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Requirement of Calmodulin Binding by HIV-1 gp160 for Enhanced
FAS-mediated Apoptosis *

Keith J.

Micoli ,

George

Pan ,

Yong

Wu ,

John P.

Williams ,

William J.

Cook , and

Jay M.

McDonald § ¶




From the Department of Pathology, University of
Alabama at Birmingham, Birmingham, Alabama 35294and the

§ Veterans Administration Medical Center,
Birmingham, Alabama 35233



ABSTRACT

TOP

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



Accelerated apoptosis is one mechanism proposed
for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type
1 (HIV-1 ) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of
gp...

in vitro calmodulin binding to a peptide corresponding to
the C-terminal calmodulin-binding domain of gp160. Stable Tet-off
Jurkat cell lines were developed that inducibly express wild type gp160
or gp160A835W. Increasing expression of wild type gp16...

INTRODUCTION

TOP

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



HIV-1 1 infection is
characterized by immune system hyperactivation and dysfunction that
increases with disease progression until immune function is lost.
During the long asymptomatic phase, a state of equilibrium appears
to be achieved, consisting o...



One proposed mechanism for the accelerated loss of CD4+ cells is an
increased rate of apoptosis ( 4 ). Apoptosis, or programmed cell death,
differs from necrosis in that dying cells participate actively in their
own death and generally do not induce ...



The importance of apoptosis in AIDS is controversial although there is
abundant in vitro evidence supporting a role for apoptosis in the pathogenesis of HIV-1 ( 12 ), and although mechanisms regulating this apoptosis in vivo are not clear, current ev...



The coat glycoprotein of HIV-1, gp160, is post-translationally cleaved
to an extracellular subunit, gp120, and a transmembrane subunit, gp41.
The subunits are non-covalently associated, and both are required for
viral entry into the cell. Two calmodu...



We demonstrate here that gp160 expression enhances FAS-mediated
apoptosis in Jurkat cells by increasing calmodulin expression and
accelerating caspase 3activation and that these effects that require
calmodulin binding to gp160 are blocked by a single...

EXPERIMENTAL PROCEDURES

TOP

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



 Cells and Cell Culture--

Jurkat cells were purchased from the
ATCC (Mannasas, VA) and grown at 37°C in 5% CO 2 in
RPMI 1640medium supplemented with 10% heat-inactivated fetal bovine
serum, 100units/ml penicillin, 100µg/ml streptomycin, and 2m M L -glutamine.



 Plasmids--

Plasmid pSRHS containing the HIV-1 envelope gene,
gp160, and the truncated forms of gp160, 147, which lacks the
C-terminal 147amino acids of gp41, and 67, which lacks the
C-terminal 67 amino acids of gp41, as well as the plasmid pKS8,
containing the ...



 Transfections--

All transient transfections were performed
using the cationic lipid, DMRIE-C (Life Technologies, Inc.). Briefly,
lipid-DNA complexes were allowed to form for 45min at room temperature in serum-free medium. Cells were added to the complex in serum-fre...



 Antibodies and Reagents--

Monoclonal antibody to calmodulin
was developed as described previously ( 20 ) and is available from
Upstate Biotechnology Inc. Lake Placid, NY. Mouse anti-human FAS
monoclonal antibody (Upstate Biotechnology Inc.) was used to induce
apoptosis. Mouse...



 Site-directed Mutagenesis--

Stratagene's (La Jolla, CA)
Quikchange Site-directed Mutagenesis Kit was used to make point
mutations of gp160 according to manufacturer's instructions. Primers
for the mutagenesis were purchased from Life Technologies, Inc., and
correspond to the de...

strain HXB2 as a template.



 Creation of gp160- and gp160A835W-expressing Cell
Lines--

Tet-Off Jurkat cells (, Palo
Alto, CA) were transfected with the pTRE expression vector containing
gp160 or gp160A835W cDNA by electroporation. Selection of stable
cell lines was initiated 48h after transfection using 100µg/ml
geneticin, 300µg/ml hyg...



 Northern Blot of gp160 and gp160A835W in Tet-off Jurkat Clones
Induced by Removal of Tetracycline--

Jurkat clones expressing HIV-1

gp160 and mutant gp160A835W were incubated with decreasing doses of
tetracycline for 48h. Total RNA was isolated by the guanidinium
thiocyanate/phenol/chloroform method and separated by formaldehyde gel
electrophoresis. RNA was blotted onto Hybond N ...



 Peptide Synthesis--

Peptides corresponding to the C-terminal
calmodulin-binding site of gp160 (residues 826-843) were
synthesized at the University of Alabama at Birmingham Comprehensive
Cancer Center Peptide Synthesis and Analysis Shared Facility and
purified by high p...



 TdT Apoptosis Staining--

In situ apoptosis
staining was performed using terminal deoxynucleotide transferase
(Roche Molecular Biochemicals). Briefly, cells were collected after
treatments and cytospun onto microscope slides and fixed in 10%
formalin in PBS. Slides were then ...

cobalt chloride). After a brief wash, slides were blocked in 1% bovine
serum albumin, 0.1% gelatin in PBS for 15min at room temperature.
Slides were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody for 1h at room temperature, ...



 Western Blot for Caspase 3and Calmodulin--

Jurkat cells were
treated as indicated and lysed in buffer containing 50m M

HEPES, pH 7.4,150m M NaCl, 10% glycerol, 1% Triton X-100, 1m M sodium orthovanadate, 10m M EDTA,
10m M EGTA, 1m M ammonium molybdate, 50m M NaF, 0.5µ M okadaic acid, 5m M

benzamidine, and 50µg/ml pepstatin. Equivalent amounts of protein
(100µg) were separated on 12.5% SDS-PAGE and transferred to
Immobilon P membrane (Millipore, Bedford, MA). Membranes were fixed in
0.2% glutaraldehyde in Tris-buffered saline (TBS ) fo...



 Dansyl-Calmodulin Binding--

The binding of calmodulin to
peptides corresponding to the C-terminal calmodulin-binding domain of
gp160 or the A835W mutant was determined fluorimetrically with
dansyl-calmodulin (Ocean Biologics, Corvalis, OR) as reported
previously ( 21 ). Briefly...

KCl, and 1m M CaCl 2 with or without 5 m M EGTA. Emission spectra were obtained with concentrations
of peptides ranging from 0to 1µ M, and mellitin was used
as a positive control for binding.

RESULTS

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ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



 Enhancement of Ceramide- and FAS-mediated Apoptosis by
gp160--

Jurkat cells were transfected with gp160 or
mock-transfected with empty vector and treated with a monoclonal FAS
antibody (clone CH11), and apoptosis was determined as described under "Experimental Procedures." Similar levels of apoptosis (11% in moc...

A ). Cells transfected with
gp160 undergo significantly more apoptosis (35±3%) than vector-transfected cells (25±1%) after treatment with FAS
antibody ( n =8, p <0.01). There was
no difference in basal or FAS-mediated apoptosis between
vector-transfe...





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Fig. 1.

Apoptosis in control Jurkat cells and
gp160-transfected Jurkat cells treated with anti-FAS or
C 2 -ceramide. Jurkat cells were transfected using
DMRIE-C with empty vector, pSRHS, or pSHRSgp160. Forty-eight hours
post-transfection, cells were treated ...




Previous studies have shown that ceramide levels increase in
HIV-1-infected cells in vitro ( 22 ) and that treatment of
latently infected cells with ceramide induces viral production ( 23 ). Furthermore, ceramide is a reported second messenger in FA...



 Effect of Calmodulin Antagonists on gp160-enhanced Ceramide-
and FAS-mediated Apoptosis--

Jurkat cells were transfected with
gp160 as described above and pretreated with 10µ M

tamoxifen (TMX ) or trifluoperazine (TFP ) for 30min prior to addition
of FAS antibody (Fig. ). Although TMX is
widely used as an anti-estrogen, it is as potent a calmodulin antagonist as TFP in the range of 1-10 µ M ( 25 ). TMX

( n =3) and TFP ( n =5) inhibited the
FAS-mediated apoptosis in gp160-transfected cells by 75±10and
90±2%, respectively (Fig. A ).

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FAS-mediated apoptosis was tested in the Tet-off cell
lines, both in the presence of tetracycline and 48h after removal
of tetracycline from the culture medium (Fig.
). In the presence of tetracycline, when
glycoprotein expression is repressed, both ...

A ). This level of apoptosis is consistent with the effect of anti-FAS treatment on mock-transfected Jurkat cells (Fig.

A ). In cells treated with anti-FAS for 3h following
removal of tetracycline, 70% of wild type gp160-expressing cells
undergo apoptosis, an increase of 2.5-fold over uninduced cells,
whereas the gp160A835W-expressing cells show no significant increas...





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Fig. 8.

Apoptosis and caspase 3activation in Tet-off
Jurkat cells expressing gp160 or gp160A835W. A, Tet-off
Jurkat cells transfected with either gp160 or gp160A835W were treated
with anti-FAS for 3h following 48h incubation in the
presence or absence of 2µg...

Tet-off Jurkat cells transfected with gp160 or gp160A835W or
untransfected Jurkat cells were incubated for 48h in the absence
of tetracycline to induce expression of glycoprotein and then treated
with 500ng/ml anti-FAS for the times indicated and lys...




Parallel effects on caspase 3activation were observed. Tet-off Jurkat
cells expressing gp160 or gp160A835W were treated with anti-FAS for the
indicated times, washed, and lysed, and equivalent protein was resolved
by SDS-PAGE and Western-blotted for...

DISCUSSION

TOP

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



Research on HIV-1 and AIDS has recently led to development of
treatments that have significantly decreased mortality in the United
States. However, the current highly active anti-retroviral treatment
does not eliminate the virus from the body ( 28 ) ...







Earlier work has shown that expression of HIV-1 envelope glycoprotein,
gp160, overcomes a block in the FAS pathway in Molt4 cells ( 19 ), a
FAS-resistant human T-cell line deficient in hematopoietic stem cell
phosphatase. The results presented here s...







The long cytoplasmic tail of gp160 is a feature conserved among HIV-1,
HIV-2, and simian immunodeficiency virus. No specific function has been
attributed to the cytoplasmic region, and despite considerable
variability in the overall sequence, all kno...







To determine whether the mechanism of gp160-dependent
enhanced apoptosis occurs at the level of the FAS receptor, we
investigated whether transfection of gp160 enhanced
ceramide-mediated apoptosis. Ceramide is a proposed mediator of
apoptosis produce...







Caspase 3,or caspase 3-like proteases, are critical effectors that
play an important role in most types of apoptosis ( 8 ). During
activation, caspase 3is first cleaved by an upstream caspase (caspase
8in the FAS pathway) and then undergoes a second ...

block gp160-enhanced FAS- and ceramide-induced apoptosis,
suggesting a calmodulin-dependent mechanism. The molecular site of action of calmodulin antagonists in gp160-enhanced apoptosis is
currently under investigation. However, the data presented he...







To confirm the effect of calmodulin binding by gp160 on FAS-mediated
apoptosis, we created a point mutation in the C-terminal CaM-binding
domain based on comparisons with the calmodulin-binding domain of MLCK

( 37-38 ). The sequence of the calmodulin-binding domains of both smooth
muscle and skeletal muscle MLCK were compared with the gp160
calmodulin-binding sequence. However, the smooth muscle MLCK was used
for computer modeling because its complex with...

( 18 ) reported that changing several basic residues to negatively
charged glutamic acid residues and single hydrophobic residues changed
to polar residues all reduced calmodulin binding and the lytic function of these peptides. Other reports have sh...







Although the mechanism by which gp160 up-regulates calmodulin is not
known, the simplest explanation is that gp160 may be acting as a
calmodulin sink, binding available calmodulin, and that cells
compensate by increasing expression of calmodulin. Cal...







Expression of gp160 has been reported to induce apoptosis without
stimulation by FAS antibody ( 40 ). Induction of apoptosis was
dependent on calmodulin binding to gp160, as cells expressing C-terminal truncations of as few as five amino acids did no...







There are also reports of HIV-1 and simian immunodeficiency virus
proteins other than gp160 that increase apoptosis, including Vpu ( 41 ),
Nef ( 42 ), and Tat ( 43 ). Whereas the proposed mechanisms that these
proteins use to enhance apoptosis vary, ...







Currently, there are no known calmodulin-dependent enzymes
directly involved in the FAS pathway, but data presented here suggest that at a minimum, calmodulin binding is required to mediate
gp160-enhanced apoptosis. It remains to be determined whethe...







In conclusion, these investigations provide strong evidence that gp160
expression enhances the cellular response to anti-FAS and increases
caspase 3activity and that these events require calmodulin binding to
gp160. The possibility that it is the inc...

FOOTNOTES



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