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Journal of Virology, November 1998, p. 9045-9053, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Neuronal Death Induced by Brain-Derived Human
Immunodeficiency Virus Type 1 Envelope Genes Differs between Demented
and Nondemented AIDS Patients
C.
Power,1,2,*
J. C.
McArthur,3,4
A.
Nath,5
K.
Wehrly,6
M.
Mayne,1
J.
Nishio,6
T.
Langelier,1
R. T.
Johnson,3 and
B.
Chesebro6
Departments of Medical
Microbiology1 and
Internal
Medicine,2 University of Manitoba, Winnipeg,
Manitoba R3E 0W3, Canada;
Departments of
Neurology3 and
Epidemiology,4 Johns Hopkins University,
Baltimore, Maryland 21287;
Department of Neurology, University
of Kentucky, Lexington, Kentucky 405365; and
Laboratory of Persistent Viral Diseases, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, Montana
598406
Received 12 March 1998/Accepted 28 July 1998
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ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) infection of the brain
results in viral replication primarily in macrophages and microglia.
Despite frequent detection of viral genome and proteins in the brains
of AIDS patients with and without HIV dementia, only 20% of AIDS
patients become demented. To investigate the role of viral envelope
gene variation in the occurrence of dementia, we examined regions of
variability in the viral envelope gene isolated from brains of AIDS
patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven
demented and six nondemented AIDS patients displayed significant
sequence differences between clinical groups, and by phylogenetic
analysis, sequences from the demented group showed clustering.
Infectious recombinant viruses containing brain-derived V3 sequences
from both clinical groups were macrophagetropic, and viruses containing
brain-derived V1, V2, and V3 sequences from both clinical groups spread
efficiently in macrophages. In an indirect in vitro neurotoxicity assay
using supernatant fluid from HIV-1-infected macrophages, recombinant
viruses from demented patients induced greater neuronal death than
viruses from nondemented patients. Thus, the HIV-1 envelope diversity
observed in these patient groups appeared to influence the release of
neurotoxic molecules from macrophages and might account in part for the
variability in occurrence of dementia in AIDS patients.
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INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) is frequently detectable in the brains of HIV-infected
individuals at all stages of infection (4), but only 20% of
AIDS patients develop HIV dementia (HIV-D) (35). In both
HIV-D and HIV nondemented (HIV-ND) AIDS patients, productive HIV-1
infection in the brain is limited to nonneuronal cells, primarily
perivascular macrophages and microglia (20, 26, 58) and, to
a lesser extent, astrocytes (61, 70). Only certain HIV-1
isolates can infect macrophages (16), and tropism for these
cell types is influenced by specific amino acids within and adjacent to
the V3 hypervariable region (Fig. 1) of
the HIV-1 envelope (10, 41, 54, 69). Similar sequences influence tropism for microglia (23), and not surprisingly, V3 sequences from brain-derived HIV-1 closely resemble those of previously described blood-derived macrophagetropic viruses (48, 51). The extent of HIV-1 infection in macrophages is also
affected by other viral envelope regions including the V1 and V2
hypervariable regions (27), which appear to modulate the
efficiency of viral spread in macrophages (64). Although
altered V3 sequences associated with the syncytium-inducing
nonmacrophagetropic phenotype frequently arise during the progression
of clinical AIDS (24, 53), there are conflicting results
regarding the generation of variant V1-V2 sequences during progression
of clinical disease (21, 55, 67).
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FIG. 1.
The entire HIV-1 gp120 sequence (511 amino acids)
showing the regions of interest, including the V1-V2 and C2-V3
fragments and restriction sites used in this study. The V1 and V2
regions lie between the DraIII and StuI sites (76 amino acids), and the C2 and V3 regions are within the
StuI-to-NheI fragment (143 amino acids).
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Viral envelope gene variation is known to influence incidence of
neurological disease in a variety of retroviral and nonretroviral systems (13, 33, 60, 65). In several neurotropic murine retrovirus models, one or more changes in envelope amino acids are
sufficient to cause a switch from a virulent to a nonvirulent phenotype
(34, 42, 45, 71). Among HIV-1-infected humans, previous
studies of HIV-D and HIV-ND AIDS patients indicated that specific
brain-derived V3 sequences correlate with the occurrence of dementia
(48). Some of these sequences may influence specialized adaptation to growth in microglial cells as opposed to macrophages in
general (59), while other sequence differences might
determine neurotoxic effects, subsequent to macrophage or microglial
cell infection. The induction of neuronal injury or death may proceed from a complex cascade of events involving viral and host molecules to
cause the clinical manifestations of HIV-D. Nonetheless, how different
viral proteins might contribute to neural damage remains uncertain. It
has been proposed that HIV-1 envelope protein might be directly toxic
to neurons (14) or might mediate neuronal injury indirectly
through induction of toxic cytokines or other host molecules released
from infected microglia (32). However, other viral proteins,
including Tat (40) and gp41 (2), have also
been implicated in HIV-1 neuropathogenesis.
In the present study, brain-derived V1 and V2 envelope sequences from
prospectively studied individuals with or without HIV-D were examined
(47, 48), and significant differences were detected between
sequences of demented and nondemented groups. Recombinant viruses,
constructed by using portions of envelope genes obtained from both
clinical groups, showed similar patterns of infectivity and spread in
macrophages. However, neuronal death induced by supernatants from
HIV-infected macrophages was significantly greater among HIV-D-derived
viruses than among HIV-ND-derived viruses, suggesting that viral
envelope variability might be important in the pathogenesis of HIV-D.
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MATERIALS AND METHODS |
Sequence amplification and analysis.
V1-V2 envelope
sequences were obtained by using previously reported conditions in a
nested PCR protocol (48). Briefly, cDNA was synthesized from
total RNA, obtained directly from infected brains of patients described
previously (68), and was used as a template for the first
PCR. Two microliters of the product was added to the second PCR.
Oligonucleotides 6575/7330C (9) and 6575/6804C
(5'-ACAGGCCTATATAATGACT-3') were included in the first and
second PCRs, respectively. This amplification yielded a fragment of 230 bp that included both the V1 and V2 regions of the HIV-1 envelope gene.
Products from multiple amplifications of the same cDNA template were
cloned into the pCRII vector (Invitrogen), and the sequence was
analyzed by dideoxy sequencing. Sequence fragments were aligned with
CLUSTAL (DNAStar) by identity comparison of each residue, and
phylogenetic tree construction was performed by neighbor-joining and
maximal parsimony analyses. To ensure that contaminant viruses had not
been amplified, all sequences were compared to previously reported
HIV-1 V1-V2 sequences (38).
Construction of recombinant viruses.
Recombinant HIV-1
clones containing V1, V2, and V3 regions from each patient were
generated with a two-step construction process. First, brain-derived
V1-V2 sequences from patients were excised from the pCRII cloning
sites, DraIII and StuI (Fig. 1). These fragments
were ligated into the DraIII and StuI sites of a
plasmid which contained the envelope sequence from the EcoRI
(5743) to DraIII (6591) sites of the HIV-1 clone NL4-3, with
an adjacent polylinker encoding
DraIII-StuI-XbaI-MluI-NheI-BsuI-BamHI
sites. This cassette was in a vector, p4-8b, derived from pBluescript KS(+) but lacking the DraIII site at position 230, thus
making the DraIII site in the HIV-1 envelope unique. Next,
HIV-1 sequences from EcoRI to StuI were excised
from clones generated from each patient's cDNA and were ligated into
plasmids described previously (47), containing C2-V3
sequences from StuI and NheI sites from each
patient in the NL4-3 background. To facilitate this cloning step, each
of these vectors was previously modified by replacement of the original
NL4-3 sequence from EcoRI to StuI by a synthetic oligonucleotide. The resulting vectors were transfected into
CD4-expressing HeLa cells that were then cocultivated for 24 h
with uninfected phytohemagglutinin-stimulated human peripheral blood
mononuclear cells (PBMC). PBMC were then maintained in
interleukin-2-containing medium to obtain infectious virus stocks as
previously described (6).
In vitro infectivity assays.
All viruses derived from
transfection studies were titrated in PBMC as previously reported
(6). HIV-1 infectivity studies were performed in
CD4-positive HeLa (HeLa-CD4) cells (clone 1022) (8),
CD4-positive, CKR5-positive HeLa (HeLa-CD4/CKR5) cells (clone JC37)
(44), and primary human macrophages by using established methods (6). Viral infection and replication were measured as supernatant p24 levels or by staining with anti-p24 antibody in a
focal immunoassay (7). Controls included uninfected cells or
cells infected with viruses of known cell tropism, including NL4-3 and
JR-FL.
Neurotoxicity assay.
Neuronal cultures were prepared from
12- to 15-week gestational fetuses with approval of the Human Ethics
Committee at the University of Manitoba as previously reported
(40). Briefly, the meninges and blood vessels were removed,
the tissue was mechanically dissociated, cells were resuspended in
Opti-MEM (GIBCO) with 1% heat-inactivated fetal bovine serum, 1% N2
supplement (GIBCO), and 1% antibiotic solution (104 U of
penicillin G per ml and 10 mg of streptomycin B per ml in 0.9% NaCl),
seeded in 96-well microtiter plates at 105 per well, and
maintained for a minimum of 4 weeks prior to use. Sample wells were
immunostained for the neuronal marker microtubule-associated protein 2, and only cultures in which >70% of the cells stained positive for the
marker were used for experiments. The remaining cells were principally
astrocytes, as indicated by glial acidic fibrillary protein
immunopositivity with rare microglia (<1%) which immunostained with
EBM-11 (46).
Primary human macrophages were infected with 10
2 to
10
3 50% tissue culture infective doses
(TCID
50) of different HIV recombinants
containing
brain-derived C2-V3 or V1-V3 sequences or of JR-FL.
Culture supernatant
was harvested as conditioned medium (CM) at
days 3, 7, and 10 postinfection and stored at
80°C. Prior to
application to neuronal
cultures, CM was centrifuged at 13,000
rpm for 10 min to clear cellular
debris, mixed at several dilutions
with Opti-MEM containing 0.1% fetal
bovine serum, and applied
to the cultured neurons for 3 to 24 h.
The neuronal cultures were
stained subsequently with trypan blue to
identify dead cells and
fixed in 4% paraformaldehyde, and the number
of neurons with trypan
blue-positive nuclei per unit area was
determined over five randomly
chosen fields by an examiner unaware of
the specific treatment.
Neuronal death was expressed as the percentage
of trypan blue-stained
neurons to total number of neurons counted.
Background neuronal
death in untreated fetal neuronal cultures varied 4 to 8%, depending
on the age of the fetus, and thus was subtracted from
total neuronal
death for each experiment. To assess the role of the
glutamate
receptors in the neurotoxicity observed, neuronal cultures
were
pretreated with the
N-methyl-
D-aspartate
(NMDA) receptor antagonist
AP5 and the
amino-3-hydroxy-5-methyl-4-isoazole propionate (AMPA)
receptor
antagonist CNQX (RBI, Natick, Mass.) before application
of the CM.
Individual experiments were conducted in triplicate
wells and repeated
at least twice; means and standard errors of
the means (SEM) were
determined.
Statistical tests.
Statistical analyses comparing groups
were made by using nonparametric (Mann-Whitney U),
parametric (Student's t), or Fisher's exact test.
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RESULTS |
HIV-1 envelope sequence analysis.
We compared the V1-V2
sequences (Fig. 1) derived from brains of HIV-D and HIV-ND patients in
our prospectively studied population (2, 20, 48, 68) (Table
1). Phylogenetic analysis of V1-V2
sequences from HIV-D and HIV-ND patients showed that clones from the
same patient were closely related, but individual patients displayed
marked sequence divergence with low bootstrap values (Fig.
2). V1-V2 sequences in five of the seven
HIV-D patients whose severity of dementia was measured by the Memorial
Sloan-Kettering (MSK) scale (49) were clustered together,
while no clusters of three or more patients within the HIV-ND group
were observed. When specific residues at each position of all
brain-derived sequences were compared (Fig.
3), a lysine at position 130 predominated in the HIV-D group in 7 of 11 clones and was never present at the same
position in the ND group. There were numerous positions at which two or
more patients in one group shared an identical residue that was not
present at the same position in the other clinical group. These were
termed unique amino acids (Fig. 3, circled) and were more frequently
observed among clones from HIV-D clones. Thus, the above findings
resembled those previously observed in brain-derived C2-V3 envelope
sequences in which HIV-D and HIV-ND sequences differed significantly at
specific positions and the number of unique amino acids was greater in
the HIV-D group (48). Together with the phylogenetic
analysis indicating the clustering of five of seven HIV-D sequences,
these observations suggest that specific V1 and V2 sequences were also
closely associated with the development of HIV-D.
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FIG. 2.
Phylogenetic comparison of brain-derived V1-V2 sequences
obtained from HIV-D (D) and HIV-ND (ND) individuals with AIDS and the
consensus sequence from clade D (con.D clade) (38), using
neighbor-joining analysis (30). Numbers refer to individual
patients, and letters (A and B) refer to clones from the same patient.
Two clones were analyzed for each patient. Clones from five of seven
HIV-D patients clustered together, while none of the HIV-ND patients
showed close associations. The horizontal axis represents the number of
substitution events. Analysis of the V1 and V2 sequences separately
revealed no clustering of sequences from the same clinical group.
Similar topologies were obtained with different phylogenetic methods,
but bootstrap values were low (<70) between individuals.
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FIG. 3.
Brain-derived V1-V2 envelope sequences from HIV-D (D)
and HIV-ND (ND) patients aligned with the brain consensus sequence and
corresponding sequences from established macrophagetropic viruses.
HIV-D sequences are presented in order of severity of dementia
according to MSK score shown at the right. Two clones were sequenced
from each patient except for patients 68, 20, 19, and 69. Position 130 (boxed) shows significant difference between HIV-D and HIV-ND groups
(Fisher's exact, P < 0.01), with a lysine
predominating in the HIV-D group and a variable amino acid in the
HIV-ND group. Comparison of the frequency of the Lys130 in established
databases (38) revealed that it was detected significantly
more frequently in the HIV-D sequences than in the database (Fisher's
exact, P < 0.01). Circled amino acids identified in
two or more patients in one group, but not present in the other group
at a specific position, were termed unique. The mean number of unique
amino acids per clone ± SEM was significantly greater in the
HIV-D group (3.0 ± 0.44) than in the HIV-ND group (1.6 ± 0.20) (Student's t test, P < 0.05). In
patients with two or more clones, the difference between clones varied
from zero to three amino acids. The length of the V2 region, the number
of positively charged amino acids, and number of potential
glycosylation sites did not differ between HIV-D and HIV-ND groups.
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Tropism of brain-derived recombinant HIV clones.
Previous
studies of recombinant viruses containing brain-derived C2-V3 envelope
sequences indicate that these sequences influence the ability of HIV-1
to infect macrophages and mixed glial cells (47). The V1-V2
envelope regions of HIV-1 appear to enhance the efficiency of
replication in macrophages in vitro, at least in part, by increasing
viral spread (64). To further examine the effect of
brain-derived V1-V2 sequences, V1-V2 sequences from two HIV-D patients
and three HIV-ND patients were inserted into recombinant viruses
already containing C2-V3 sequences from these same patients. These
clones were compared to the recombinant viruses containing only the
C2-V3 envelope sequences from the same patients. All recombinant
viruses with either C2-V3 or V1-V3 brain-derived envelope sequences
infected HeLa-CD4/CKR5 cells efficiently but failed to infect HeLa-CD4
cells lacking CKR5 expression (Table 2).
Thus, by these criteria, all recombinant viruses utilized the
CKR5 coreceptor, similar to what occurs with other
macrophagetropic viruses (5, 56), but no
difference between recombinant viruses containing brain-derived C2-V3
versus V1-V3 sequences was seen.
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TABLE 2.
Comparison of infectivities of recombinant HIV-1 clones
containing brain-derived V1-V3 and C2-V3 sequences in different
cell types
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In experiments where infectivity of these clones was also analyzed in
human macrophages, V1-V3 recombinant viruses showed
a large increase in
p24 levels and the number of positive cells
with time postinfection. In
contrast, the recombinant viruses
containing only C2-V3 sequences from
patients produced p24 levels
and numbers of infected macrophages that
were lower and failed
to increase with time after infection even when
10-fold-higher
amounts of virus were used to infect cells (Fig.
4). Thus, the
addition of the V1 and V2
regions of brain-derived HIV-1 to the
C2-V3 containing clones from each
patient enhanced viral replication
and spread in macrophages. However,
infectivity of the V1-V3 clones
did not differ significantly between
HIV-D- and HIV-ND-derived
viruses. These results indicated that the
ability to replicate
efficiently and spread in macrophages might be a
property common
to all brain-derived HIV-1 envelope sequences but did
not correlate
with the development of HIV-D.
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FIG. 4.
Infectivity of brain-derived C2-V3- and V1-V3-containing
recombinant viruses measured by p24 levels in macrophage culture
supernatant (A) or p24-positive foci (B) over time. At an input titer
of 102 TCID50/0.1 ml, V1-V3 clones (open
circles) replicated to a greater extent in macrophages, reflected by
higher p24 levels in supernatant (A) and progressively increasing
numbers of p24-positive cells (B), compared to the matched C2-V3 clones
(closed circles) for both HIV-D- and HIV-ND-derived viruses. When a
10-fold-higher input of C2-V3 viruses was used (closed squares), higher
p24 values and focus counts were observed for all viruses, confirming
that these C2-V3 clones were macrophagetropic. The increasing p24 level
in patient 17 did not reflect an increase in focus number and probably
was due to accumulation of p24 in the medium, since all medium was not
removed when cells were fed every third day.
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Virus-induced neuronal toxicity.
Since neurons are rarely, if
ever, infected by HIV-1, the mechanism of neuronal injury and/or death
resulting from HIV-1 infection of the brain is assumed to be indirect,
possibly due to the release of neurotoxic molecules by HIV-1-infected
macrophages or microglia (17, 50). Therefore, we examined
human fetal neuronal cultures after treatment with CM from macrophage
cultures infected with different HIV-1 clones. CM from macrophages
infected with JR-FL, a brain-derived isolate from a patient with HIV-D
(29), produced a maximum neuronal death when diluted to 50 to 66% with fresh medium, but 100% CM produced a reduced neuronal
death rate (Fig. 5A). Comparison of
neurotoxicity induced by CM harvested at days 1, 3, 7, and 10 after
infection of macrophages with JR-FL revealed that maximal neuronal
death was induced by CM from days 1 and 3 (Fig. 5B). Consistent with
previous experiments, viral reverse transcriptase assays indicated that
background values were detected on days 1 and 3 and significantly
higher virus release occurred on days 7 and 10 (data not shown). Thus,
there was no correlation between neurotoxicity and the presence of
virus and viral proteins in supernatant fluids. To determine if the
duration of exposure to CM influenced the extent of neuronal death
induced by CM, neuronal death rates after treatment of neuronal
cultures for 3, 12, and 24 h were compared. Neuronal death did not
differ significantly at the three time points tested (Fig. 5C),
suggesting that neuronal death occurred rapidly and that only a
subpopulation of neurons were susceptible to killing in this assay.
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FIG. 5.
Comparison of mean percentage (±SEM) of neuronal death
induced by CM from macrophages infected with HIV-1 JR-FL, uninfected
macrophages (control), or NMDA treatment (A), CM harvested from
different time points after infection (B), and different times of
exposure of neuronal cultures to CM (C). JR-FL induced neurotoxicity at
various dilutions of CM (A), with maximum neuronal death at a 50 to
66% dilution. Neuronal cultures treated with 50% CM from uninfected
macrophages showed significantly less neuronal death than CM at the
same concentration from JR-FL-infected macrophages (Student's
t test, P < 0.001). A decline in neuronal
killing was observed with 50% CM harvested at later time points
postinfection (B). The extent of neuronal death did not change with
different times of exposure to CM, harvested at 3 days postinfection
(C). Macrophages were infected at an input titer of 103
TCID50/0.1 ml.
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With respect to neuronal death induced by CM from macrophage cultures
infected with recombinant HIV-1 clones containing brain-derived
envelope sequences, the viruses containing the brain-derived C2-V3
regions from the HIV-D group induced significantly higher levels
of
neuronal death than the HIV-ND group (Fig.
6). Comparison of
individual
HIV-D-derived and individual HIV-ND-derived viruses
showed that each
HIV-D-derived virus induced significantly higher
levels of neuronal
death than each HIV-ND-derived virus. Comparing
the five clones
containing brain-derived V1-V3 sequences in the
neurotoxicity assay, we
found a trend toward increased neuronal
death induced by the two HIV-D
clones compared to the HIV-ND clones,
but too few clones were tested to
give this observation statistical
significance (Fig.
7). Nevertheless, the percentage of
neuronal
death induced by all the V1-V3-containing clones (9 to 14%)
was
higher than that induced by the C2-V3-containing clones (1 to
8.5%), implying that neuronal death may be influenced by more
than one
region of the HIV-1 envelope. This latter effect may
be due to the fact
that more macrophages were infected on day
3 by the brain-derived
V1-V3-containing clones than by the C2-V3-containing
clones (Fig.
4).
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FIG. 6.
Mean percentage of neuronal death (±SEM) caused by CM
from control (uninfected) or JR-FL-infected macrophage cultures or
macrophage cultures infected with recombinant viruses containing
brain-derived C2-V3 sequences from HIV-D and HIV-ND individuals. Mean
neuronal death caused by all recombinant HIV-D viruses was
significantly greater than neuronal death caused by all HIV-ND viruses
(Student's t test, P < 0.0001). Each
HIV-D-derived virus caused significantly more neuronal death than each
HIV-ND-derived virus when the isolates were compared individually
(P < 0.05). Macrophages were infected at an input
titer of 103 TCID50/0.1 ml.
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FIG. 7.
Comparison of mean percentages (±SEM) of neuronal death
induced by CM for brain-derived V1-V3-containing recombinant viruses
from two HIV-D and three HIV-ND patients compared to CM from control
uninfected CM. For all five patients, the recombinant viruses caused
greater neuronal death than the uninfected CM (Student's t
test, P < 0.001). HIV-D clones caused greater
neurotoxicity than corresponding HIV-ND clones. Macrophages were
infected at an input titer of 102 TCID50/0.1
ml.
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Several neurotoxic molecules that vary in stability and mechanism of
action have been implicated in different assays of HIV-induced
neuronal
death (
19,
40). To characterize the neurotoxin released
by
macrophages in the present assay, we determined the stability
of the CM
from macrophages infected by a recombinant virus from
an HIV-D patient
(clone 62-1). CM was boiled for 10 min, brought
to 37°C, and applied
to neuronal cultures. The percentage of neurotoxicity
of the boiled CM
did not differ significantly from that of the
untreated supernatant,
indicating that the neurotoxic molecule(s)
was heat stable (Fig.
8). Since glutamate receptor-mediated
mechanisms
have been suggested to participate in HIV-induced
neurotoxicity
(
3,
14), we pretreated the neuronal cultures
with an NMDA
(AP5) or AMPA (CNQX) receptor antagonist. Application of
CM from
macrophages infected with the virus clone 62-1 revealed that
after
the NMDA receptor was blocked with 0.5 mM AP5, neuronal death
was
significantly reduced (Fig.
8). In contrast, blocking the
AMPA receptor
with CNQX caused no inhibition of neurotoxicity.
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FIG. 8.
Comparison of mean neuronal death rates induced by CM
from macrophages infected by the recombinant virus 62-1, containing the
brain-derived C2-V3 sequences from HIV-D patient 62, after boiling for
10 min or pretreatment with AP5 or CNQX. AP5 (0.5 mM) significantly
reduced neuronal killing by CM (Student's t test,
P < 0.01). AP5 or CNQX at identical concentrations
without CM did not influence neuronal death rates (data not shown).
Macrophages were infected at an input titer of 103
TCID50/0.1 ml.
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DISCUSSION |
The results presented above indicate that HIV-1 envelope
sequence diversity influenced induction of neuronal death. In addition, the ability of a recombinant virus to cause neuronal death was associated with the clinical status of the individual patient from
which the envelope sequence was derived. These findings suggest that envelope protein itself may be important in causing neurological disease and that envelope sequence variation may account in part for
the variation in occurrence of clinical HIV-D. The current studies also
indicate that in both HIV-D and HIV-ND patients, brain infection
appears to select for viruses that express a V3 envelope region capable
of mediating infection of macrophages and microglia and using CCR5 as a
coreceptor. Furthermore, the patients in both groups also selected
viruses with V1 and V2 regions that could cause a spreading infection
in macrophage cultures in vitro. Thus, a spreading infection in
macrophages and microglia may be a necessary feature of HIV infection
in brain, although the spreading phenotype by itself is not sufficient
to cause clinical HIV-D. In addition, induction of dementia may require
the presence of specific viral sequences such as the
dementia-associated sequences detected in the V1 and V3 regions (Fig.
3) (48).
The phylogenetic clustering observed in the V1-V2 sequences of many of
the demented patients in this study was unexpected. The HIV-1 strains
which appear early after seroconversion resemble macrophagetropic
viruses (36), suggesting that such viruses are selected
during transmission between individuals. However, no similar selection
for common V1 or V2 sequences has yet been observed. HIV replication in
patients is known to give rise to a wide diversity of mutant or variant
viruses which would be subjected to many selective pressures during the
course of the infection. The finding of a cluster among V1-V2 sequences
from HIV-D patients might suggest either that these patients were
initially infected with a similar and somewhat unique virus or that
these patients have exerted similar selective pressures on their
viruses during infection. This could be due to similar immune response
genes leading to selection for or against certain viral variants, or it
could be due to other host genetic or nongenetic factors which these
patients have in common. Whatever viral or host factors account for
this cluster may also have the capability of influencing the occurrence
of clinical dementia.
Since HIV does not directly infect neurons, the pathogenesis of HIV-D
is likely a complex indirect multistep process extending from virus
entry into the brain to infection of brain microglial cells and
production of molecules capable of damaging neurons (32).
Many different viral and host factors could simultaneously influence
the extent and/or tempo of the disease. In fact, even a single viral
gene such as env could have several different effects that
might map to similar or different regions of the envelope protein. For
example, certain V3 amino acid residues affect macrophage tropism
(10), V1 and V2 sequences affect virus spread in macrophages (64), and certain V1 and V3 sequences correlate with
dementia (48). In the present study, the addition of the
brain-derived V1-V2 fragment increased replication in macrophages,
resulting in greater induction of neurotoxicity by viruses from both
nondemented and demented patients. Hence, replication level and
neurotoxicity potential are interrelated viral features that may
influence the occurrence of neurological disease. Similar phenomena
have been observed in brain disease induced by other retroviruses.
Tropism of simian immunodeficiency virus (SIV) for brain microglial
cells is correlated with the selection of particular sequences in
several different envelope regions (25, 31); however,
additional specific envelope sequences in the transmembrane region may
also be required for induction of clinical brain disease
(33). Similarly, the murine polytropic retrovirus Fr98 has
two separate regions of the envelope gene which contain determinants of
neurovirulence that may act by different mechanisms to facilitate the
same clinical neurological disease (22, 45, 52).
These studies used an indirect neurotoxicity assay to determine the
cytotoxic effects of viral envelope sequences derived from brain tissue
of AIDS patients. Although viruses from the demented patients induced
significantly more neuronal killing than those from the nondemented
patients, this analysis may underestimate the extent of true neuronal
damage, as some cells might be injured, but not killed, by exposure to
supernatant fluid of infected macrophages. The percentage of neuronal
death in the present assay was maximal at 50 to 66% macrophage CM. The
reduced toxicity observed with 100% CM might be due to a possible
neuroprotective effect of higher serum concentrations (15).
In addition, the extent of neuronal killing was greater for
supernatants derived from macrophage cultures infected with recombinant
viruses containing brain-derived V1-V3 sequences that also displayed
higher levels of viral spreading, indicating that an increased number
of infected cells results in enhanced neurotoxicity. However, the
levels of neurotoxicity in the present study were lower than those in
other assays of neurotoxicity (12, 18). Our findings were
based on the use of human neuronal cultures in which a subpopulation of
neurons may be susceptible to injury and/or death, and rates of
neuronal death are low with several different neurotoxins
(40). In our assay of neurotoxicity, the rate of neuronal
death declined with supernatants harvested from later time points
postinfection, indicating that perhaps the macrophage cultures change
in their capacity to release the neurotoxic molecule(s) with time. It
is not yet clear which molecules released by macrophages are
responsible for the observed neuronal death, although many reports
indicate that host molecules released by cells infected by HIV-1
(18, 50), feline immunodeficiency virus (37), and
SIV (1, 39) contribute to neuronal damage. The present study
showed that the neurotoxic molecule(s) released by infected macrophages
was heat stable, and in agreement with earlier studies (3,
12), our results implicate the NMDA receptor in the mechanism of
neurotoxicity. Several candidate molecules that are released from
macrophages are heat stable and influence NMDA receptor-mediated
neuronal death. These include quinolinic acid (43), Ntox
(19), and nitric oxide and its metabolites (12).
Individual HIV-1 strains with differing envelope sequences have been
shown to vary in the ability to induce quinolinic acid (11)
and NO (28) production. Thus, it is conceivable that
brain-derived HIV-1 strains vary in pathogenic potential, and some of
this variability could be due to variation in envelope protein
sequences as documented in the present and previous studies
(48).
Our findings of an association between clinical status, viral envelope
sequence, and in vitro biological effects conferred by the viral
sequences suggest a possible relationship between viral protein
variation and occurrence of clinical dementia. However, the actual in
vivo mechanisms influencing the development of dementia in AIDS
patients remain unclear. To determine the true pathogenic effects of
different HIV-1 envelope sequences in the brain, in vivo assays will be
essential. Potential models include SCID mice inoculated with
HIV-infected macrophages in the brain (66), transgenic
animals (62, 63), or SIV/HIV recombinant viruses constructed
for testing in primates (57).
| |
ACKNOWLEDGMENTS |
We thank John Portis, Jonathan Geiger, and Kevin Coombs for
helpful discussions, Jonathan Glass for assistance with collection of
human tissues, and Carol Martin and Mark Bernier for preparation of the
neuronal cultures.
C.P. is an NHRDP/MRC Scholar. This study was supported by the MHRC,
NHRDP/MRC, and grants NS26643, AI35042, and RR00722.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Clinical Neurosciences, University of Calgary, 107-3330 Hospital Dr., Calgary, Alberta T2N 4N1, Canada. Phone: (403) 220-5011. Fax: (403)
283-8731. E-mail: power{at}ucalgary.ca.
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Journal of Virology, November 1998, p. 9045-9053, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
