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Journal of Virology, June 2001, p. 5646-5655, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5646-5655.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Membrane-Fusing Capacity of the Human Immunodeficiency Virus
Envelope Proteins Determines the Efficiency of CD4+ T-Cell
Depletion in Macaques Infected by a Simian-Human Immunodeficiency
Virus
Bijan
Etemad-Moghadam,1
Daniela
Rhone,2
Tavis
Steenbeke,2
Ying
Sun,1
Judith
Manola,3
Rebecca
Gelman,3
John W.
Fanton,4
Paul
Racz,5
Klara
Tenner-Racz,5
Michael K.
Axthelm,4
Norman L.
Letvin,2 and
Joseph
Sodroski1,6,*
Department of Cancer Immunology and
AIDS1 and Department of Biostatistical
Sciences,3 Dana-Farber Cancer Institute,
Department of Pathology, Division of Viral Pathogenesis, Beth Israel
Deaconess Medical Center,2 Harvard Medical
School, and Department of Immunology and Infectious
Diseases, Harvard School of Public Health,6
Boston, Massachusetts 02115; Oregon Regional Primate
Research Center, Beaverton, Oregon 97006-34994;
and Department of Pathology and Korber Laboratory,
Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany
203595
Received 17 November 2000/Accepted 16 March 2001
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ABSTRACT |
The mechanism of the progressive loss of CD4+ T
lymphocytes, which underlies the development of AIDS in human
immunodeficiency virus (HIV-1)-infected individuals, is unknown. Animal
models, such as the infection of Old World monkeys by simian-human
immunodeficiency virus (SHIV) chimerae, can assist studies of HIV-1
pathogenesis. Serial in vivo passage of the nonpathogenic SHIV-89.6
generated a virus, SHIV-89.6P, that causes rapid depletion of
CD4+ T lymphocytes and AIDS-like illness in monkeys.
SHIV-KB9, a molecularly cloned virus derived from SHIV-89.6P, also
caused CD4+ T-cell decline and AIDS in inoculated monkeys.
It has been demonstrated that changes in the envelope glycoproteins of
SHIV-89.6 and SHIV-KB9 determine the degree of CD4+ T-cell
loss that accompanies a given level of virus replication in the host
animals (G. B. Karlsson et. al., J. Exp. Med. 188:1159-1171, 1998). The envelope glycoproteins of the pathogenic SHIV mediated membrane fusion more efficiently than those of the parental,
nonpathogenic virus. Here we show that the minimal envelope
glycoprotein region that specifies this increase in membrane-fusing
capacity is sufficient to convert SHIV-89.6 into a virus that causes
profound CD4+ T-lymphocyte depletion in monkeys. We also
studied two single amino acid changes that decrease the membrane-fusing
ability of the SHIV-KB9 envelope glycoproteins by different mechanisms.
Each of these changes attenuated the CD4+ T-cell
destruction that accompanied a given level of virus replication in
SHIV-infected monkeys. Thus, the ability of the HIV-1 envelope glycoproteins to fuse membranes, which has been implicated in the
induction of viral cytopathic effects in vitro, contributes to the
capacity of the pathogenic SHIV to deplete CD4+ T
lymphocytes in vivo.
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INTRODUCTION |
Human immunodeficiency viruses
(HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIV)
cause AIDS in humans and Old World monkeys, respectively (3, 10,
13, 14, 19, 41). AIDS results from the progressive loss of
CD4+ T lymphocytes (16, 17). The mechanisms
underlying the destruction of CD4+ T lymphocytes in HIV- or
SIV-infected primate hosts have not been elucidated. The specific
destruction of CD4+ T cells in infected hosts should be
distinguished from the generalized state of immune system activation
and the resultant increase in the number of apoptotic lymphocytes that
accompany these persistent viral infections (21, 22, 50).
This increased level of apoptosis is not limited to CD4+ T
cells, but also involves CD8+ T lymphocytes and B
lymphocytes (18, 21, 22, 50, 52), which are not typically
decreased in number in HIV- or SIV-infected hosts (16,
17). The degree of apoptosis does not necessarily correlate with
viral burden (50, 52), in contrast to the rate of
CD4+ T-cell depletion, which is strongly dependent upon the
level of virus replication (7, 14, 46, 49, 63). Studies of virus and cell turnover in HIV-1-infected humans (25, 74) suggest that virus-producing cells, but not latently infected cells,
undergo rapid destruction, apparently through nonapoptotic pathways
(18). Thus, virus-driven mechanisms such as virus-induced cytopathic effects and immunologic clearance of infected cells may make
important contributions to the specific loss of CD4+ T
lymphocytes in HIV-1-infected individuals.
Several HIV-1 components have been associated with cytotoxic effects in
tissue-cultured cells. The acute cytopathic changes observed in
virus-infected cultures include the formation of multinucleated syncytia and the lysis of single cells (12, 19, 66, 68). Both of these forms of cytopathic effect can be mediated by the viral
envelope glycoproteins (6, 34, 38, 40, 45, 65). The HIV-1
envelope glycoproteins, gp120 and gp41, support virus entry by binding
to the viral receptors, CD4 and chemokine receptors, on the target cell
and fusing the viral and the target cell membranes (8, 51, 70,
75, 77). HIV-1 envelope glycoproteins expressed on the infected
cell surface fuse these cells with receptor-bearing, uninfected cells,
leading to the formation of lethal syncytia (45, 65).
Intracellular envelope glycoprotein-receptor complexes (27) are capable of mediating membrane fusion reactions
that lead to the lysis of single cells (6). Recent studies
indicate that the majority of primary CD4+ T lymphocytes
expressing HIV-1 envelope glycoproteins lyse as single cells, due to a
process dependent upon the membrane-fusing capacity of the envelope
glycoproteins (40).
Other HIV-1 proteins can influence cell viability. The regulatory
protein Tat has been reported to induce both apoptotic and antiapoptotic effects in T cells (4, 43, 48, 56, 78). The
expression of Vpr results in cytostatic effects and apoptosis in some
cell lines (69). Vpr, however, is not required for
CD4+ T-cell depletion and the induction of AIDS in
SIV-infected monkeys (20, 26). The HIV-1 protease can also
cause some cytotoxic effects, but studies of viral mutants indicate
that most of the cytopathic effects associated with HIV-1 infection of
tissue-cultured cells are not mediated by this enzyme
(35). Nef has been reported to induce cytolysis of some
murine lymphoid cells (54) but, like Vpr, is not
absolutely required for the destruction of T cells and the development
of AIDS in SIV-infected monkeys (1).
The study of HIV-1 pathogenesis has been advanced by the availability
of animal models in which viral or host variables can be controlled.
Because HIV-1 does not replicate efficiently in species other than
humans and chimpanzees, chimeric simian-human immunodeficiency viruses
(SHIVs) have been created that can infect Old World monkeys (28,
44, 47, 64). SHIVs contain HIV-1-derived segments encoding the
viral envelope glycoproteins and the Tat, Rev, and Vpu regulatory
proteins in a SIV background (28, 44, 47, 64). A SHIV
containing the envelope glycoproteins of a primary HIV-1 isolate, 89.6, replicated efficiently in rhesus monkeys but did not deplete
CD4+ T lymphocytes or induce disease in these animals
(60). Serial transfer of blood from SHIV-89.6-infected
monkeys to naive monkeys generated a virus, SHIV-89.6P, that exhibited
only modest increases in replication in infected monkeys compared with
SHIV-89.6 (61). However, SHIV-89.6P caused rapid loss of
CD4+ T lymphocytes and, subsequently, AIDS-like illness in
inoculated monkeys (61). The risk of developing
AIDS-related disease in monkeys infected with SHIV-89.6P variants is
strongly influenced by the degree of decline in CD4+ T
lymphocytes during the acute phase of infection (2, 62).
SHIV-KB9 is a molecularly cloned, pathogenic virus derived from the
SHIV-89.6P isolate (31). The proviruses of the
nonpathogenic SHIV-89.6 and the pathogenic SHIV-KB9 differ in the
tat and env genes and in the long terminal
repeats (LTRs) (31). A recombinant virus, SHIV-89.6*,
which is identical to SHIV-89.6 except that it contains the
passage-associated changes in the LTR and tat gene,
replicated in monkeys but was not pathogenic (32). This result demonstrated that the passage-associated changes in the LTRs and
tat gene were not sufficient for the profound ability of
SHIV-KB9 to deplete CD4+ T lymphocytes in monkeys. The
passage-associated env changes alter the ectodomain of the
gp120 and gp41 envelope glycoproteins and the cytoplasmic tail of the
gp41 glycoprotein (31). The contribution of these
passage-associated changes in env to pathogenesis was
investigated by studying recombinant SHIVs, SHIV-KB9ecto and SHIV-KB9ct, that, in addition to the passage-associated LTR and tat changes, contain the changes in the envelope
glycoprotein ectodomains or cytoplasmic tail, respectively
(32). Both ectodomain and cytoplasmic tail changes were
shown to contribute to small increases in the average level of virus
replication achieved in infected monkeys (32).
Importantly, the envelope glycoprotein ectodomain changes, but not the
cytoplasmic tail changes, increase the degree of CD4+
T-cell depletion associated with a given level of virus replication in
the host animal (32). In vitro studies indicated that the 89.6 and KB9 envelope glycoproteins exhibited indistinguishable levels
of expression, processing, subunit association, representation on the
cell and virion surface, and CD4-binding ability (15, 32).
The KB9 envelope glycoproteins demonstrated increased chemokine receptor-binding affinity and membrane-fusing ability compared with the
89.6 envelope glycoproteins (15, 32). Both of these properties were determined by the gp120 and gp41 ectodomains (15, 32). Twelve amino acid differences exist between the 89.6 and KB9 envelope glycoprotein ectodomains (15, 31). The amino acid differences responsible for the increased chemokine
receptor-binding affinity and membrane-fusing ability of the KB9
envelope glycoproteins have been defined (15). This study
utilizes this genetic information to test the hypothesis that augmented
membrane-fusing activity is important for the ability of SHIV-KB9 to
deplete CD4+ T lymphocytes efficiently in vivo.
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MATERIALS AND METHODS |
Viruses.
Mutations were introduced into the env
sequence of the SHIV-KB9 3' proviral half by using the QuickChange
site-directed mutagenesis kit (Stratagene) and were confirmed by DNA
sequencing. Infectious viruses were produced by transfection of CEMx174
cells with proviral DNA as described previously (32, 44).
SHIV-KB9(
225) and SHIV-KB9(305) displayed replication kinetics
comparable to those of SHIV-KB9 and SHIV-89.6 in both CEMx174 cells and
rhesus monkey peripheral blood mononuclear cells (data not shown).
Animals.
Rhesus macaques were divided into groups according
to the inoculated virus; the groups exhibited similar average
CD4+ T-lymphocyte counts prior to virus inoculation. Rhesus
macaques were inoculated intravenously with 106 reverse
transcriptase units of the stock viruses. The rhesus monkeys used in
this study were maintained in accordance with established guidelines
(53). Monkeys were anesthetized with ketamine-HCl for all
inoculations and blood sampling. Peripheral lymph node biopsies were
performed under Telazol anesthesia.
Lymphocyte phenotyping and p27 quantification.
Blood was
obtained from the infected animals on days 3, 7 or 8, 10, 14, 17, 21, 29, and 36 after virus inoculation. Peripheral blood lymphocytes were
phenotyped for CD3/CD4 and CD3/CD8, as previously described
(59). Absolute lymphocyte counts in blood were determined
with an automated hematology analyzer (T540; Coulter Corp., Hialeah,
Fla.) that provided a partial differential count. The concentration of
SIVmac p27 core antigen in the plasma was determined by
using a commercial enzyme-linked immunosorbent assay kit (SIV core
[p27] antigen EIA kit; Coulter Corp.).
Fresh lymph node biopsies were obtained on days 10, 14, 21, and 43 after virus inoculation; individual cells were teased out, filtered,
and phenotyped as above.
Statistical analysis.
Cumulative p27 antigenemia was used as
a measure of viral replication; the area under the p27-versus-time
curve (days 0 to 21 following infection) was estimated using the
trapezoidal method. The "set point" for CD4+ T
lymphocytes for each animal was defined as the median of the absolute
CD4+ T-lymphocyte counts in the peripheral blood recorded
between days 14 and 36 following infection. Several mathematical models (linear and nonlinear exponential decay models, an inverse model, and a
hyperbolic model) were considered to be plausible candidates for
explaining the relationships between virus replication and CD4+ T-lymphocyte counts. The adjusted
R2 value was used to select the best-fitting
model (23). To test differences between the effect of each
virus group and its interaction term on CD4+ T-lymphocyte
set points, we used a partial F test with a
Bonferroni-adjusted alpha value of 0.025 (23, 33).
In situ hybridization for SHIV RNA in lymph nodes.
In situ
hybridization of lymph nodes obtained on day 10 after virus inoculation
was carried out as previously described (32, 71). Cells
with at least 20 silver grains, which corresponded to a sixfold
increase in silver grains over the background level, were scored as
viral RNA positive. By using epiluminescent illumination, viral
RNA-positive cells in 10 standard areas (450 by 700 µm) of the
T-cell-dependent zones in the lymph nodes were counted with a 20×
objective and the mean values were calculated.
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RESULTS |
SHIV envelope glycoprotein variants and phenotypes.
The
envelope glycoprotein ectodomains of the SHIV variants used in this
study are depicted in Fig. 1. SHIV-89.6*,
SHIV-KB9ct, and SHIV-KB9ecto are recombinants between the parental
SHIV-89.6 and the pathogenic SHIV-KB9 (31). SHIV-89.6*
is identical to SHIV-89.6 except that it contains the
passage-associated changes in the LTR and tat gene.
SHIV-KB9ct contains, in addition, the passage-associated changes in the
gp41 cytoplasmic tail. Thus, the envelope glycoprotein ectodomains of
SHIV-89.6, SHIV-89.6*, and SHIV-KB9ct are identical. As the
membrane-fusing capacity of SHIV-89.6/SHIV-KB9 recombinants is
determined by the envelope glycoprotein ectodomains (15,
32), SHIV-89.6* and SHIV-KB9ct exhibit a membrane-fusing ability
comparable to that of the parental SHIV-89.6. SHIV-KB9ecto contains the
passage-associated changes in the LTR, tat gene, and the
envelope glycoprotein ectodomains. Thus SHIV-KB9ecto and SHIV-KB9 share
envelope glycoprotein ectodomains and exhibit a high level of
membrane-fusing ability (15, 32).
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FIG. 1.
Structure and biological properties of the HIV-1
envelope glycoproteins used in this study. The envelope glycoprotein
ectodomains of the SHIV variants used in this study are depicted. The
gp41 ectodomain is shaded gray. The vertical bars represent the
positions of amino acids that are altered in the pathogenic SHIV-KB9,
compared with the parental SHIV-89.6 (31). The residue
number is indicated beneath the diagram, with numbering according to
recommended convention (37). The gp120 variable (V1 to -4)
and conserved (C2) regions in which the residues are located are
indicated. The SHIV-89.6, SHIV-89.6*, and SHIV-KB9ct envelope
glycoprotein ectodomains are identical, as are those of SHIV-KB9 and
SHIV-KB9ecto. The relative membrane-fusing ability of the envelope
glycoproteins was determined by cocultivating cells expressing
comparable levels of surface envelope glycoproteins with CEMx174
lymphocytes for 6 h at 37°C. The number of syncytia formed was
counted and normalized to that seen for the 89.6 envelope glycoproteins
(15). The relative ability of the gp120 glycoproteins to
bind to cells expressing the CCR5 chemokine receptor in the presence of
soluble CD4 was determined as described previously (15,
32). The binding obtained for the 89.6 gp120 glycoprotein was
assigned a value of 1. Relative CD4-binding ability was evaluated as
described previously (32) and was normalized to that of
the 89.6 gp120 glycoprotein.
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The SHIV-KB9 gp120 and gp41 ectodomains differ from those of SHIV-89.6
in 12 amino acid residues (
15,
31,
32). In a
previous
study (
15), each of these 12 amino acid residues in
the
KB9 envelope glycoproteins was changed back to the residue
found in the
89.6 envelope glycoproteins, and the effect of the
changes on
membrane-fusing ability was evaluated. We found that
two mutants,
KB9(
225) and KB9(
305), with single amino acid changes
in KB9 gp120
residues isoleucine 225 and glutamic acid 305, respectively,
exhibited
membrane fusion activity similar to that of the 89.6
envelope
glycoproteins (Fig.
1). The ability of the 89.6, KB9,
KB9(
225), and
KB9(
305) envelope glycoproteins to induce the
formation of syncytia
in a CD4
+ lymphocyte line is illustrated in Fig.
2. The KB9 envelope glycoproteins
induced
approximately five to six times as many syncytia as the
89.6 envelope
glycoproteins. The syncytium-forming abilities of
the KB9(
225) and
KB9(
305) mutants were comparable to that of
the 89.6 envelope
glycoproteins. These results are similar to
those previously obtained
in other CD4
+ cell lines and in primary CD4
+ T
lymphocytes (
15,
40). Some mechanistic insights into these
phenotypes have been obtained (
15) and are summarized in
Fig.
1. The change in isoleucine 225, within the second conserved (C2)
gp120 region, does not affect the interactions of the envelope
glycoproteins with either CD4 or the chemokine receptors
(
15).
Instead, this change apparently alters gp120-gp41
interactions
that are important for achieving fusion-competent
conformations
of the envelope glycoprotein complex (
15).
The change in glutamic
acid 305, within the gp120 third variable (V3)
loop, affects membrane
fusogenic capacity by decreasing the affinity of
gp120 for the
chemokine receptors (
15). Thus, changes in
gp120 residues 225
and 305, which are located on opposite faces of the
native gp120
glycoprotein (
39), exert quantitatively
similar effects on membrane-fusing
activity by different mechanisms.
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FIG. 2.
Syncytium-forming ability of the HIV-1 envelope
glycoprotein variants. COS-1 cells expressing equivalent surface levels
of the indicated HIV-1 envelope glycoproteins were cocultivated at
37°C with CEMx174 CD4+ lymphocytes, and syncytia were
scored after 6 h as described previously (15).
Control COS-1 cells were transfected with the  KS plasmid, which does
not express functional envelope glycoproteins. Means and
standard deviations of duplicate experiments are indicated.
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Replacement of the other 10 KB9 envelope glycoprotein residues that
were altered during virus passage with the amino acid
residues found in
the 89.6 envelope glycoproteins either did not
attenuate membrane
fusogenic capacity or exerted less dramatic
effects on this property
(
15). As an example of the former phenotype,
the five
passage-associated changes in the KB9 gp120 V1/V2 variable
loops were
not necessary for efficient syncytium-forming ability
in tissue culture
[see the 89.6(+225-567) recombinant in Fig.
1 and
2]. Each of the
seven passage-associated amino acid residues
remaining in the
89.6(+225-567) envelope glycoproteins was found
to contribute to the
full syncytium-forming ability of this glycoprotein
(
15).
Investigation of passage-associated changes sufficient for
CD4+ T-cell depletion.
The SHIV-89.6(+225/567)
envelope glycoproteins retain all of the KB9 envelope glycoprotein
residues, including isoleucine 225 and glutamic acid 305, shown to
contribute to enhanced membrane-fusing activity (15). To
examine whether the changes in these residues were sufficient for the
ability of SHIVs to cause CD4+ T-cell depletion in vivo,
two rhesus monkeys were inoculated with SHIV-89.6*, SHIV-KB9, or
SHIV-89.6(+225-567). As mentioned above, SHIV-89.6* is identical to
the parental SHIV-89.6 except that it contains the passage-associated
changes in the LTR and tat gene (32).
SHIV-89.6(+225-567) is identical to SHIV-89.6 except that it contains
the seven passage-associated amino acid changes spanning the gp120
carboxy-terminal half and the gp41 ectodomain (Fig. 1). The absolute
CD4+ T-lymphocyte counts in the inoculated monkeys are
shown in Fig. 3. Precipitous and profound
decreases in CD4+ T lymphocytes were observed in the
monkeys infected with SHIV-KB9 and SHIV-89.6(+225-567), but not in the
monkeys infected with SHIV-89.6*. These results indicate that the seven
amino acid changes in the gp120 and gp41 ectodomain segment
encompassing residues 225 to 567 are sufficient to convert SHIV-89.6
into a virus capable of causing rapid CD4+ T-lymphocyte
decline in rhesus monkeys. The results also show that the
passage-associated changes in the SHIV-KB9 gp120 V1/V2 loops (and in
the LTR, Tat protein, and gp41 cytoplasmic tail) are not required for
efficient CD4+ T-cell depletion in SHIV-infected monkeys.
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FIG. 3.
CD4+ T-lymphocyte counts in rhesus monkeys
infected with SHIV variants. Two rhesus monkeys were inoculated
intravenously with SHIV-89.6* ( ,  ), SHIV-KB9 ( ,  ), or
SHIV-89.6(+225-567) ( ,  ). The absolute CD4+
T-lymphocyte counts in the peripheral blood of the infected monkeys are
shown, with normal values in uninfected animals ranging from 1,000 to
1,400 cells per microliter.
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Effects of membrane fusion attenuation on CD4+
T-cell-depleting ability of SHIVs.
We wished to investigate the
effects of the membrane fusion-attenuating changes in gp120 residues
225 and 305 on the intrinsic CD4+ T-cell-depleting ability
of SHIV-KB9, i.e., the efficiency with which SHIV-KB9 depletes
CD4+ T lymphocytes at a given level of virus replication in
vivo. The degree of CD4+ T-cell loss in SHIV-infected
monkeys is influenced by the level of virus replication, which can vary
widely even among animals inoculated with identical viruses
(32). Thus, an assessment of the intrinsic
CD4+ T-cell-depleting ability of SHIV-KB9 variants requires
the evaluation of virus replication levels and CD4+ T-cell
counts in numerous monkeys. Previous studies defined the relationship
between the level of virus replication and the degree of
CD4+ T-lymphocyte depletion in rhesus monkeys infected by
SHIV-KB9/SHIV-89.6 recombinants that differ only in the sequences of
the envelope glycoprotein ectodomains (32). Analysis of
this data using a nonlinear exponential decay model indicated that, at
a given level of replication, viruses (SHIV-KB9 and SHIV-KB9ecto) with
KB9 ectodomain sequences deplete CD4+ T lymphocytes more
efficiently than viruses (SHIV-89.6* and SHIV-KB9ct) with 89.6 ectodomain sequences (P < 0.001) (32).
The only sequence differences between SHIV-KB9 and SHIV-KB9ecto, or
between SHIV-89.6* and SHIV-KB9ct, occur in the gp41 cytoplasmic tail
and do not contribute significantly to the efficiency of
CD4+ T-cell loss (32). To examine the
potential contribution of membrane fusogenicity to increased CD4+
T-cell-depleting ability, SHIV-KB9 derivatives altered in gp120
residues 225 and 305 [SHIV-KB9(225) and SHIV-KB9(305),
respectively] were created and inoculated intravenously into 11 rhesus
monkeys each. Additional monkeys were infected with SHIV-KB9 as
controls. The data obtained from these animals and from the animals in
the aforementioned study (32) were included in the
analysis of virus replication and CD4+ T-cell counts (Table
1). The level of virus replication
achieved in each monkey was described by the cumulative antigenemia
detected over the initial 3 weeks of infection, by which time the major decline in CD4+ T lymphocytes occurs in monkeys infected
with pathogenic SHIVs (Fig. 3). The CD4+ T-lymphocyte set
point was defined as the median of the absolute CD4+
T-lymphocyte counts in the peripheral blood between days 14 and 36 after virus inoculation, when CD4+ T-cell counts typically
stabilize in SHIV-infected monkeys (31, 32, 61). The
relationship between cumulative antigenemia and the CD4+
T-lymphocyte set point in the SHIV-infected monkeys is shown in Fig.
4. For the analysis of these data, the
infecting viruses were organized into four groups: viruses with 89.6 envelope glycoprotein ectodomains (SHIV-89.6* and SHIV-KB9ct), viruses
with KB9 envelope glycoprotein ectodomains (SHIV-KB9 and SHIV-KB9ecto),
SHIV-KB9(225), and SHIV-KB9(305). The data were analyzed using a
nonlinear exponential decay model; this model was chosen based on the
adjusted R2 value, which represents the
proportion of variability in CD4+ T-lymphocyte levels
explained by the model (23, 33, 67) (Table
2). The chosen model contains a virus
group-replication interaction term in addition to the main effect
terms, thus allowing the shapes of the curves to vary independently by
virus group. The interaction term improved the model's ability to
capture the sharp changes in CD4+ T-lymphocyte set points
occurring at low levels of cumulative antigenemia. Partial F
tests revealed significant differences in CD4+
T-cell-depleting ability between either SHIV-KB9(225) or
SHIV-KB9(305) and the SHIVs with KB9 ectodomain sequences (Table
3). The mean values for cumulative
antigenemia did not differ significantly among these three groups of
viruses [mean values were 21.6, 16.6, and 17.5 ng/day/ml for
SHIV-KB9(225), SHIV-KB9(305), and the SHIV-KB9/SHIV-KB9ecto group,
respectively]. There were no significant differences in
CD4+ T-cell-depleting ability between SHIV-KB9(225) and
SHIV-KB9(305) (P = 0.62) or between either of these
viruses and the viruses with the 89.6 ectodomain sequences. Analysis of
the data using the next best model, an inverse model with interaction
terms, yielded the same conclusions (Tables 2 and 3). Thus, the
passage-associated changes in residues 225 and 305 each significantly
contribute to the increased intrinsic efficiency with which viruses
with KB9 envelope glycoprotein ectodomains deplete CD4+ T
lymphocytes in SHIV-infected monkeys.
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FIG. 4.
Relationship between viremia and CD4+
T-lymphocyte counts in monkeys infected with SHIV variants. The set
point value for the peripheral blood CD4+ T-cell counts was
plotted against the cumulative p27 antigenemia for each infected
animal. The curves were fitted to the data using a nonlinear
exponential decay model with a viral group-replication interaction term
(23, 67). A single curve was fitted to the data for
SHIV-KB9 and SHIV-KB9ecto, which both contain KB9 envelope glycoprotein
ectodomains; likewise, a single curve was fitted to the data for
SHIV-89.6* and SHIV-KB9ct, which contain the 89.6 envelope glycoprotein
ectodomains.
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Analysis of lymph nodes from SHIV-infected monkeys.
The lymph
nodes of all of the macaques infected by SHIV variants in this study
and the majority of macaques infected in a previous study
(32) were examined. Analysis of lymph nodes taken from the
monkeys on days 10, 14, 21, and 43 after virus inoculation revealed
that the percentage of CD4+ T lymphocytes in the lymph
nodes corresponded to the value observed in the peripheral blood at all
of these time points (data not shown). Close inspection of the
T-cell-dependent areas of the lymph nodes did not reveal the presence
of syncytia (data not shown).
The number of cells in the T-cell-dependent areas of the lymph nodes
that were expressing SHIV RNA on day 10 after virus inoculation
was
determined by in situ hybridization (
71) (Table
1).
Previous
studies of SHIV-KB9 variants showed that the number of
virus-expressing
cells in the lymph nodes peaked around day 10 of
infection (
32,
62). To investigate the relationship
between the number of viral
RNA-positive cells in the lymph nodes and
the peripheral blood
CD4
+ T-lymphocyte set point, a
nonlinear exponential decay model was
again employed (
23,
67). The model had an
R2 value of 65%,
indicating that it explained a relatively large
proportion of the
variability. There was a statistically significant
exponential decline
in the CD4
+ T-cell set point as the number of viral
RNA-positive cells increased
(
P = 0.03). Thus, in
monkeys infected by the SHIVs studied herein,
there is a close
relationship between the number of virus-expressing
cells and the
overall decline in CD4
+ T
lymphocytes.
The observed effects of
env changes on the efficiency of
CD4
+ T-cell depletion and the relationship between the
number of virus-producing
cells and CD4
+ T-cell loss imply
that changes in the membrane fusogenicity of
the infecting SHIV might
alter the balance between the level of
viremia and the number of
virus-producing cells. To examine this,
we used the number of viral
RNA-positive cells in the lymph nodes
on day 10 after infection as an
indicator of the virus-producing
cell burden in the animals. The ratio
of cumulative antigenemia
to the number of viral RNA-positive cells was
compared between
groups of viruses with membrane fusogenicity
equivalent to that
of either SHIV-89.6 or SHIV-KB9. Figure
5 shows that this ratio
was significantly
lower for SHIV variants with greater membrane
fusogenicity
(
P = 0.0006; Wilcoxon rank sum test).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 5.
Ratio of viremia to the number of virus-producing cells
in macaques infected with SHIV variants differing in membrane
fusogenicity. The ratio of the cumulative antigenemia to the number of
viral RNA-positive cells measured in the lymph nodes on day 10 after
infection is shown for macaques infected with SHIV variants with less
fusogenic envelope glycoproteins [89.6*, KB9ct, KB9(225), and
KB9(305)], compared with that for SHIV variants with more fusogenic
envelope glycoproteins (KB9 and KB9ecto). In the three instances in
which viral RNA-positive cells in the lymph nodes were undetectable,
the lowest detectable value, 0.12, was used to calculate the ratio. The
median values for the two groups are 1.96 and 0.32, respectively
(P = 0.0006; Wilcoxon rank sum test).
|
|
| |
DISCUSSION |
The ability of the HIV-1 envelope glycoproteins to fuse cellular
membranes is critical for in vitro cytopathic effects (6, 38, 40,
45, 65), which include syncytium formation and single cell
lysis. The use of an animal model in which primate hosts are infected
with defined viruses allowed us to test the hypothesis that the
membrane-fusing capacity of the HIV-1 envelope glycoproteins
contributes to CD4+ T-lymphocyte-depleting ability in vivo.
In support of this hypothesis, a close relationship between the
membrane fusogenicity and in vivo CD4+ T-cell-depleting
ability of SHIV variants was observed. A SHIV identical to the
nonpathogenic parental virus, except for the seven envelope
glycoprotein residues required for efficient membrane-fusing capacity,
caused rapid and profound CD4+ T-cell depletion in the
monkeys. Several of the changes that arose during in vivo passage of
the pathogenic SHIV-KB9, for example the changes in the gp120 V1/V2
variable loops or the gp41 cytoplasmic tail, did not increase membrane
fusogenicity (15, 32) and were dispensable for efficient
CD4+ T-cell destruction in SHIV-infected monkeys. By
contrast, the changes in gp120 residues 225 and 305 affected both in
vitro membrane fusion activity and the efficiency with which SHIV-KB9
depleted CD4+ T lymphocytes at a given level of in vivo
replication. Because of the possibility of reversion of the introduced
mutations during the experiments, the actual impact of the changes in
residues 225 and 305 on in vivo CD4+ T-cell-depleting
ability may be greater than that observed. Nonetheless, both of these
mutant SHIVs exhibited relationships between CD4+ T
lymphocyte depletion and virus replication that were statistically indistinguishable from those seen for the group of nonpathogenic SHIV
variants (SHIV-89.6* and KB9ct). Both gp120 changes alter membrane
fusogenicity by different mechanisms, but do not affect envelope
glycoprotein processing, cell surface expression, subunit association,
CD4 binding affinity, or coreceptor choice (15, 32). The
buried position of residue 225 in the gp120 structure (39)
essentially restricts the potential interactions of this residue to
those involving other elements of the envelope glycoproteins. These
observations strongly argue that the ability to fuse membranes, and not
another fortuitously altered property of the envelope glycoproteins, is
important for the destruction of CD4+ T cells in
SHIV-infected animals.
A close relationship between the number of virus-producing cells and
the degree of CD4+ T-lymphocyte decline exists in
SHIV-infected monkeys (32). Enhanced membrane fusogenicity
of the viral envelope glycoproteins would be expected to increase the
efficiency of new infections and to decrease the vitality of at least
the virus-producing cells. This would increase the pool of infected
cells destined for destruction without necessarily modifying the level
of viremia. Thus, changes in the intrinsic ability of the virus to
damage virus-producing cells would alter the usual relationship
(55, 58, 71, 76) between the number of cells expressing
viral products and viremia. Consistent with this model, the ratio of
cumulative viremia to the number of viral RNA-positive cells in lymph
nodes was lower for SHIVs with more fusogenic envelope glycoproteins
than for the SHIVs with less fusogenic envelope glycoproteins.
As in most HIV-1-infected humans and SIV-infected monkeys (42,
52, 55, 57), syncytia are not prominent in the lymph nodes of
SHIV-infected animals. The rarity of multinucleated cells in vivo is
not inconsistent with a contribution of envelope glycoprotein membrane
fusogenicity to the demise of virus-producing cells. Recent studies
indicate that the HIV-1 envelope glycoproteins lyse most primary
CD4+ T lymphocytes as single cells, through a membrane
fusion-dependent process (40). The formation of
intracellular envelope glycoprotein-receptor complexes in the Golgi
apparatus apparently initiates membrane fusion events that compromise
the integrity of the cell membrane (6, 27).
The contribution of the death of uninfected cells to CD4+
T-lymphocyte depletion in SHIV-infected monkeys is uncertain
(32). If the death of uninfected cells contributes in a
quantitatively significant way to CD4+ T-cell decline in
this model, our results suggest that most of this death must depend
upon proximity to virus-producing cells or fusogenic virions. Assuming
that viral proteins would be secreted from virus-producing cells in
proportion to virions, they are less likely to contribute significantly
to the observed differences in the CD4+ T-cell-depleting
ability of SHIV-89.6 and SHIV-KB9. Further studies are needed to
clarify whether the death of uninfected cells or pathogenic processes
other than viral membrane fusion contribute to the loss of
CD4+ T lymphocytes in SHIV-infected monkeys.
SHIV-infected monkeys resemble HIV-1-infected humans with respect to
the relatedness of the infecting viruses, the evolutionary proximity of
the host species, the patterns and levels of virus replication, the
abnormalities of T-cell subsets that accompany infection, and the
eventual disease manifestations (24, 28-32, 44, 47, 60, 61,
64). Studies of viral replication and T-cell turnover in
HIV-1-infected individuals (26, 74) are consistent with
the possibility that viral cytopathic effects contribute to
CD4+ T-lymphocyte decline. SHIV-KB9 resembles the highly
cytopathic primary HIV-1 isolates that typically emerge later in
infection and that can utilize a broader range of chemokine receptors
(9, 11, 32, 36, 72, 73), thereby placing a greater
proportion of CD4+ T lymphocytes at risk of destruction
(5). These similarities make it likely that envelope
glycoprotein-mediated membrane fusion contributes to HIV-1
immunopathogenesis in humans. Intervention strategies targeting the
fusogenic function of the HIV-1 envelope glycoproteins might
therefore dually impact virus replication and CD4+ T-cell destruction.
| |
ACKNOWLEDGMENTS |
We thank Sheri Farnum and Yvette McLaughlin for manuscript preparation.
This work was supported by grants from the National Institutes of
Health (no. AI33832 and Center for AIDS Research Award no. AI28691) and
the European Commission and by the G. Harold and Leila Mathers
Foundation, the Friends 10, the late William F. McCarty-Cooper, and
Douglas and Judith Krupp.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dana-Farber
Cancer Institute, 44 Binney St., JFB 824, Boston, MA 02115. Phone:
(617) 632-3371. Fax: (617) 632-4338. E-mail:
joseph_sodroski{at}dfci.harvard.edu.
| |
REFERENCES |
| 1.
|
Baba, T.,
V. Liska,
A. Kimani,
N. Ray,
P. Dailey,
D. Penninck,
R. Bronson,
M. Greene,
H. McClure,
L. Martin, and R. Ruprecht.
1999.
Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.
Nat. Med.
5:194-203[CrossRef][Medline].
|
| 2.
|
Barouch, D. H.,
S. Santra,
J. E. Schmitz,
J. J. Kuroda,
T. M. Fu,
W. Wagner,
M. Bilska,
A. Craiu,
X. X. Zheng,
G. R. Krivulka,
K. Beaudry,
M. A. Lifton,
C. E. Nickerson,
W. L. Trigona,
K. Punt,
D. C. Freed,
L. Guan,
S. Dubey,
D. Casimiro,
A. Simon,
M. E. Davies,
M. Chastain,
T. B. Strom,
R. S. Gelman,
D. C. Montefiori,
M. G. Lewis,
J. Shiver, and N. Letvin.
2000.
Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.
Science
290:486-492[Abstract/Free Full Text].
|
| 3.
|
Barre-Sinoussi, F.,
J. C. Chermann,
F. Rey,
M. T. Nugeyre,
S. Chamaret,
J. Gruest,
C. Dauguet,
C. Axler-Blin,
F. Vezinet-Brun,
C. Rouzioux,
W. Rozenbaum, and L. Montagnier.
1983.
Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).
Science
220:868-871[Abstract/Free Full Text].
|
| 4.
|
Bartz, S. R., and M. Emerman.
1999.
Human immunodeficiency virus type 1 Tat induces apoptosis and increases sensitivity to apoptotic signals by up-regulating FLICE/caspase-8.
J. Virol.
73:1956-1963[Abstract/Free Full Text].
|
| 5.
|
Bleul, C. C.,
L. Wu,
J. A. Hoxie,
T. A. Springer, and C. R. Mackay.
1997.
The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA
94:1925-1930[Abstract/Free Full Text].
|
| 6.
|
Cao, J.,
I. W. Park,
A. Cooper, and J. Sodroski.
1996.
Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.
J. Virol.
70:1340-1354[Abstract].
|
| 7.
|
Chakrabarti, L.,
M. C. Cumont,
L. Montagnier, and B. Hurtrel.
1994.
Variable course of primary simian immunodeficiency virus infection in lymph nodes: relation to disease progression.
J. Virol.
68:6634-6642[Abstract/Free Full Text].
|
| 8.
|
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].
|
| 9.
|
Cheng-Mayer, C.,
D. Seto,
M. Tateno, and J. A. Levy.
1988.
Biologic features of HIV-1 that correlate with virulence in the host.
Science
240:80-82[Abstract/Free Full Text].
|
| 10.
|
Clavel, F.
1987.
HIV-2, the West African AIDS virus.
AIDS
1:135-140[Medline].
|
| 11.
|
Connor, R. I.,
K. E. Sheridan,
D. Ceradini,
S. Choe, and N. Landau.
1997.
Change in coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185:621-628[Abstract/Free Full Text].
|
| 12.
|
Dedera, D., and L. Ratner.
1991.
Demonstration of two distinct cytopathic effects with syncytium formation-defective human deficiency virus type 1 mutants.
J. Virol.
65:6129-6139[Abstract/Free Full Text].
|
| 13.
|
Desrosiers, R. C.
1990.
The simian immunodeficiency viruses.
Annu. Rev. Immunol.
8:557-578[CrossRef][Medline].
|
| 14.
|
Desrosiers, R. C.,
J. Lifson,
J. Gibbs,
S. Czajak,
A. Howe,
L. Arthur, and R. P. Johnson.
1998.
Identification of highly attenuated mutants of simian immunodeficiency virus.
J. Virol.
72:1431-1437[Abstract/Free Full Text].
|
| 15.
|
Eternad-Moghadam, B.,
Y. Sun,
E. K. Nicholson,
M. Fernandes,
K. Liou,
J. Lee, and J. Sodroski.
2000.
Envelope glycoprotein determinants of increased fusogenicity in a pathogenic simian-human immunodeficiency virus (SHIV-KB9) passaged in vivo.
J. Virol.
74:4433-4440[Abstract/Free Full Text].
|
| 16.
|
Fauci, A. S.
1988.
The human immunodeficiency virus: infectivity and mechanisms of pathogenesis.
Science
239:617-622[Abstract/Free Full Text].
|
| 17.
|
Fauci, A. S.,
A. M. Macher,
D. L. Longo,
H. C. Lane,
A. H. Rook,
H. Masur, and E. P. Gelmann.
1984.
NIH conference. Acquired immunodeficiency syndrome: epidemiologic, clinical, immunologic, and therapeutic considerations.
Ann. Intern. Med.
100:92-106.
|
| 18.
|
Finkel, T.,
G. Tudor-Williams,
N. Banda,
M. Cotton,
T. Curriel,
C. Monks,
T. Baba,
R. Ruprecht, and A. Kuppler.
1995.
Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.
Nat. Med.
1:129-134[CrossRef][Medline].
|
| 19.
|
Gallo, R. C.,
S. Z. Salahuddin,
M. Popovic,
G. M. Shearer,
M. Kaplan,
B. F. Haynes,
T. J. Palker,
R. Redfield,
J. Oleske,
B. Safai,
G. White,
P. Foster, and P. D. Markham.
1984.
Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.
Science
224:500-503[Abstract/Free Full Text].
|
| 20.
|
Gibbs, J.,
A. Lackner,
S. Lang,
M. Simon,
P. Sehgal,
M. Daniel, and R. Desrosiers.
1995.
Progression to AIDS in the absence of a gene for vpr or vpx.
J. Virol.
69:2378-2383[Abstract].
|
| 21.
|
Gougeon, M. L.,
S. Garcia,
J. Henney,
R. Tscfhopp,
H. Lecoeur,
D. Guetard,
V. Rame,
C. Dauguet, and L. Montagnier.
1993.
Programmed cell death in AIDS-related HIV and SIV infections.
AIDS Res. Hum. Retrovir.
9:553-563[Medline].
|
| 22.
|
Groux, H.,
G. Torpier,
D. Monte,
Y. Mouton,
A. Capron, and J. C. Amieson.
1992.
Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.
J. Exp. Med.
175:331-340[Abstract/Free Full Text].
|
| 23.
|
Hamilton, L.
1992.
Regression with graphics: a second course in applied statistics, p. 41.
Duxbury Press, Belmont, Calif.
|
| 24.
|
Harouse, J. M.,
A. Gettie,
R. C. Tan,
J. Blanchard, and C. Cheng-Mayer.
1999.
Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs.
Science
284:816-819[Abstract/Free Full Text].
|
| 25.
|
Ho, D. D.,
A. U. Neumann,
A. Perelson,
W. Chen,
J. M. Leonard, and M. Markowitz.
1995.
Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection.
Nature
373:123-126[CrossRef][Medline].
|
| 26.
|
Hoch, J.,
S. M. Lang,
M. Weeger,
C. Stahl-Hennig,
C. Coulibaly,
U. Dittmer,
G. Hunsmann,
D. Fuchs,
J. Muller,
S. Sopper,
B. Fleckenstein, and K. T. Uberla.
1995.
vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys.
J. Virol.
69:4807-4813[Abstract].
|
| 27.
|
Hoxie, J. A.,
J. D. Alpers,
J. Rackowski,
K. Huebner,
B. Haggarty,
A. Cedarbaum, and J. C. Reed.
1986.
Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV.
Science
234:1123-1127[Abstract/Free Full Text].
|
| 28.
|
Igarashi, T.,
R. Shibata,
F. Hasebe,
Y. Ami,
K. Shinohara,
T. Komatsu,
C. Stahl-Hennig,
H. Petry,
G. Hunsmann,
T. Kuwata,
M. Jin,
A. Adachi,
T. Kurimura,
M. Okada,
T. Miura, and M. Hayami.
1994.
Persistent infection with SIVmac chimeric virus having tat, rev, vpu, env and nef of HIV type 1 in macaque monkeys.
AIDS Res. Hum. Retrovir.
10:1021-1029[Medline].
|
| 29.
|
Joag, S. V.,
Z. Li,
L. Foresman,
E. B. Stephens,
L. Zhao,
I. Adany,
D. M. Pinson,
H. M. McClure, and O. Narayan.
1996.
Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques.
J. Virol.
70:3189-3197[Abstract].
|
| 30.
|
Joag, S. V.,
Z. Li,
L. Foresman,
D. M. Pinson,
R. Raghavan,
W. Zhuge,
I. Adany,
C. Wang,
F. Jia,
D. Sheffer,
I. Ranchalis,
A. Watson, and O. Narayan.
1997.
Characterization of the pathogenic KU-SHIV model of acquired immunodeficiency syndrome in macaques.
AIDS Res. Hum. Retrovir.
13:635-645[Medline].
|
| 31.
|
Karisson, G. B.,
M. Halloran,
J. Li,
I.-W. Park,
R. Gomila,
K. 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].
|
| 32.
|
Karlsson, G. B.,
M. Halloran,
D. Schenten,
J. Lee,
P. Racz,
K. Tenner-Racz,
J. Manola,
R. Gelman,
B. Etemad-Moghadam,
E. Desjardins,
R. Wyatt,
N. P. Gerard,
L. Marcon,
D. Margolin,
J. Fanton,
M. K. Axthelm,
N. L. Letvin, and J. Sodroski.
1998.
The envelope glycoprotein ectodomains determine the efficiency of CD4+ T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques.
J. Exp. Med.
188:1159-1171[Abstract/Free Full Text].
|
| 33.
|
Kleinbaum, D. G.,
L. L. Kupper, and K. E. Muller.
1988.
Applied regression analysis and other multivariable methods, 2nd ed., p. 126-131.
Duxbury Press, Belmont, Calif.
|
| 34.
|
Koga, Y.,
M. Sasaki,
K. Nakamura,
G. Kimura, and K. Nornoto.
1990.
Intracellular distribution of the envelope glycoprotein of human immunodeficiency virus and its role in the production of cytopathic effect in CD4+ and CD4 human cell lines.
J. Virol.
64:4661-4671[Abstract/Free Full Text].
|
| 35.
|
Konvalinka, J.,
M. Litterst,
R. Welker,
H. Kottler,
F. Rippmann,
A. Heuser, and H. Krausslich.
1995.
An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.
J. Virol.
69:7180-7186[Abstract].
|
| 36.
|
Koot, M.,
A. H. Vos,
R. P. Keet,
R. E. de Goede,
M. W. Dercksen,
F. G. Terpstra,
R. A. Coutinho,
F. Miedema, and M. Tersmette.
1992.
HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay.
AIDS
6:49-54[Medline].
|
| 37.
|
Korber, B. T.,
B. Foley,
C. Kuiken,
S. Pillai, and J. G. Sodroski.
1999.
Numbering position in HIV relative to HXB2. Human retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences.
Los Alamos National Laboratories, Los Alamos, N.Mex.
|
| 38.
|
Kowalski, M.,
L. Bergeron,
T. Dorfman,
W. Haseltine, and J. Sodroski.
1991.
Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein.
J. Virol.
65:281-291[Abstract/Free Full Text].
|
| 39.
|
Kwong, P. D.,
R. Wyatt,
J. Robinson,
R. W. Sweet,
J. Sodroski, and W. A. Hendrickson.
1998.
Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.
Nature
393:648-659[CrossRef][Medline].
|
| 40.
|
LaBonte, J.,
T. Patel,
W. Hofmann, and J. Sodroski.
2000.
Importance of membrane fusion mediated by the human immunodeficiency virus envelope glycoproteins for lysis of primary CD4-positive T cells.
J. Virol.
74:10690-10698[Abstract/Free Full Text].
|
| 41.
|
Letvin, N. L.,
M. D. Daniel,
P. K. Sehgal,
R. C. Desrosiers,
R. D. Hunt,
L. M. Waldron,
J. J. MacKey,
D. K. Schmidt,
L. V. Chalifoux, and N. W. King.
1985.
Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III.
Science
230:71-73[Abstract/Free Full Text].
|
| 42.
|
Lewin-Smith, M.,
S. Wahl, and J. Orenstein.
1999.
Human immunodeficiency virus-rich multinucleated giant cells in the colon: a case report with transmission electron microscopy, immunohistochemistry, and in situ hybridization.
Mod. Pathol.
12:75-81[Medline].
|
| 43.
|
Li, C.,
D. Friedman,
C. Wang,
V. Metelev, and A. Pardee.
1995.
Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.
Science
268:429-431[Abstract/Free Full Text].
|
| 44.
|
Li, J.,
C. I. Lord,
W. Haseltine,
N. L. Letvin, and J. Sodroski.
1992.
Infection of cynomolgus monkeys with a chimeric HIV-1/SIVmac virus that expresses the HIV-1 envelope glycoproteins.
J. Acquir. Immune Defic. Syndr.
5:639-646.
|
| 45.
|
Lifson, J. D.,
M. B. Feinberg,
G. R. Reyes,
L. Rabin,
B. Banapour,
S. Chakrabarti,
B. Moss,
F. Wong-Staal,
K. S. Steimer, and E. G. Engelman.
1986.
Induction of CD4-dependent cell fusion by the HTLV-III/LAV glycoprotein.
Nature
323:725-728[CrossRef][Medline].
|
| 46.
|
Lifson, J. D.,
M. A. Nowak,
S. Goldstein,
J. L. Rossio,
A. Kinter,
G. Vasquez,
T. A. Wiltrout,
C. Brown,
D. Schneider,
L. Wahl,
A. L. Lloyd,
J. Williams,
W. R. Elkins,
A. S. Fauci, and V. M. Hirsch.
1997.
The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.
J. Virol.
71:9508-9514[Abstract].
|
| 47.
|
Luciw, P. A.,
E. Pratt-Lowe,
K. E. Shaw,
J. A. Levy, and C. Cheng-Mayer.
1995.
Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).
Proc. Natl. Acad. Sci. USA
92:7490-7494[Abstract/Free Full Text].
|
| 48.
|
McCloskey, T. W.,
M. Ott,
E. Tribble,
S. A. Khan,
S. Teichberg,
M. O. Paul,
S. Pahwa,
E. Verdin, and N. Chirmule.
1997.
Dual role of HIV Tat in regulation of apoptosis in T cells.
J. Immunol.
158:1014-1019[Abstract].
|
| 49.
|
Mellors, J. W.,
C. Rinaldo,
P. Gupta,
R. White,
J. Todd, and L. A. Kingsley.
1996.
Prognosis in HIV-1 infection predicted by the quantity of virus in plasma.
Science
272:1167-1170[Abstract].
|
| 50.
|
Meyaard, L.,
S. A. Otto,
R. R. Jonker,
M. J. Mijnster,
R. P. Keet, and F. Miedema.
1992.
Programmed death of T cells in HIV-1 infection.
Science
257:217-219[Abstract/Free Full Text].
|
| 51.
|
Moore, J. P.,
A. Trkola, and T. Dragic.
1997.
Co-receptors for HIV-1 entry.
Curr. Opin. Immunol.
9:551-562[CrossRef][Medline].
|
| 52.
|
Muro-Cacho, C. A.,
G. Pantaleo, and A. S. Fauci.
1995.
Analysis of apoptosis in lymph nodes of HIV-infected persons. Intensity of apoptosis correlates with the general state of activation of the lymphoid tissue and not with stage of disease or viral burden.
J. Immunol.
154:5555-5566[Abstract].
|
| 53.
|
National Institutes of Health.
1985.
Guide for the care and use of laboratory animals, rev. ed. Department of Health and Human Services publication no. (NIH) 85-23.
National Institutes of Health, Bethesda, Md.
|
| 54.
|
Okada, H.,
R. Takei, and M. Tashiro.
1997.
Nef protein of HIV-1 induces apoptotic cytolysis of murine lymphoid cells independently of CD95 (Fas) and its suppression serine/threonine protein kinase inhibitors.
FEBS Lett.
417:61-64[CrossRef][Medline].
|
| 55.
|
Pantaleo, G.,
C. Graziosi,
J. F. Demarest,
O. J. Cohen,
M. Vaccarezza,
K. Gantt,
C. Muro-Cacho, and A. S. Fauci.
1994.
Role of lymphoid organs in the pathogenesis of human immunodeficiency virus (HIV) infection.
Immunol. Rev.
140:105-130[CrossRef][Medline].
|
| 56.
|
Purvis, S.,
J. Jacobberger,
M. Sramkoski,
A. Patki, and M. Lederman.
1995.
HIV type 1 Tat protein induces apoptosis and death in Jurkat cells.
AIDS Res. Hum. Retrovir.
4:443-450.
|
| 57.
|
Racz, P.,
K. Tenner-Racz,
F. van Vloten,
H. Schmidt,
M. Dietrich,
J. C. Gluckman,
N. Letvin, and G. Janossy.
1990.
Lymphatic tissue changes in AIDS and other retrovirus infections: tools and insights.
Lymphology
23:85-91[Medline].
|
| 58.
|
Reimann, K. A.,
K. Tenner-Racz,
P. Racz,
D. Montefiori,
Y. Yasutomi,
W. Lin,
B. Ransil, and N. Letvin.
1994.
Immunopathogenic events in acute infection of rhesus monkeys with simian immunodeficiency virus of macaques.
J. Virol.
68:2362-2370[Abstract/Free Full Text].
|
| 59.
|
Reimann, K. A.,
B. C. Waite,
D. E. Lee-Parritz,
W. Lin,
B. Uchanska-Ziegler,
M. J. O'Connell, and N. L. Letvin.
1994.
Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys.
Cytometry
17:102-108[CrossRef][Medline].
|
| 60.
|
Reimann, K. A.,
J. Li,
G. Voss,
C. Lekutis,
K. Tenner-Racz,
P. Racz,
W. Lin,
D. Montefiori,
D. Lee-Parritz,
R. Collman,
J. Sodroski, and N. Letvin.
1996.
An env gene derived from a primary HIV-1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.
J. Virol.
70:3198-3206[Abstract].
|
| 61.
|
Reimann, K. A.,
J. T. Li,
R. Veazey,
M. Halloran,
I.-W. Park,
G. B. Karlsson,
J. Sodroski, and N. L. Letvin.
1996.
A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys.
J. Virol.
70:6922-6928[Abstract/Free Full Text].
|
| 62.
|
Reimann, K. A.,
A. Watson,
P. J. Dailey,
W. Lin,
C. I. Lord,
T. D. Steenbeke,
R. A. Parker,
M. K. Axthelm, and G. B. Karlsson.
1999.
Viral burden and disease progression in rhesus monkeys infected with chimeric simian-human immunodeficiency viruses.
Virology
256:15-21[CrossRef][Medline].
|
| 63.
|
Ruprecht, R. M.,
T. Baba,
R. Rasmussen,
Y. Hu, and P. Sharma.
1996.
Murine and simian retrovirus models: the threshold hypothesis.
AIDS
10:S33-S40.
|
| 64.
|
Shibata, R.,
F. Maldarelli,
C. Siemon,
T. Matano,
M. Parta,
G. Miller,
T. Frederickson, and M. A. Martin.
1997.
Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing.
J. Infect. Dis.
176:362-373[Medline].
|
| 65.
|
Sodroski, J.,
W. C. Goh,
C. Rosen,
K. Campbell, and W. A. Haseltine.
1986.
Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity.
Nature
322:470-474[CrossRef][Medline].
|
| 66.
|
Somasundaran, M., and H. Robinson.
1987.
A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion.
J. Virol.
61:3114-3119[Abstract/Free Full Text].
|
| 67.
|
Stata Corporation.
1987.
Stata reference manual, release 5, p. 574-584.
Stata Corporation, College Station, Tex.
|
| 68.
|
Stevenson, M.,
C. Meier,
A. M. Mann,
N. Chapman, and A. Wasiak.
1988.
Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for persistence in AIDS.
Cell
53:483-496[CrossRef][Medline].
|
| 69.
|
Stewart, S. A.,
B. Poon,
J. Y. Song, and I. S. Y. Chen.
2000.
Human immunodeficiency virus type 1 Vpr induces apoptosis through caspase activation.
J. Virol.
74:3105-3111[Abstract/Free Full Text].
|
| 70.
|
Tan, K.,
J. Liu,
J. Wang,
S. Shen, and M. Lu.
1997.
Atomic structure of a thermostable subdomain of HIV-1 gp41.
Proc. Natl. Acad. Sci. USA
94:12303-12308[Abstract/Free Full Text].
|
| 71.
|
Tenner-Racz, K.,
H.-J. Stellbrink,
J. van Lunzen,
C. Schneider,
H.-P. Jacobs,
B. Raschdorff,
G. Großschupff,
R. M. Steinman, and P. Racz.
1998.
The unenlarged lymph nodes of HIV-1-infected, asymptomatic patients with high CD4 T cell counts are sites for virus replication and CD4 T cell proliferation. The impact of highly active antiretroviral therapy.
J. Exp. Med.
187:949-959[Abstract/Free Full Text].
|
| 72.
|
Tersmette, M.,
R. A. Gruters,
F. de Wolf,
R. E. de Goede,
J. M. Lange,
P. T. Schellekens,
J. Goudsmit,
H. G. Huisman, and F. Miedema.
1989.
Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates.
J. Virol.
63:2118-2125[Abstract/Free Full Text].
|
| 73.
|
Tersmette, M.,
J. M. Lange,
R. E. de Goede,
F. de Wolfe,
J. K. Eeftink-Schattenkerk,
P. T. Schellekens,
R. A. Coutinho,
J. G. Huisman,
J. Goudsmit, and F. Miedema.
1989.
Association between biological properties of human immunodeficiency virus variants and risk for AIDS and AIDS mortality.
Lancet
i:983-985.
|
| 74.
|
Wei, X.,
S. Ghosh,
M. Taylor,
V. Johnson,
E. Emini,
P. Deutsch,
J. Lifson,
S. Bonhoeffer,
M. Nowak,
B. Hahn,
M. Saag, and G. Shaw.
1995.
Viral dynamics in human immunodeficiency virus type 1 infection.
Nature
373:117-122[CrossRef][Medline].
|
| 75.
|
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].
|
| 76.
|
Wong, J. K.,
H. F. Gunthard,
D. V. Havlir,
Z. Q. Zhang,
A. T. Haase,
C. C. Ignacio,
S. Kwok,
E. Emini, and D. D. Richman.
1997.
Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure.
Proc. Natl. Acad. Sci. USA
94:12574-12579[Abstract/Free Full Text].
|
| 77.
|
Wyatt, R., and J. Sodroski.
1998.
The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.
Science
280:1884-1888[Abstract/Free Full Text].
|
| 78.
|
Zauli, G.,
D. Gibellini,
D. Milani,
M. Mazzoni,
P. Borgatti,
M. LaPlaca, and S. Capitani.
1993.
Human immunodeficiency virus type 1 Tat protein protects lymphoid, epithelial, and neuronal cell lines from death by apoptosis.
Cancer Res.
53:4481-4485[Abstract/Free Full Text].
|
Journal of Virology, June 2001, p. 5646-5655, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5646-5655.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
