag [35S]methionine (PerkinElmer Life
Sciences) (specific activity >1000 Ci/mmol) was added into each plate
for the periods indicated. For pulse chasing, cultures were labeled
with [35S]methionine at 37 °C for 30 min, and then
unlabeled methionine at five times the normal concentration found in
Dulbecco's modified Eagle's medium was added to cultures to initiate
the chase. At indicated chase times, culture supernatants were
centrifuged at 10,000 × g in an Eppendorf
microcentrifuge at 4 °C for 3 min to pellet unattached cells and
cell debris. The extracellular fractions were supplemented with Nonidet
P-40 and sodium deoxycholate each at a final concentration of 1%.
Cells were washed twice with cold PBS and lysed with 1 ml of PBS
containing 1% Nonidet P-40, 1% sodium deoxycholate, and the complete
protease inhibitor mixture. After standing at 4 °C for 10 min, the
cell lysates were cleared by centrifugation at 10,000 × g at 4 °C for 10 min. Equal volumes of cell lysates and
culture supernatants were precipitated with 5 µl of mAb 902 ascitic
fluid, 5 µl of sheep anti-gp120, or 10 µl of anti-HIV-1 antisera
pooled from HIV-1-infected patients. These antibodies were prebound to
a mixture containing 30 µl each of protein A- and protein G-Sepharose
4B (Amersham Biosciences). After incubation at 4 °C for 2-3 h, the
immune complexes were washed six times with RIPA buffer (10 mM Tris-HCl (pH 7.2), containing 1% Triton X-100, 0.1%
SDS, 1% sodium deoxycholate, 5 mM EDTA, and 0.15 M NaCl) and then boiled in 50 µl of Laemmli buffer. The proteins released from the beads were resolved by SDS-7.5% PAGE followed by fluorography.
In Vitro Transcription/Translation--
In vitro wt
and mutant Env synthesis was performed using the T7 polymerase-based
reticulocyte TNT quick-coupled transcription/translation kit (Promega).
Half a microgram each of the wt and mutant pCDNA3/env plasmids was used in a total volume of 25 µl of reaction mixture in
the absence of canine pancreatic microsomal membrane as described previously (41). Reactions were performed in a radiolabeled format
using [35S]methionine or a non-radiolabeled format using
Transcend biotinylated lysyl-tRNA according to the protocols provided
by the manufacturer. Equal volumes of the 35S-labeled or
biotinylated wt and mutant reaction mixtures were directly analyzed by
SDS-PAGE or subjected to immunoprecipitation with mAb 902 (for
radiolabeled proteins) or Western blotting using various mAb mixtures
(for biotinylated proteins).
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RESULTS |
Deletion of Val-833 in the gp41 LLP-1 Motif Impairs Virus
Replication--
To delineate the role of the LLP-1 sequence in the
virus life cycle, single point deletions were individually introduced
at residues Val-833, Ile-843, Ile-847, and Ile-854 in the Env protein of the HXB2 strain (Fig. 1A).
Among the residues deleted, two Val residues are located adjacent to
each other at positions 832 and 833 of the Env protein. The resultant
Val deletion mutant is referred to as the 833 mutant for simplicity.
These mutants were constructed using HXB2R3 as the backbone provirus by
overlap PCR mutagenesis. As a control for the effects of deletions in the LLP-1 sequence, Thr-818, which is located outside the LLP-1 sequence, was also deleted (Fig. 1A). These deleted residues
were located in neither the tat nor the rev open
reading frames.
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Fig. 1.
Construction of HIV-1 LLP-1 point deletion
and substitution mutants. The amino acid sequence in single-letter
codes from residues 816-856 located in the C terminus of the HIV-1
gp41 cytoplasmic tail of the HXB2 strain is shown at the
top. Single point deletions and substitutions were
individually introduced into the residues underlined as
described under "Experimental Procedures." Dashes
indicate that the amino acids in the mutant proviruses are identical to
those of the wt provirus. Deleted amino acids are indicated by
.
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To examine whether these single deletions exert an effect on virus
infectivity, cell-free viruses produced from 293 cells transfected with
the wt provirus or with each of the mutant proviruses and normalized by
RT activity were used to challenge CEM-SS cells. The wt and all mutant
viruses except the 833 mutant virus showed productive replication
with a peak of RT production 11-15 days after infection (Fig.
2A). The 833 mutant did not
replicate at all in CEM-SS cells even 30 days after challenge (Fig.
2A). When virus infectivity was assayed in PM1 cells,
mutants 843, 847, and 854 all exhibited viral replication
kinetics similar to that of the wt virus, although the 818 mutant
showed replication kinetics slightly slower than that of the wt virus
(Fig. 2B). The slightly reduced viral replication in the
818 mutant was much less significant compared with the inability of
the 833 mutant virus to establish an infection in PM1 cells even 30 days after challenge (Fig. 2B).
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Fig. 2.
Replication of viruses encoding point
deletions in the LLP-1 sequence. Cell-free viruses containing
5 × 104 cpm of RT activity obtained from 293 cells
transfected with the wt or mutant proviruses were used to challenge
CEM-SS (A) or PM1 (B) cells. Virus production as
measured by RT activity was assessed postinfection.
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The 833 Mutant Virus Exhibits a Reduced Steady-state Env
Expression Phenotype--
To delineate the nature of the defect of the
833 mutant virus, the expression of viral proteins was assessed by
transfection of 293 cells with wt or mutant proviruses. Equal volumes
of lysates obtained from transfected cells were separated by SDS-10%
PAGE and analyzed by Western blotting using mAbs 902, Chessie 13, Chessie 8, and 183. Hybridoma 902 secretes a mouse mAb specific for the gp120 V3 region of the LAV and IIIB strains of HIV-1. Chessie 13 and
Chessie 8 mAbs recognize residues 252-273 and 727-732, respectively,
of the Env protein of the HIV-1LAI strain. These Env-specific mAbs are not known to recognize
conformation-dependent epitopes in Env. Hybridoma 183 is a
mAb specific for the HIV-1 capsid (CA) protein p24. These mAbs in
combination offer an advantage in that gp160, gp120, gp41, and Gag
proteins can be specifically and simultaneously detected in the same
blot through one single operation. The wt and all mutant proviruses
showed similar levels of cell-associated Gag precursor Pr55 and its
cleaved products p41 and p24 upon transfection (Fig.
3A, top panel,
lanes 2-7). All mutant proviruses except the 833 mutant
produced comparable amounts of cell-associated gp160 precursor and
gp120 to those produced by the wt provirus upon transfection (Fig.
3A, top panel, lanes 2-7). In
contrast, the 833 mutant produced much lower levels of
cell-associated gp160 and gp120 than did the wt (Fig. 3A,
compare lane 4 to lane 2 in the top
panel). When virions obtained from each transfection were
analyzed, similar amounts of p24, p41, and uncleaved Pr55 were detected
in the wt and all mutant virions (Fig. 3A, top
panel, lanes 9-14). However, gp120 was found to be
associated with the wt and all mutant virions but not the 833 mutant
virion (Fig. 3A, top panel, lanes
9-14). To better separate gp160 and gp120, additional aliquots of
cell and virion lysates were resolved by SDS-7.5% PAGE followed by
Western blotting using mAbs 902 and Chessie 13. Similar patterns of
gp160 and gp120 expressions to those shown in the top panel
of Fig. 3A were also observed (Fig. 3A,
bottom panel).
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Fig. 3.
Expression of viral proteins encoded by
deletion mutants assessed by DNA transfection. A,
expression in 293 cells. 293 cells were transfected with 10 µg each
of the wt or mutant proviruses by the standard calcium phosphate
coprecipitation method. Two days after transfection, cultures were
processed for cell and virion lysates. Equal aliquots of cell and
virion lysates were subjected to SDS-10% PAGE followed by Western
blotting using mAbs 902, Chessie 13, Chessie 8, and 183 (top
panel). The migration positions of molecular mass markers
in kDa are also shown. Additional aliquots of cell and virion lysates
were resolved by SDS-7.5% PAGE followed by Western blotting using mAbs
902 and Chessie 13 (bottom panel). B, expression
in HeLa cells. HeLa cells were transfected with 10 µg each of the wt
or mutant proviruses by the LipofectAMINE transfection method. Cell and
virion lysates were then resolved by SDS-10% PAGE followed by Western
blotting using mAbs 902, Chessie 13, Chessie 8, and 183.
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To determine whether the reduced Env steady-state expression observed
in the 833 mutant is cell type-specific, viral protein expression in
HeLa cells was examined. The 833 mutant provirus produced lower
levels of cell-associated gp160 and gp120 compared with the wt and
other mutant proviruses upon transfection, even though the production
of Gag products in this mutant was normal (Fig. 3B,
left panel). Again, no detectable amount of gp120 was found
to be associated with the 833 mutant virion, although the amounts of
p24, p41, and uncleaved Pr55 associated with this mutant virion were
comparable with those detected in the wt and other mutant virions (Fig.
3B, right panel). The ratio of intracellular gp160 to gp120 observed in the 833 mutant was similar to that observed in the wt and other mutants (Fig. 3B, left
panel), indicating that the 833 mutant Env precursor is
normally processed into gp120 and mutant gp41.
We next determined whether the 833 mutant also exhibits a reduced
steady-state Env expression pattern in human CD4+ T cells,
which are natural host cells for HIV-1 infection. Because T cells are
usually transfected less efficiently than required for biochemical
analyses of viral protein expression and Env incorporation, a high
level, transient HIV-1 expression system based on pseudotyping with VSV
G protein (19, 49) was employed. For this, 293 cells were cotransfected
with pHCMV-VSV G along with the wt provirus and each of the mutant
proviruses. Cell-free VSV G-pseudotyped virus stocks were harvested,
normalized by RT activity, and used to challenge CEM-SS. Equal portions
of cell and virion lysates were analyzed by Western blotting using the
mAb mixture containing the Env- and CA-specific mAbs. Infection with
the wt and all mutant pseudotypes showed comparable steady-state levels
of cell-associated Gag products (Fig.
4A, lanes 1-6).
Similar levels of p24 were associated with the wt and all mutant
virions (Fig. 4A, lanes 7-12). Again, the 833
mutant showed significantly lower levels of cell-associated gp160
precursor and gp120 than did the wt and other mutants (Fig.
4A, lanes 1-6), and gp120 and gp41 could not be
detected in this mutant virion (Fig. 4A, lane 9).
When the same samples were analyzed by Western blotting using sheep
anti-gp120 antisera, similar patterns of Env steady-state expression
and incorporation into virions to those observed in Fig. 4A
were obtained for the wt and mutant viruses (Fig. 4B).
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Fig. 4.
Synthesis of viral proteins encoded by
deletion mutants. A and B, assessment of
viral protein expression by VSV G-pseudotype infection. 293 cells were
cotransfected with 7.5 µg of pHCMV-VSV G and 7.5 µg each of the wt
or mutant proviruses. Cell-free, VSV G-pseudotyped recombinant viruses
containing 7 × 105 cpm of RT activity obtained from
each transfection were used to challenge 2 × 106
CEM-SS cells. After overnight incubation, cells were washed with PBS
and allowed to incubate at 37 °C for an additional 2 days. Equal
portions of cell and virion lysates were analyzed by SDS-10% PAGE
followed by Western blotting using mAbs 902, Chessie 13, Chessie 8, and
183 (A). The same samples were also analyzed by Western
blotting using sheep anti-gp120 (B). C, analysis
of mutant Env proteins encoded by the pSVE7-puro expression
vector. HeLa cells were cotransfected with pIIIextat
together with the wt or mutant pSVE7-puro plasmids.
Cotransfection with pIIIextat and
pSVE7(KS)-puro (40), a derivative of
pSVE7-puro in which the KpnI site (at nucleotide
position 6351 of the HXB2 sequence) to the StuI site
(positioned at 6834) is deleted, was used as a negative control. Two
days after transfection, lysates obtained from each transfection were
resolved by SDS-7.5% PAGE followed by Western blotting using mAbs 902, Chessie 13, and Chessie 8.
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Expression of the 833 Mutant Env from an Env Subgenomic
Expression Vector--
To determine whether the reduced Env
steady-state expression phenotype observed in the 833 mutant is an
intrinsic characteristic of the mutant Env and is independent of other
viral protein expression, the env genes in all of the mutant
proviruses were subcloned into the pSVE7-puro env expression
vector. HeLa cells were cotransfected with pIIIextat along
with either the wt plasmid or each of the mutant pSVE7-puro
plasmids. Again, the 833 mutant produced lower levels of
intracellular gp160, gp120, and gp41 than did the wt and other mutants
(Fig. 4C).
Characterization of Viruses Encoding Point Deletions in the N
Terminus of the LLP-1 Region--
To determine further whether the
N-terminal, but not the C-terminal, segment of the LLP-1 sequence is
critical for Env steady-state expression, additional point deletions
were introduced into residues at Ile-830, Ala-836, and Ile-840 (Fig.
1B). The 830 mutant virus, like the 833 mutant virus,
did not replicate at all in CEM-SS cells even 35 days after virus
challenge (Fig. 5A). The
840 mutant virus replicated with delayed kinetics compared with the
wt virus with a peak of RT production between days 25 and 28. This was in contrast to the peak of RT production on day 11 observed for the wt
virus (Fig. 5A). Although the 836 mutant virus replicated with kinetics slower than that of the wt virus, the delay in
replication with this mutant was less significant than those with the
830 and 840 mutant viruses (Fig. 5A).
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Fig. 5.
Characterization of mutants with point
deletions in the N terminus of the LLP-1 motif. A,
infectivity assay. Cell-free, wt, and mutant virus stocks were prepared
from 293 cells transfected with wt or mutant proviruses as indicated.
Viruses containing 2 × 104 cpm of RT activity from
each stock were used to challenge CEM-SS cells, and virus production
measured by RT activity was monitored after infection. B,
viral protein expression in CEM-SS cells. VSV-G-pseudotyped virus
stocks were prepared from 293 cells transfected with pHCMV-VSV G along
with the wt or mutant proviruses. Cell-free viruses containing
106 cpm of RT activity obtained from each pseudotype stock
were used to infect CEM-SS cells. Cell lysates and virions were
analyzed by Western blotting using mAbs 902, Chessie 13, Chessie 8, and
183.
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Viral proteins expressed by these mutants were then examined in CEM-SS
cells infected with the wt or mutant VSV G pseudotypes. Deletions of
residues 830, 836, and 840 did not affect the synthesis and processing
of intracellular Pr55, nor did they affect virus assembly/budding (Fig.
5B). The 836 mutant produced comparable amounts of
cell-associated gp160, gp120, and gp41 to those produced by the wt
virus (Fig. 5B, compare lane 4 with lane
1). Both the 830 and 840 mutants produced smaller amounts of
intracellular Env proteins than did the wt virus (Fig. 5B,
compare lanes 3 and 5 with lane 1),
with the 830 mutant showing a greater reducing effect on Env
steady-state expression than did the 840 mutant. However, the 830
mutant produced slightly larger amounts of cell-associated Env proteins
than did the 833 mutant (Fig. 5B and data not shown). Comparable amounts of gp120 and gp41 to those detected in the wt virion
were found to be associated with the 836 mutant virion (Fig.
5B, compare lane 9 with lane 6),
whereas no gp120 or gp41 could be detected in the 830 mutant virion
(Fig. 5B, lane 8). gp120 and gp41 were barely
detectable in the 840 mutant virion (Fig. 5B, lane
10).
Effects of Amino Acid Substitutions at Val-832 and Val-833 on Env
Steady-state Expression--
Because deletion of one of the two
adjacent Val residues located at positions 832 and 833 displayed the
most significantly reduced Env steady-state expression phenotype among
the deletion mutants analyzed, we next determined the amino acid
specificity of Val-832 and Val-833 on Env steady-state expression.
Mutant viruses encoding Env in which Val-832 and Val-833 were each
replaced by the nonconserved residues Ala, Ser, and Pro were examined
(Fig. 1C). These substitutions were expected to alter the
-helical structure in the increasingly severe order of Pro > Ser > Ala (50). The 832A and 832S mutants showed wt virus-like
replication kinetics, whereas the 832P mutant exhibited slightly
delayed replication kinetics compared with that of the wt virus (Fig.
6A). The 833A and 833S mutants
also displayed wt virus-like replication kinetics (Fig. 6B).
Like the 833 mutant, the 833P mutant did not replicate at all even
31 days after challenge to CEM-SS cells (Fig. 6B).
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Fig. 6.
Characteristics of Val-832 and Val-833
substitution mutants. A, infectivity assay of Val-832
substitution mutants. 293 cells were transfected with the wt or 832 mutant proviruses as indicated. Cell-free viruses containing 2 × 104 cpm of RT activity obtained from each transfection were
used to challenge CEM-SS cells, and RT activity was monitored
postinfection. B, infectivity assay of Val-833 substitution
mutants. 293 cells were transfected with the wt or 833 mutant
proviruses, and virus infectivity was assayed as described in
A. C, expression of viral proteins encoded
by point substitution mutants. Cell-free, VSV G-pseudotyped
recombinant viruses as indicated containing 106 cpm of RT
activity were used to challenge CEM-SS cells. Equal portions of cell
lysates and virion fractions were then analyzed by Western blotting
using mAbs 902, Chessie 13, Chessie 8, and 183.
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VSV G-pseudotype infection of CEM-SS cells was employed to assess viral
protein expression in these substitution mutants. All of the Val-832
and Val-833 substitution mutants produced similar amounts of
intracellular and virion-associated gag gene products to
those produced by the wt virus (Fig. 6C). Among these
mutants, only the 833P mutant showed significantly reduced levels of
intracellular gp160, gp120, and gp41 compared with the wt and other
mutants (Fig. 6C, lanes 1-8). In addition, gp120
and gp41 could not be detected in this mutant virion (Fig.
6C, lane 16).
The 833 Mutant Env Rapidly Degrades After Biosynthesis--
We
then explored the mechanism responsible for the reduced Env
steady-state expression of the 833 mutant. First, cells
cotransfected with the wt or 833 mutant pSVE7-puro
plasmid along with pIIIextat were labeled with
[35S]methionine at 37 °C for 10 min. Cell lysates
containing the wt or 833 mutant Env were individually precipitated
with sheep anti-gp120, pooled human anti-HIV-1, and mAb 902. A similar
or even a slightly greater amount of the 833 mutant gp160 precursor than that of the wt precursor was precipitated by mAb 902 (Fig. 7A). These observations
indicated that mAb 902, like polyclonal anti-gp120 and anti-HIV-1,
reacts with the 833 mutant gp160 precursor as effectively as with
the wt gp160. Because of the availability of hybridoma 902 and the
lower background precipitated by this mAb compared with other
polyclonal antibodies, mAb 902 was used in later studies. Next, HeLa
cells expressing the wt or 833 mutant Env were metabolically labeled
at 37 °C for different times, and cell lysates were
immunoprecipitated with mAb 902. Similar amounts of gp160 precursor
were detected in both the wt and 833 mutant for each labeling time
(Fig. 7B), indicating that deletion of Val-833 does not
affect de novo Env biosynthesis.
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Fig. 7.
Assessment of the synthesis and degradation
of 833 mutant Env. A,
immunoprecipitation of Env proteins by various antibodies. Two days
after transfection, HeLa cells expressing wt or 833 mutant Env were
labeled with [35S]methionine at 37 °C for 10 min.
Equal volumes of cell lysates were precipitated with sheep anti-gp120,
pooled human anti-HIV-1 antisera, or mAb 902 as indicated, and
precipitated proteins were analyzed by SDS-7.5% PAGE followed by
fluorography. B, analysis of Env synthesis. HeLa cells
expressing wt or 833 mutant Env were labeled with
[35S]methionine at 37 °C for different times, and
equal volumes of cell lysates were immunoprecipitated with mAb 902 followed by SDS-7.5% PAGE. C, pulse chasing of Env
proteins. HeLa cells expressing wt or 833 mutant Env were
metabolically labeled at 37 °C for 30 min and then chased for
different times in the presence of excess cold methionine. Equal
volumes of cell lysates and extracellular fractions obtained from each
chase time were precipitated with mAb 902 followed by SDS-PAGE.
D, kinetics of processing and extracellular secretion of
35S-labeled wt and mutant Env proteins. The gels shown in
C were scanned with an Instant Imager (Packard Instrument
Co.). The bands corresponded to intracellular gp160 and gp120, and
extracellular gp120 of the wt and 833 mutant were quantitated. The
radioactivity of each gp120 band was calibrated by multiplying by a
factor of 4/3 because the numbers of methionine residues present in
gp120 and gp41 are 12 and 4, respectively. In each gel, the relative
percentage of the radioactivity of each Env species for each chase time
to the radioactivity of intracellular gp160 at chase time 0 was
calculated and plotted versus the chase time.
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To determine whether the reduced steady-state Env expression of the
833 mutant is due to the rapid turnover of this mutant after
synthesis, a pulse-chase experiment was performed. The levels of the
intracellular 833 mutant Env precursor declined more rapidly than
those of the wt Env as the chase proceeded (Fig. 7C). The percentage of radioactivity of each species at each indicated chase
time relative to the radioactivity of the gp160 precursor at chase time
0 was quantitated by an instant imager and then calculated. The 833
mutant Env precursor displayed a more rapid turnover rate than the wt
precursor (Fig. 7D). However, the faster decrease in
intracellular 833 mutant gp160 compared with that of intracellular
wt gp160 was not concomitant with the more rapid appearance of
intracellular or extracellular gp120 in this mutant (Fig.
7D, right panel). This observation indicates that
the greatly reduced Env steady-state expression observed in the 833
mutant is not due to the enhanced dissociation of gp120 from the
gp120-mutant gp41 complex nor is it due to the subsequent rapid
secretion of gp120 into the culture medium.
Rapid Degradation of 830 and 833P Mutant Env Proteins in Cells
After Synthesis--
Because the two mutants 830 and 833P also
exhibited reduced Env steady-state expression compared with the wt
virus, the efficiency of de novo Env synthesis in these two
mutants was compared with that of the wt Env in a study utilizing
10-min pulse labeling. Similar amounts of the wt, 830, and 833P
gp160 precursors were precipitated by either anti-HIV-1 or mAb 902 (Fig. 8A). When a pulse-chase
experiment was performed, the 830 and 833P mutant Env proteins, like
that of the 833 mutant Env, showed faster turnover kinetics than did
the wt Env (Fig. 8, B and C).
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Fig. 8.
Analysis of the synthesis and degradation
of 830 and 833P mutant Env proteins.
A, immunoprecipitation of mutant Env proteins. HeLa cells
expressing wt or mutant Env proteins were pulse-labeled with
[35S]methionine at 37 °C for 10 min, and cell lysates
were immunoprecipitated with pooled anti-HIV-1 antisera or mAb 902. B, pulse chasing of mutant Env proteins. Cells expressing
Env proteins as indicated were pulse-labeled and chased, and cell
lysates and extracellular fractions were immunoprecipitated with mAb
902. C, kinetics of the turnover of 35S-labeled
wt and mutant Env proteins. Bands corresponding to each Env species as
shown in B were quantitated and calibrated. The relative
percentage of the radioactivity of each Env species at each chase time
to the radioactivity of intracellular gp160 at chase time 0 was plotted
versus the chase time.
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Different mAb Mixtures Equivalently Recognize the wt and Mutant Env
Proteins--
To address directly the issue of whether mAb 902 precipitates the wt, 830, 833, and 833P Env proteins equivalently
in labeling and pulse-chase studies, in vitro coupled
transcription/translation in the absence of microsomal membranes was
performed in a radiolabeled format using pCDNA3/env
plasmids that encode the full-length wt or 830, 833, and 833P
mutant env genes downstream of a T7 promoter. Comparable
levels of trichloroacetic acid-precipitable counts were obtained in
equal volumes of the wt and mutant reaction mixtures (data not shown).
When equal volumes of the [35S]methionine-labeled wt and
mutant reaction mixtures were directly subjected to SDS-PAGE, similar
amounts of the wt and mutant proteins, which all migrated as a
predominant band with an apparent molecular mass of 90 kDa, were
detected (Fig. 9A). This
molecular mass was consistent with the molecular weight of the
unglycosylated form of Env. These observations also indicate that
highly homogenous wt and mutant proteins are comparably synthesized
under this in vitro protein synthesis condition. Next, equal
volumes of the wt and mutant reaction mixtures were immunoprecipitated
with pooled anti-HIV or mAb 902. mAb 902, as well as anti-HIV-1,
reacted equivalently with the wt, 830, 833, and 833P mutant
proteins synthesized in vitro (Fig. 9B).
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Fig. 9.
Characterization of Env proteins synthesized
in vitro and assessment of Rev synthesis in
pSVE7-puro transfections. A and
B, analysis of [35S]methionine-labeled wt and
mutant proteins. pCDNA3/env plasmids encoding wt or
mutant proteins as indicated were used in in vitro coupled
transcription/translation reactions in the absence of microsomal
membranes to produce [35S]methionine-labeled proteins.
After synthesis, excess cold methionine was added to the mixtures, and
the reaction mixtures were allowed to incubate at 30 °C for an
additional 30 min. Equal volumes (2 µl) of the wt and mutant reaction
mixtures were directly separated by SDS-7.5% PAGE (A).
Another aliquot (7 µl) of each reaction mixture was first
immunoprecipitated with pooled anti-HIV-1 antisera or mAb 902, and the
precipitated proteins were then resolved by SDS-7.5% PAGE
(B). C and D, analyses of biotinylated
wt and mutant proteins. Equal volumes (5 µl) of biotinylated wt and
mutant in vitro reaction mixtures were directly separated by
SDS-7.5% PAGE followed by Western blotting using
streptavidin-conjugated HRP to detect total in vitro
synthesized proteins (C). After stripping off
streptavidin-linked HRP from the immune complexes, the blot shown in
C was reprobed with mAbs 902 + Chessie 13 + Chessie 8 + 183 (D, top panel). Other aliquots (5 µl) of the wt
and mutant reaction mixtures were analyzed by Western blotting using
mAbs 902 + Chessie 13 + Chessie 8 (D, middle
panel). After stripping off antibodies from the immune complexes,
the blot shown in the middle panel of D was
reprobed with mAbs 902 + Chessie 13 (D, bottom
panel). In each case, HRP-conjugated antibody against mouse IgG
was used as a second antibody to detect the nonglycosylated wt and
mutant proteins. E, analysis of Rev synthesis. Equal volumes
of lysates from HeLa cells expressing wt or mutant Env proteins were
subjected to SDS-15% PAGE followed by Western blot analysis using an
HIV-1 Rev mAb. The same cell lysates were also resolved on a 10% gel
followed by Western blotting using a -actin mAb.
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To address whether the mAbs mixtures used in Western blot analyses
recognize the wt and mutant proteins equivalently, in vitro protein synthesis was performed in a non-radiolabeled format using Transcend biotinylated lysyl-tRNA. Equal volumes of the wt and mutant
reaction mixtures were analyzed by Western blotting using streptavidin-conjugated HRP to detect total proteins synthesized in vitro. Similar amounts of the wt and mutant proteins were
detected (Fig. 9C). Equal volumes of the wt and mutant
reaction mixtures were then analyzed by Western blotting using various
combinations of mAbs. After the first antibody incubation, blots were
incubated with HRP-conjugated second antibody directed against mouse
IgG. All mAb mixtures, i.e. 902 + Chessie 13 + Chessie 8 + 183, 902 + Chessie 13 + Chessie 8, and 902 + Chessie 13, reacted with
the wt protein and each of the mutant proteins synthesized in
vitro equivalently (Fig. 9D).
Rev Was Comparably Synthesized in the wt and Mutant pSVE7-puro
Transfections--
To rule out the possibility that the decreased
levels of the 830, 833, and 833P mutant precursors are due to
reduced Rev synthesis by these three mutant pSVE7-puro
transfections in cells, equal volumes of cell lysates from each
transfection, which contained comparable amounts of -actin (Fig.
9E, bottom panel), were analyzed by Western
blotting using an HIV-1 Rev mAb. Similar amounts of the Rev protein,
which migrated as a 16.5-kDa species, were detected in the wt
transfection and all mutant pSVE7-puro transfections (Fig.
9E, top panel).
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DISCUSSION |
Although previous truncation and deletion analyses have implicated
involvement of the cytoplasmic tail, or possibly the LLP-1 region, of
gp41 in Env stability, the precise effects of such large truncations or
deletions on Env stability are not fully understood. Large truncations
or deletions in a protein inevitably result in drastic structural and
folding alterations and/or disruption in interactions with cellular
factors. The observed phenotypic changes of these deletion Env mutants
cannot be simply attributed to a loss of functions that results from
the lack of target sequences in Env. In our initial study to address
the importance of amino acid residues located in the LLP-1 region in
virus infection, we examined HIV-1 mutant viruses that encoded point
deletions at Thr-818, Val-833, Ile-843, Ile-847, and Ile-854 in Env of
the HXB2 strain. Only the 833 mutant greatly reduced Env
steady-state expression, which, in turn, resulted in impaired virus
infectivity and gp120 incorporation into this mutant virion (Fig. 2,
Fig. 3, and Fig. 4, A and B). The 833 mutant
also showed significantly reduced steady-state Env expression in the
absence of other viral proteins (Fig. 4C), indicating that
Env expressed from the pSVE7-puro plasmid represents that in
the context of replication of the entire HIV-1 genome. The reduced Env
steady-state expression profile of the 833 mutant was observed in
various cell types including 293, CEM-SS, and HeLa cells (Fig. 3 and
Fig. 4), implying that this feature of the 833 mutant Env is not a
cell type-specific phenomenon.
To define further the involvement of the N terminus of the LLP-1
sequence in Env steady-state expression, we examined the 830,
836, and 840 mutants. The 840 mutant reduced steady-state Env
expression compared with the wt virus, but the reduction in the Env
steady-state level in this mutant was less prominent than that observed
in the 830 mutant (Fig. 5B). Parallel to these observations, gp120 and gp41 could not be detected in the 830 mutant
virion and were barely detectable in the 840 mutant virion (Fig.
5B). The reduced Env steady-state expression thus delays or
impairs the viral infectivity of these two mutants (Fig.
5A). Although deletion of Ala-836 did not greatly affect the
intracellular levels of Env proteins nor alter gp120 and gp41
incorporation into the mutant virion (Fig. 5B), this mutant
replicated with kinetics slightly slower than that of the wt virus
(Fig. 5A). Because the cytoplasmic tail is not essential for
Env-mediated syncytia formation (4, 5, 51), it appears that deletion of
Ala-836 may marginally affect some step that occurs after receptor binding and membrane fusion.
To delineate which Val residue located adjacent to positions 832 and
833 is critical for steady-state Env expression, mutants with Val-832
and Val-833 individually replaced by the nonconserved amino acids Ala,
Ser, and Pro were examined. Among all substitution mutants examined,
only a Val-to-Pro substitution at residue 833 profoundly reduced Env
steady-state expression, which is concomitant with severely impaired
Env incorporation into virions and infectivity of the mutant virus
(Fig. 6). The effect on Env steady-state expression of Pro substitution
at Val-833 is specific because Pro substitution for Val-832 did not
alter Env steady-state expression (Fig. 6C).
The possibility that mutations in the N terminus of the LLP-1 motif
induce a conformational change that hinders recognition of the mutant
Env proteins by Env-specific mAbs used can be eliminated based on the
following studies. First, to address the issue of whether the mAb
mixtures used recognized the wt and mutant proteins equivalently,
in vitro transcription/translation assays were performed in
the absence of microsomal membranes. This in vitro coupled transcription/translation provides a ready protein synthesis system to
express sufficient amounts of [35S]methionine or
biotinylated wt and mutant proteins with a high degree of purity for
biochemical analyses without further purification. Unlike mutant Env
synthesis in cells, in vitro production of Env proteins in
the absence of microsomal membranes results in non-glycosylated Env
species that do not undergo precursor processing and degradation. Also,
the amounts of in vitro synthesized Env proteins can be directly assessed by detection of the radiolabeled or biotinylated proteins in a given volume. In fact, equal volumes of the wt and mutant
reaction mixtures contained comparable amounts of the
35S-labeled or biotinylated wt and mutant proteins (Fig. 9,
A and C), indicating that the wt and mutant
proteins are comparably synthesized in vitro. In addition,
these in vitro synthesized proteins were highly homogenous,
as judged by the detection of a predominant band of the wt and mutant
proteins when the reaction mixtures were directly resolved by SDS-PAGE
(Fig. 9, A and C). mAb 902 precipitated
equivalently the wt, 830, 833, and 833P mutant proteins
synthesized in vitro (Fig. 9B). Also, all mAb mixtures used in the study, i.e. 902 + Chessie 13 + Chessie
8 + 183, 902 + Chessie 13 + Chessie 8, and 902 + Chessie 13, equivalently recognized the in vitro synthesized wt, 830,
833, and 833P mutant proteins (Fig. 9D). We observed that
the amounts of 830, 833, and 833P mutant gp160 precursors
precipitated by mAb 902 were comparable with the amount of wt gp160
precipitated when cells expressing these mutant proteins were labeled
for a short period, ranging from 5 to 30 min (Fig. 7, A and
B, and Fig. 8A). This is also consistent with the
notion that mAb 902 precipitates the wt and mutant proteins equivalently.
Env expression from the pSVE7-puro plasmid is
Rev-dependent. Because all of the mutations examined in the
present study are not located in the tat or rev
open reading frame, the rev-coding sequence in all mutant
pSVE7-puro plasmids should remain intact, and Rev should
presumably be expressed comparably by the wt and all mutant plasmids.
Because Gag expression in HIV-1-infected cells is also
Rev-dependent, the observations that all of the mutants
examined in this study produced comparable amounts of Gag proteins to
those produced by the wt virus (Figs. 3-6) indicate that mutations in
the LLP-1 motif do not affect Rev expression. This notion is directly
proven by the observation that comparable amounts of Rev were detected
in cells transfected with the wt or mutant pSVE7-puro along
with pIIIextat (Fig. 9E). Therefore, the
time-dependent decrease in intracellular gp160 levels
observed in these mutants at later chase periods, but not during pulse labeling (Figs. 7 and 8), reveals that the mutant Env proteins are
unstable after biosynthesis, presumably due to their
time-dependent transport to an intracellular site where
they degrade rapidly.
The conserved residues located in the LLP-1 region are not necessarily
crucial for Env stability. Ile-830, Ile-843, and Ile-847 are highly
conserved among different HIV-1 clades (Fig. 10) (52). However,
only deletion of Ile-830 significantly reduced Env stability (Figs. 5
and 8). The two Val residues located adjacent to each other at
positions 832 and 833 are not conserved among different HIV-1 clades.
In addition to the Val residue, Gly, Ala, Leu, and Ile are also found
at these positions in different HIV-1 subtypes (Fig.
10). The non-conservative feature of
residues 832 and 833 of the HXB2 strain implies that the primary
sequence in these two positions may not be critical for Env
stability.
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Fig. 10.
Sequence comparison of the LLP-1 region of
different HIV-1 serotypes. The amino acid sequences in
single-letter codes of representative isolates from different clades of
HIV-1 M, N, and O are aligned, and the residues conserved in all HIV-1
isolates are shaded. The numbers shown at the
top of the amino acid sequences indicate the residues
located in the HXB2 strain that were mutated and analyzed in the
present study. The sequences of the isolates listed here are as
follows: serotype A, K99; B, HXB2;
C, ETH2220; D, ELI; AE,
TH920149; F, 93BR0201; G, LBV217; H,
90CF056; J, SE91733; N, YBF30; and O,
CM4974.
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A deletion in an -helix is expected to skew the structure in a way
that residues originally aligned on one face of the helix become
displaced. The differential effect of deletions in the N-terminal and
C-terminal regions of LLP-1 on Env stability suggests that the
structure defined by the N terminus of the LLP-1 motif is critical for
maintaining Env stability, but that the functions of Env can tolerate
alterations to the C-terminal region of the LLP-1 sequence. Moreover,
substitutions by the nonconserved amino acids Ala, Ser, and Pro at
Val-833 are expected to disrupt the local -helical structure of the
N terminus of the LLP-1 motif in an increasingly severe manner. This
hypothesis is supported by previous peptide modeling studies on the
leucine zipper-like motif of HIV-1 gp41. Substitution of Ile-573 by a
Ser residue in a peptide mimicking this domain more greatly decreases
the stability of the coiled-coil structure of the peptide than that which occurs by an Ala residue, whereas a peptide containing an Ile-to-Pro substitution exhibits no stable -helical structure in
solution (50). There appears to be a correlation between the degree of
destabilization of Env by Ala, Ser, and Pro substitutions for Val-833
and the ability of these substitutions to alter the -helical
structure of the N terminus of the LLP-1 region (Fig. 6C and
Fig. 8C). These studies further substantiate the proposal that the -helical structure, but not the primary sequence, of the N
terminus of the LLP-1 motif is critical for Env stability.
The mechanism underlying the rapid degradation of Env proteins with
mutations in the N terminus of the LLP-1 motif is not yet understood.
It is likely that the local structural perturbation in the N terminus
of the LLP-1 sequence induced by Ile-830 or Val-833 deletion or a
Val-to-Pro substitution at residue 833 may alter the overall folding of
Env, thus causing these mutant proteins to be routed to an
intracellular compartment where the mutant Env proteins rapidly
degrade. There is a precedent for this hypothesis. Truncation of the
cytoplasmic tail of the simian immunodeficiency virus Env results in an
alteration in the conformation of the ectodomain of the TM protein on
the cell surface (53). Alternatively, the N terminus of the LLP-1 motif
may be directly involved in interactions with cellular factors that
modulate Env folding, transport, and/or stability. Deletions of these
residues thus alter Env trafficking and/or stability.
Previous studies (5, 9, 10, 12, 13, 51, 54) showed that deletions or
truncations of regions encompassing residues 830-833 do not apparently
affect Env stability. Hunter and co-workers (5, 8) also examined
mutants with truncations from the C terminus of the cytoplasmic tail or
with deletions of multiple consecutive residues or multiple
substitutions at different sites located in the conserved C terminus of
the gp41 cytoplasmic tail. Except for the C-terminal 19-amino acid
deletion mutant that is unstable and rapidly degrades after
biosynthesis, all other mutants do not seem to affect Env stability,
even though these mutations may reduce virus infectivity and Env
incorporation into virions. Even the longest deletion mutant SD1,
which lacks the sequence between Ile-820 and Ala-839, still replicates
in CD4+ T cells but with delayed kinetics (8). In the
present study, we demonstrate that Ile-830 and Val-833 located in the N
terminus of the gp41 LLP-1 motif have a critical role in modulating
HIV-1 Env stability. The Ile-830 and Val-833 mutations likely induce a
change in Env conformation that targets mutant Env by a cellular degradation pathway. This study has implications toward understanding how HIV-1 Env expression is modulated by the structure of an
intracytoplasmic domain in gp41 during virus infection.
| |
ACKNOWLEDGEMENTS |
The pHCMV-VSV G plasmid was a kind gift from
J. C. Burns (University of California School of Medicine, San
Diego). We acknowledge the help of the National Institutes of Health
AIDS Research and Reference Reagent Program in providing the reagents
listed below, especially P. L. Nara (CEM-SS), M. Reitz, Jr. (PM1),
B. Chesebro (hybridoma 902 and 183), G. K. Lewis (hybridoma
Chessie 13 and Chessie 8), and M. Phelan (sheep anti-gp120). The HIV-1
Rev mAb, donated by J. Karn, was provided by the EU Program EVA/MRC
Centralized Facility for AIDS Reagents, National Institute for
Biological Standards and Control, UK (supported by Grant
QLK2-CT-1999-00609 and GP828102).
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FOOTNOTES |
*
This work was supported by National Science Council Grants
90-2320-B-001-067 and the Institute of Biomedical Sciences, Academia Sinica, Republic of China.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Div. of Infectious
Diseases, Institute of Biomedical Sciences, Academia Sinica, 128, Section 2, Yen-Chiu-Yuan Road, Taipei 11529, Taiwan, Republic of China.
Tel.: 886-2-2652-3933; Fax: 886-2-2785-8847; E-mail: schen@ibms.
sinica.edu.tw.
Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M201479200
| |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus, type 1;
mAb, monoclonal antibody;
TM, transmembrane;
gp, glycoprotein;
LLP, lentivirus lytic peptide;
wt, wild type;
HRP, horseradish peroxidase;
LTR, long terminal repeat;
HCMV, human cytomegalovirus virus;
VSV, vesicular stomatitis virus;
RT, reverse transcriptase;
PBS, phosphate-buffered saline.
| |
REFERENCES |
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|
Fisher, A. G.,
Ratner, L.,
Mitsuya, H.,
Marselle, L. M.,
Harper, M. E.,
Broders, S.,
Gallo, R. C.,
and Wong-Staal, F.
(1986)
Science
233,
655-659[Abstract/Free Full Text]
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Terwilliger, E.,
Sodroski, J. G.,
Rosen, C. A.,
and Haseltine, W. A.
(1986)
J. Virol.
60,
754-760[Abstract/Free Full Text]
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Lee, S.-J., Hu, W.,
Fi |
TOP
ABSTRACT
INTRODUCTION
