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Natural variants of human immunodeficiency virus from patients with neurological disorders do not kill T4+ Cells.

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Natural Variants of Human
Immunodeficiency Virus from Patients
with Neurological Disorders Do Not Kdl
Rita Anand, P h D
Human immunodeficiency virus (HIV) has selective T4cell tropism and is cytocidal to cells with the helper-inducer
phenotype. Central nervous system dysfunctions can complicate full-blown acquired immunodeficiency syndrome
(AIDS) but can also be present either in isolation or in the context of AIDSrelated complex. Remarkably bland
histopathological findings have been reported in some patients with AIDS dementia in the presence of severe clinical
dysfunction. Thus, to understand the cytopathic properties of HIV, we recovered five viral isolates from 4 patients
with neurological symptoms of AIDS and identified them as HIVs. T h e replication and cytocidal properties of these
isolates were compared with l y m p h a d e n o p a t h y - a i a t e d virus in vitro. All five isolates exhibited replication
efficiency equivalent to lymphadenopathy-associated virus, but four isolates did not kill CD4 (T4 + ) cells. These
findings provide evidence for the existence of replication-competent noncytocidal natural variants of HIV and raise
the possibility that, in some AIDS patients, neurological disorders might be caused by HIV variants that are noncytocidal to T4 cells.
Anand R. Natural variants of human immunodeficiency virus from patients with neurological
disorders do not kill T4 + cells. A n n Neurol 1')88;23(suppl):Sbb-S70
Infection by human immunodeficiency virus (HIV),
previously called either lymphadenopathy-associated
virus (LAV) or human T-lymphotropic virus type 111
(HTLV-111) 11-31, results in a spectrum of clinical
manifestations ranging from asymptomatic seroconversion to full-blown acquired immunodeficiency syndrome (AIDS) 141. There is increasing evidence that
AIDS is frequently involved with central nervous system (CNS) dysfunctions [S-111. Although this disorder usually develops after other complications of
AIDS 112- 161 it often is seen as the first major manifestation of H I V infection 17).
Heterogeneity in AIDS virus genomes has been
well documented 111, 18, 191. However, structural or
functional subtypes of H I V within the spectrum of
heterogeneity have not been characterized. The mechanism of HIV-mediated induction of neurological disorders in AIDS patients is not known. To investigate
whether HIVs infecting neurologically affected AIDS
patients could be classified in o n e functional group,
and also prompted by reports of minimal neurohistopathology in many AIDS dementia patients ClS], we
isolated and studied cytocidal properties of H I V from
patients with CNS disorders as their primary manifestation of disease.
From the Neuropsychiatry Branch, National Institute of Mental
Health, Saint Elizabeth's Hospital, WAW Building. Washington,
Dc 20032.
Patients and Methods
Five isolates (N1 through N5) were recovered from 4 patients (Table l). Isolate N l was obtained by cocultivation of
autopsied brain tissue of a patient who died of dementia 181.
Isolates N2 through N5 were recovered from patients when
they were in the early stages of AIDS and had primarily
neurological manifestations.The N2 isolate was from peripheral blood mononuclear cells (PMNCs)of a 4 1-year-oldmale
intravenous drug abuser suffering from dementia. His T4
celLT8 cell ratio was not tested, but he was not lymphopenic. The N3 isolate was from PMNCs of a 40-year-old
homosexual with headache, oral thrush, and reduced T4
cells. The patient was later found to have a B-cell lymphoma.
Isolates N4 and N5 were recovered from PMNCs and cerebrospinal fluid. respectively, of a 28-year-old male heavy intravenous drug abuser with myelopathy. His PMNCs did not
show a decrease in the number of T4-positive (T4') cells.
The patients' sera were positive for antibodies to HIV by
ELISA (Table 2).
Virus Isolation
HIV isolates were recovered by cocultivating patients' cells
or minced tissue with 3-day-old normal adult PMNCs stimu-
lated with phytohemagglutinin (PHA) [20]. Briefly, PMNCs
were separated by Ficoll-Hypaque gradients and stimulated
with 10 &ml PHA in RPMI-1640 medium containing 10%
Address correspondence t o Dr Anand.
Table I . Historical Data about AIDS Patients with Neurological Disordtn
Risk Factor
SexfAge (Yr)
M yelopathy
Not known
M/5 7
M/4 1
New York
New York
New York
New York
PMNC = peripheral blood mononuclear cells; IVDA
CSF = ceribkspid fluid.
intravenous drug abuser;
Table 2. Characterization of N IsoIates and Patients’ Sera
The presence of antibodies specific for human immunodeficiency
virus (HIV) in sera of these patients was tested by ELISA kits ( A b
bon Laboratories, Inc), Chicago, IL. ELISA was not performed on
the patient from whom isolate N 1 was recovered because he was not
a suspected AIDS patient before death. Capture assay was performed on tissue culture supernatant fluids to test the presence of
HIV by using antigen test kits (Abbott Laboratories, Inc).
fetal bovine serum. Cocultivation was performed at 1 x lo6
celldml in medium B (RPMI-1640 medium with 10% T-cell
growth factor, and 1:500 diluted goat antibody to human
interferon) [20). The presence of virus in culture supernatant
fluids was tested by reverse transcriptase assay [21]. Concentrated culture fluids were assayed for particulate reverse transcriptase activity with the synthetic template primer poly
(rA) . (dt) in the presence of 10 mmol magnesium. Once
the culture was positive, 200 ml fresh PMNCs were infected
with each isolate, and virus stocks (passage l), at the peak of
reverse transcriptase activity, in aliquots of 5 ml per tube
were prepared and frozen at - 80°C for further experiments.
Continued transmissibility of each isolate in freshly cultured
PMNCs was tested for ten generations, and the isolates were
found to be continuously transmissible.
Dot-Blot and Southwn Blot Methods
For dot-blot analysis, culture supernatant fluids were used as
a source of viral RNA. Ten milliliters of culture supernatant
fluids (when reverse transcriptase activity was more than 10
x lo4cpm/ml) were clarified at 1,000 rpm for 10 minutes to
clear the fluids of cells and cell debris. Virus was pelleted by
centrifugation of cleared supernatants at 40,000 rpm for 30
minutes. Virus pellets were dissolved in 100 ~1 of water,
boiled for 4 minutes, and dottcd on nitrocellulose filters.
Filters were then hybridized to 32[P)-labeled HIV-probe
D N A representative of the full genome [22]. DNA-DNA
hybridizations were performed by Southern blot method.
High-molecular-weight D N A was extracted from infected
PMNCs with phenol/mcresol, as described [22]. Briefly,
D N A was digested with different restriction enzymes ac-
cording to the instructions supplied by the manufacturer
(Bethesda Research Laboratories, Inc, Gaithersburg, MD).
Digested D N A was run on 0.8% agarose gels, transferred to
nitrocellulose filters, and hybridized with nick translated
D N A probe.
MeasuremPnt of Replication and Cytocidzl Pmpertiu
Virus stocks were titrated by reverse transcriptase method
prior to setting up replication-cytopathicity experiments.
Threeday-old PHA-stimulated PMNCs were infected with
equal amounts (15,000 reverse transcriptase cpmlml) of each
isolate at 1 x lo6 cells/ml. After 4 hours of incubation at
37°C in 5% carbon dioxide, cultures were centrifuged to
remove extracellular virus and the cells were resuspended in
fresh medium B and incubated at 37°C and 5% C 0 2 in a
humidified atmosphere. Virus replication was measured by
assaying reverse transcriptase activity in culture supernatant
fluids by taking sequential aliquots of the cultures o n certain
days after infection.
The cytocidal effect was tested by enumerating the frequency of T 4 + and T8+ cells in culture, using an indirect
immunofluorescence with monoclonal antibddies and
fluorescence-activated cell sorter (FACS) analysis 123.). The
ratio of T4’ to T8+ cells was derived from FACS-qssisted
values obtained on the frequencies of each T-cell subppulation in sequential aliquots of the cultures on the &me day
virus replication was tested. Frequency of T-cell
tions and virus replication were measured f r q ~ $
cultures. The experiments were repeated three rupes, using
PMNCs from different donors, and the reqdts obtained
were consistent. LAV was used as a control in these experiments. The absolute number of T4’ and T8+ cells in cultures were derived from the values obtained by trypan blue
exclusion test on live cells in culture and from frequencies of
T 4 + and T8+ cells by FACS analyses.
Isohtion by Corultivation
Isolates N1 through N5 were recovered by cocultivation with normal adult PMNCs. The cocultures became reverse uanscriptase-positive within 4 to 15
days. The isolates were infectious and transmissible in
PMNCs and were filterable through 0.2-pm filters.
The morphology of the virus particles was similar to
that of prototype HIV as tested by elecuonmicrographic studies of cells infected with LAV and N
Anand: Noncytocidal Variants of H I V
Antigenic and Molecular ldentjfication of N Isolates
To idenufy these retrovital isolates as HIV, two HIV-
Table 3. Virus Replication and Ratio of T4' and T8' Celh
in PMNC Cultures 9 Days a f w lnfiction
specific tests were performed. The immunological nature of the N 1 through N5 isolates was established by
an HIV-specific antigen capture assay (Abbott Laboratories, Inc, Chicago, IL) by testing culture supernatant
fluids in antigen-ELISA. All isolates were shown to be
positive, with an optical density reading of more than
1.667 and a negative cutoff reading of 0.123 in the
test. Appropriate positive and negative controls specified by the manufacturer were included (see Table 2).
Viral RNA of the N 1 through N5 isolates was
confirmed to be HIV-specific by the RNA-DNA datblot method. Intense hybridization was observed with
all isolates (see Table 2). Culturc fluids from parallel
uninfected cultures did not show positive hybridization. In another control experiment, HTLV-I and
HTLV-I1 viral RNA was found not to hybridize with
HIV DNA probe under similar conditions. The
genomic relatedness of DNA from the N1 through
N5-infected PMNCs to HIV was demonstrated by the
Southern blot method. Hgh-molecular-weight DNA
(containing integrated viral DNA) was extracted from
infected PMNC, digested with restriction enzymes,
and hybridized to HIV-specific DNA probes. All isolates exhibited intense hybridization (see Table 2).
x 10 lml)
Total Cells
( x 1041ml)
Lymphocyte Ratio
(T4+:T8+ cells)
Results are an average of two replicate experiments. Normal adult
PMNCs were infected with 15,OOO RT cpm/106 celldml on day 0 as
described in the text. Virus replication and the ratio of T4 to T8
cells were studied as described in the text also. The number of total
live cells in cultures was determined by trypan blue exclusion
method. Uninfected PMNC cultures were used in parallel as a control.
PMNC = peripheral blood mononuclear cells; RT = reverse transcriptase; LAV = lymphadenopathy-associatedvirus.
RepIication Competence of N lsokates
Replication competence of the isolates was studied by
measurement of virus production (assayed by the reverse transcriptase method) in infected PMNC cultures. All isolates, N1 through N5, were replicationcompetent and showed growth potential very similar
to LAV as measured by rate and extent of replication.
A peak of reverse transcriptase activity of more than
200,000 c p d m l for each isolate was observed within
11 days, and then there was a decline by 15 days after
infection (Table 3). In experiments conducted for 30
days, reverse transcriptase activity in infected cultures
increased and remained at high levels subsequent to
the initial decline at 2 weeks after infection. Data for
the N4 and N5 isolates are shown in Figure 1.
Noncytociahl Nature of N Isolates
In contrast to the growth properties of the N 1 through
N5 isolates, the cytocidal properties of these isolates
differed strikingly from LAV. Four of five isolates did
not deplete the frequency of T4 cells in culture and
were therefore termed noncytocidal. The N2 through
N5 isolates were noncytocidal, whereas N1 was cytocidal to T4 cells (see Table 3). The differences in the
frequency of T4 cells observed in these cultures were
not secondary to the level of virus multiplication as
measured by reverse transcriptase assay. Calculations
of the absolute numbers of T4 cells in cultures based
on the live cell counts (trypan blue exclusion test
values) and frequencies of T4 + cells (FACS-assisted
Days Portinfection
Pig 1. Virus replication w e measured by monitoring reverse
transcriptase activity in the culture supernatants of hmpharknopathy-assorirrted uirns (U
and NSinfeced (U
blood mononuclear celh (PMNCs)
on &ys 7 , I I , 15, 18, 25, and 30 afer infiction. Sequential
aliquots were taken from the samejlaskc on the specrfipd&ys.
Uninfertd PMNC culture represented merse transcriptase
values of less than I0 x Id cpmlml (not shown).
values) were concordant with the lack of killing of T4'
cells in N-isolate-infected cultures (see Table 2).
A detailed analysis of the replication and noncytocidal properties of the N4 and N5 isolates and their
comparison to LAV is shown in Figures 1 through 3.
An examination of the frequency of T4' cells (Fig 2)
and frequency of T8 cells (Fig 3) showed that the N4
and N5 isolates were remarkably different from LAV.
During the culture period, the frequency of T4 and
T8+ cells and the T4 : T8 ratio were essentially unchanged in N4 and N5 cultures. In contrast, in LAVinfected cultures, there was a sgdicant drop in the
frequency of T 4 + cells (from 60% to 1.6%) and a
relative increase in the frequency of T8' cells (from
25% to 75%), and it was observed that N 4 and N5
S68 Annals of Neurology Supplement to Volume 23, 1988
Days Postinfection
Fig 2. The frequency of T4' cells in ~mphadenopathy-associated virus (u),
N4- @-A),
and N5-infected
) blood mononuclear cell (PMNC) cultures
was measured on days 7 , 1 1 , 15, 18,25, and 30. Sequential
aliquots were taken from the samejaskr on the specified days.
Uninjkted PMNC culture (-)
represents control values.
Days Postinfection
Fig 3. The frequency of T8+ cells in lymphad?nopathy-associateduirus (u
and N5- infected
) blood mononuclear cell {PMNC) cultures
was measured as described in the text on days 7, 1 1 , 15, 18, 25,
and 30. Sequential aliquots were taken from the samejaskr on
the specified days. Uninfected PMNC culture (-)
control ualues.
isolates did not deplete T4 + cells up to 30 days after
infection. This experiment was repeated three times,
using normal adult PMNCs from different donors, and
the results were reproducible. The determination of
absolute numbers of T4 and T8 cells and the numbers of live cells in PMNC cultures 25 days after the
initiation of the experiment also confirmed the noncytocidal nature of the N2 through N5 isolates.
Earlier studies of HIV isolates from AIDS patients
with CNS disorders have relied primarily on serological and genomic aspects, and the majority of the isolates characterized were from end-stage AIDS. These
investigations did not address the cytocidal properties
of the isolates. In this study, the N2 through N5 isolates were recovered from patients in early stages of
AIDS who primarily had neurological manifestations.
The presence of T4 antigen on the surface of cells
appears to be a requirement for HIV infection 124261, and since a large number of specific cell-surface
molecules are shared between lymphocytes and brain
cells 127, 281, we studied the growth and cytopathic
properties of N isolates in normal human adult
PMNCs. To accomplish this, we developed a simple
infectivity-cytopathicity assay as described in the
methods. PMNCs, with mixed populations of T4 and
T8 cells, were used in this assay, thus providing a reasonable corollary to the in vivo situation.
LAV was used as a prototype laboratory strain for
comparison with N isolates. It was found that the N1
through N5 isolates were equivalent in replication
competence to LAV, but N2 through N5 isolates were
noncytocidal to T4 cells. It is interesting that N1 was
cytocidal to T4 cells, as this was the only isolate recovered after the death of the patient. We have tested
fifteen other isolates from AIDS patients from Zaire
and the United States and have found, although there
is some variation in their ability to kill T 4 + cells, that
none are noncytocidal as N isolates [unpublished
data}. It is tempting to speculate regarding these
findings: It is possible that the noncytocidal HIVs are
present in all early AIDS patients and, owing to viral
genetic instability, cytocidal HIVs emerge in later
stages of the disease. A second possibility is that the
noncytocidal variants are present in early neurologically affected AIDS patients only. Both of these hypotheses can be tested by systematic cross-sectional
and sequential virological studies in subgroups of
AIDS and neurologically dysfunctional AIDS patients.
The isolates N4 and N5 were from PMNCs and
cerebrospinal fluid of the same patient, and both were
noncytocidal. Therefore, it appears that the noncytocidal property of the isolates is unrelated to the tissue source. Recent reports indicate that HIV isolates
from AIDS patients with neurological disorders show
preferential tropism for macrophages 129, 30). Experiments to determine whether our isolates have a similar
predilection for macrophage-monocytes have been initiated.
The observation that isolates N1 through N5 were
replication-competent and were equivalent in their
growth potential to LAV but that four of five did not
deplete T4 cells suggests that virus replication and killing of T4 cells are not always concordant functions in
HIV. The reasons for the initial decline of reverse
transcriptase activity in cultures infected with N isolates followed by a subsequent increase in this activity
are under investigation. The in vitro effect of T8+ cells
in inhibiting viral activity in infected T 4 + cells might
be one of the factors operating in our studies 131).
It has been suggested that cytocidal activity resides
in the envelope gene of wild type HIV isolates [32,
333. It is interesting that metabolic perturbations
Anand: Noncytocidal Variants of HIV S69
rather than cell killing have been reported to underlie
the pathogenesis of murine retroviruses such as polioencephalitis and other models of chronic infection 134,
351. In addition, aberrant processing of the envelope
protein in neurotropic variants of Moloney virus has
been documented [35]. The delineation of the expression of viral proteins in CNS cell types infected with
these noncytocidal isolates and the molecular analysis
of N isolates will be important to understanding the
mechanism of HIV-mediated neuropathological findings in the absence of T-cell killing.
We wish to thank Dr Steven McDougal for help with the fluorescence-activated cell sorter experiments and for helpful discussions,
and Jennifer Moore and Carol Reed for technical assistance. We also
thank the following physicians for their assistance in obtaining blood
and tissue samples from patienrs: Drs Susan Forlenza, Tony Cheung,
and Frederick P. Siegal. This study was initiated by Dr Rita Anand at
the Centers for Disease Control, Atlanta, Georgia.
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