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Journal of Medical Virology 29:10%114 (1989)
Active Replication of Human Immunodeficiency
Virus Type 1 by Peripheral Blood Mononuclear Cells
Following Coincubation With Herpes Viruses
Michel Tremblay, Mervyn Gornitsky, and Mark A. Wainberg
Lady Davis Institute for Medical Research (M.T., M.A.W.) and Department of Dentistry (M.G.),
S i r Mortimer B. D a v i s J e w i s h General Hospital and Department of Medicine, McGill University (M.T., M.A.W.),
Montreal, Quebec, Canada
Patients with acquired immunodeficiency syndrome (AIDS) commonly suffer from opportunistic infections associated with members of the
herpes virus family. To investigate whether certain of these other viruses might have an effect
on the ability of the human immunodeficiency
virus type 1 (HIV-1) t o replicate, we coincubated
peripheral blood mononuclear cells (PBMC)
from nine HIV-I-seropositive donors with live
preparations of various herpes viruses. In seven
of nine cases, exposure of PBMC to preparations
of either HSV-1, HSV-2, or CMV stimulated the
cells t o become active producers of HIV-1, as determined by reverse transcriptase activity and by
the presence of infectious progeny virus. This
increased production of HIV-1 particles appeared t o be a consequence of mitogenic proliferation and of herpes virus-encoded transacting
factors. These results supplement earlier findings o n the molecular activation of the HIV-1 genome by both HSV and CMV genetic elements
and point t o a possible role for these viruses in
the pathogenesis and ultimate clinical outcome
of HIV-1 infections.
occur over long periods in the absence of clinical symptoms and despite the presence of a n anti-HIV-1 immune response [Jaffe et al., 19851. It has been demonstrated that mitogen-stimulated T4 cells are much
more susceptible to HIV-1 infection and replication
than are unstimulated lymphocytes. Furthermore, the
replication of HIV-1 in cultured cells was itself found to
be proportional to the extent of T-cell activation [Folks
et al., 19861. Lymphocytes from healthy donors who
had been immunized with tetanus toxoid displayed enhanced susceptibility to HIV-1 infection, when the cells
were first activated in vitro by this same antigen [Margolick et al., 19871. It has thus been hypothesized that
stimulation of the immune system, by bacterial and/or
viral pathogens, may play a role as a cofactor in the
pathogenesis of AIDS [Montagnier et al., 19841 by enhancing the efficiency of HIV-1 infection [Zagury et al.,
19861. This might help to explain why only a proportion of individuals infected by HIV-1 have developed
full-blown AIDS, and i t could also account for the broad
spectrum of clinical outcomes that are seen in infected
individuals.
Members of the herpes family of viruses have been
suggested a s possible cofactors in the development of
AIDS because of their documented immunosuppressive
effects, ubiquitous presence, and, in some cases, lymKEY WORDS cytomegalovirus, lymphocyte
photropism. Moreover, herpes virus infections are very
stimulation, transacting factors
common in homosexual men, who constitute a highrisk group for development of AIDS [Siegal et al., 1981;
Quinnan et al., 19841. HSV is commonly isolated from
INTRODUCTION
AIDS patients, who often suffer from more severe HSVHIV-1 is the etiological agent of AIDS [Barre-Si- induced lesions than those of otherwise healthy indinoussi e t al., 1983; Gallo et al., 1984; Levy et al., 19841. viduals [Siegal et al., 19811. In addition, AIDS patients
The virus can selectively infect and kill T helper-inducer lymphocytes that bear the CD4 (T4) epitope at
their surface [Dalgleish et al., 1984; Gallo et al., 1984;
Levy et al., 19841.These cells, but not the T lymphocyte
subset that bears the CD8 (T8) epitope (suppressorAccepted for publication June 20, 1989.
cytotoxic cells), are preferentially destroyed following
Address reprint requests to Dr. Mark A. Wainberg, Lady Davis
infection by HIV-1, leading to a noticeable decrease in Institute for Medical Research, Sir Mortimer B. D a v i s J e w i s h
the T4iT8 ratio [Weiss et al., 19851.
General Hospital, 3755 Cote Ste-Catherine Road, Montreal, QueIn HIV-1-infected individuals, viral replication may bec, Canada H3T 1E2.
G 1989 ALAN R. LISS, INC.
Tremblay et al.
110
generally have a high frequency of CMV-associated
disease [Welch et al., 19841, elevated levels of antiCMV antibodies [Halbert et al., 19861, and are active
shedders of this virus [Polk et al., 19871.
We have therefore studied the possible role of these
viruses with regard to stimulation of HIV-1 replication
in the PBMC of HIV-1 seropositive donors. The results
indicate that HSV-1, HSV-2, and CMV are each able to
trigger overt replication of HIV-1.
MATERIALS AND METHODS
Subjects
The study population consisted of eight AIDS patients being treated a t the Dental Clinic of the Jewish
General Hospital (Montreal, Canada) and of one
asymptomatic, seropositive child followed at the Department of Pediatrics, HBpital Ste-Justine (Montreal,
Canada). All of these individuals, except for the child,
fulfilled the criteria for AIDS of the Centers for Disease
Control I1987 I. None of these patients was on therapy
a t the time of study. All patients had immunological
evaluations that included Ab titers for each of CMV
and HSV as determined by complement fixation. Levels of Ab against hepatitis B surface antigen (HB,Ag)
were determined by ELISA assay (Abbott Laboratories, North Chicago, IL).
V'iruses
Clinical isolates of HSV-1, HSV-2, and CMV were
obtained from the Virology Laboratory of the Montreal
Children's Hospital. Two different isolates of CMV
(DL-4 and LB-1) were utilized; both had been derived
from neonatal cases of congenital cytomegalic disease.
HSV-1 and HSV-2 were expanded to high titer in
Vero cells. At times of extensive cytopathology, culture
fluids from infected monolayers were clarified by lowspeed centrifugation and filtered through Millipore filters (0.45 kM). Titration of HSV-1 and HSV-2 was carried out by plaque assay on monolayers of Vero cells
[Rapp, 19631.
CMV was also passaged on Vero cells. After 5-7
days, by which time the observed cytopathology involved at least 90% of the monolayer, culture fluids
were clarified and filtered. Virus was titrated by
plaque assay IWentworth and French, 19701and stored
a t -70°C until used.
Coincubation of Cells With Various Stimuli
Peripheral blood mononuclear cells (PBMC) from
HIV-1-infected individuals were isolated by FicollPaque (Pharmacia, Piscataway, NJ) density gradient
centrifugation IBoyum, 19681. The cells were resuspended to a concentration of 1 x lo6 cellsiml in culture
medium (RPMI-1640, supplemented with L-glutamine
12 mM1, penicillin 1250 Uiml], streptomycin [250 pg/
mll, and fetal calf serum 110% viv]) and seeded into
each well (1.5 ml) of a 24 well tissue culture plate (Flow
Laboratories, McLean, VA). The cells were placed into
contact with a variety of potential viral stimuli includ-
ing HSV-1 (multiplicity of infection Imoi], 0.00012),
HSV-2 (moi, 0.00012), and CMV (moi, 0.00002). In
some experiments, we examined what effect either anticell antibodies or viral antigens might have on HIV-1
replication by coincubation of cells with anti-Leu 4 (final concentration, 1:1,000) and HB,Ag (Heptavax-B
vaccine 10.2 pg]). Culture controls were PBMC from
HIV-1-infected individuals coincubated with conditioned medium from Vero cells. The cells were incubated a t 37°C under 5% C 0 2 for 48 hr, following which
they were centrifuged and resuspended in antigen-free
culture medium, supplemented with 5% recombinant
interleukin-2 (r-IL-2, Boehringer-Mannheim, West
Germany) and polybrene (2 Fgiml). Thereafter, this
supplemented medium was used to refeed the cells
twice weekly. Beginning on the seventh day following
such coincubation, culture fluids were harvested at
regular intervals, pelleted by ultracentrifugation, and
assayed for reverse transcriptase activity by a previously published procedure [Hoffman e t al., 19851.
Lymphocyte Proliferation
PBMC from HIV-1-infected patients were cultured
in flat-bottomed microtiter plates, using three replicate samples per assay (1.5 x lo5 lymphocytes/0.15
ml/well). Either these cells were cultured in the absence of exogenous stimulant, or stimulation was attempted by including any of several live viral preparations and/or anticell monoclonal antibodies. Blastogenic
responses were measured by adding 1 p,Ci/well of
tritiated thymidine (specific activity, 30 Ciimmole;
New England Nuclear, Boston, MA), 4 h r prior to culture termination. The results are expressed a s net cpm
per well.
Infectivity of HIV-1 Recovered Following
Cell Stimulation
The infectivity of HIV-1, isolated after coincubation
of PBMC with herpes viruses, was assessed using umbilical cord blood mononuclear cells from healthy donors as targets. Such cells were isolated and stimulated
with PHA-P1 (0.1%) for 48 h r and were then treated
with polybrene (2 pgiml) for 20 min a t 37"C, resuspended a t a concentration of lo6 cellsiml in growth medium, and dispensed into a 24 well tissue culture plate
at a volume of 1.5 ml. All HIV-1 reverse transcriptasepositive samples isolated following coincubation of
PBMC with herpes viruses were inoculated onto the
previously stimulated cord blood mononuclear cells.
Supernatant fluids were harvested and assessed for reverse transcriptase activity.
RESULTS
Table I is a summary of the clinical and immunological features of the nine patients studied. All patients
but two (Nos. 4 and 9) had a positive antibody titer
against CMV. Only two patients ( 5 and 9) did not have
detectable antibodies against HSV. All patients were
seronegative for hepatitis B virus (data not shown).
Stimulation of HIV-1 Replication
111
TABLE I. Clinical and Immunological Features of Patients
Patients
1
2
3
4
5
6
7
8
9
Age
28
29
36
37
32
32
37
35
5
Clinical
features”
Anti-CMV
titerh
CA
CA, EH, GL
CA, KS
CA, HS, PcP
HS
HS
CA
CA
1:16
1:16
1:16
< 1:8
1:32
1:32
1:32
1:16
< 1:8
AS
Anti-HSV
titerb
1:16
1:16
1:8
1:8
< 1:8
1:8
1:16
1:8
< 1:8
“AS, asymptomatic; CA, Candida albicans; EH, Entemoeba hzstolytica; GL, Giartlia lamblia; HS herpes simplex; KS, Kaposi’s sarcoma;
PcP, Pneumocystis carinii pneumonia.
bCMV and HSV titers were determined by complement fixation assay.
The most common opportunistic organisms diagnosed
in the studied population were Candida albicans (six
cases) and herpes simplex virus (three cases).
The results in Table I1 show that certain of the viral
preparations employed were able to generate a lymphocyte blastogenic response when coincubation with cells
from HIV-1-seropositive donors was carried out. In
contrast, controls consisting of PBMC from HIV-l-infected patients, not so coincubated with HSV and/or
CMV preparations, showed little, if any, blastogenic
response. It is noteworthy that mononuclear cells from
six of nine patients tested showed some degree of blastogenic response to each of two CMV strains employed
as antigen. Interestingly, the only instances in which
the various CMV antigenic preparations were found to
be completely devoid of stimulatory activity were in
the case of two patients (4 and 9) who were seronegative for anti-CMV antibodies. The results in Table I1
also indicate that the various HSV antigenic preparations employed were unable to serve a s inducers of lymphocyte blastogenesis. This is despite the fact that
seven of the nine patients studied were seropositive for
HSV-1 and/or HSV-2. This finding is consistent with
previous observations from our laboratory that showed
t h a t specific HSV-mediated lymphocyte proliferation,
in the case of AIDS patients, could only be elicited after
addition to cultures of exogenous interleukin-2 [Wainberg et al., 19871. HB,Ag was similarly unable to elicit
any blastogenic response, as demonstrated in each of
three cases in which this antigen was used (data not
shown). In contrast, the use of antibodies directed to
the CD3 antigen (anti-Leu 4) gave rise, in most cases,
to a very strong blastogenic response.
The major aim of this study was to demonstrate
whether coincubation of PBMC from HIV-1-infected
individuals with viruses such as CMV and HSV might
be able to trigger production of HIV-1 on the part of
cells that had been infected by this agent but t h a t were
not actively engaged in HIV-1 replication. The results
in Table I11 show that active production of HIV-1 was
elicited in seven of nine cases in which patient cells
were coincubated with live CMV, as demonstrated by
the presence of reverse transcriptase activity in culture
fluids. Those patients for whom only one strain of CMV
is listed were able to respond to t h a t strain only but not
to the other strain of CMV utilized or to any of the
other viral preparations tested. The various CMV preparations used were able to effect active replication of
HIV-1 in the case of seven patients. One of the CMV
strains utilized, LB-1, appeared to induce HIV-1 recovery with relative efficiency and permitted the isolation
of infectious HIV-1 in each of seven cases studied. In
contrast, the other CMV strain employed, DL-4, was
able to elicit HIV-1 recovery in two cases only. Positive
recovery of HIV-1 was achieved only once, out of the
nine cases studied, when HSV-1 and HSV-2 were used
a s stimulating agents. Recovery of HIV-1 could not be
accomplished using HB,Ag as stimulus (results not
shown). HIV-1 was recovered in only one instance in
which anti-Leu 4 was used as a mitogen. The time to
HIV-1 culture positivity in these studies ranged between l l and 32 days (Table 111).
The potential infectiousness of HIV-1 recovered following coincubation of PBMC with these various
herpes virus preparations was also evaluated. The results of Table I11 show that such isolated HIV-1 was
able to infect PHA-stimulated cord blood mononuclear
cells from healthy donors in each instance.
DISCUSSION
It has been hypothesized t h a t a variety of factors
might enhance replication of HIV-1 by virus-infected
cells [Quinnan et al., 1984; Siegal et al., 19811. We
postulated that members of the herpes family of viruses might fulfill such a n enhancing role in the presence of PBMC, latently infected by HIV-1. To test this
hypothesis, PBMC from HIV-1-infected individuals
were coincubated with low-passage clinical isolates of
herpes viruses including CMV, HSV-1, and HSV-2. We
now report that such coincubation, primarily with
CMV can cause active replication of HIV-1 in latently
infected cells and/or in cells that produce low levels of
HIV-1. Such active replication was never observed
when conditioned medium from Vero cells, used to
grow herpes viruses, was employed as a stimulant.
The technique commonly used to isolate HIV from
asymptomatic, infected individuals and from patients
with AIDS and/or ARC involves a n initial stimulation
step by PHA, a lectin with polyclonal activation properties for T lymphocytes [Montagnier, 19861. The fact
that we were able to recover HIV-1 only once when
using anti-Leu 4 a s a mitogen, despite high blastogenic
responses in six cases, is intriguing. This monoclonal
antibody acts, like PHA, by causing PBMC to enter
mitosis, a n event thought to be prerequisite to retroviral replication. Early methods of HIV-1 isolation did
not use, as in the present study, coculture of PHAstimulated normal PBMC with the patient’s PHAstimulated cells. Instead, only PHA-stimulated PBMC
from AIDS and ARC patients were employed and
yielded recovery rates less than 50% [Gallo et al.,
Tremblay et al.
112
TABLE 11. Blastogenic Responses to Viral Preparations and to Monoclonal Antibodies
Patients (cpm x
Stimulant"
1
2
3
4
5
6
7
59.10
7.59 20.89
0.12 61.30 40.84 34.97
CMV (DL-4)
9.58 26.39
0.45 59.97 37.06 45.50
CMV (LB-1)
20.23
0.32
0.16
2.37
1.81 2.13
3.85 0.45
HSV-1
0.39
0.30
2.46
1.78 4.35
19.74 0.53
HSV-2
5.89
1.26
Anti-Leu 4
79.76 37.53 60.61 24.28 40.47
0.40
0.47
0.71 0.81
0.64
0.67
Unstimulated control 0.74
8
N.T.b
2.25
0.74
0.40
20.89
0.54
9
N.T.
1.42
1.57
N.T.
N.T.
0.43
"Those entities employed as stimuli of lymphocyte mitogenesis included live preparations of CMV, HSV,
and anti-Leu 4 monoclonal antibodies.
"N.T., not tested.
TABLE 111. Recovery of HIV-1 From Cultures of Patient Mononuclear Cells Following
Coincubation With Live Herpes Viruses
Patient"
1
3
4
5
7
8
9
Stimulant
CMV (LB-1)
CMV (DL-4)
CMV (LB-1)
HSV-1
HSV-2
CMV (LB-1)
CMV (DL-4)
CMV (LB-1)
CMV (LB-1)
Anti-Leu 4
CMV (LB-1)
Reverse transcriptase
No. of days
activity a t time of
reauired for
initial HIV-1 recovery
cultuie positivity
(cpm/ml x 10-61' "
25
1.20
11
0.10
11
0.09
25
0.02
32
0.07
0.05
18
0.69
25
0.22
25
0.03
18
0.02
28
0.03
12
Reverse transcriptase
activity when primary
isolates of HIV-1 were
inoculated onto cord
blood 1vmDhocvtes
(cpmiA i 1 0 ' ~
3.79
-~
2.57
2.89
4.99
4.50
1.66
3.87
2.75
3.43
2.14
2.52
"HIV-1 could not be recovered from the lymphocytes of patients 2 and 6 in spite of coincubation with each
of the CMV and HSV preparations described.
hA sample was considered positive when reverse transcriptase activity was >0.01 cpmiml x 10 ".
Unstirnulated PBMC from HIV-1-infected individuals consistently had reverse transcriptase levels
<0.0001 cpmiml x lo-'.
'Reverse transcriptase determinations were carried out 10 days after inoculation of PHA-stimulated cord
blood lymphocytes by primary isolates of HIV-1.
19841. In our study, the failure of anti-Leu 4 to elicit
HIV-1 replication could reflect the normally low levels
of infected PBMC in infected individuals LHarper et al.,
19861 and the specificity of the antibodies for the CD3
epitope.
In contrast, efficient HIV-1 recovery was achieved in
78% of cases when various herpes viruses were employed. This process may involve both immunological
recognition by HIV- 1-infected cells of herpes antigens
as nonself and molecular activation by herpes virustransacting factors. While no blastogenic responsiveness to CMV itself (strain LB-1) was detected, as expected, with each of two CMV-seronegative individuals
(subjects 4 and 9), coincubation of their lymphocytes
with CMV led to production of HIV-1, suggesting that
molecular activation had probably occurred. However,
antigenic stimulation as a mechanism responsible for
HIV-1 recovery cannot be ruled out, since most CMVseropositive individuals showed high blastogenic responses in the presence of this virus.
The discrepancy observed in recovery of HIV-1, fol-
lowing coincubation of cells with either anti-Leu 4 or
herpes viruses, may be explained by a dual effect of
these viruses. Following antigenic stimulation, T cells
that recognize herpes antigens a s nonself might be
stimulated to divide; such activation, in concert with
the effects of herpes transacting factors, could lead to
active HIV-1 replication.
It is relevant that HSV-1 can cause transcription of
latent HIV-1 [Mosca e t al., 1987a,b] by transactivating
the long terminal repeat (LTR) region of the latter virus [Gendelman, 19861. The HSV-1 genes responsible
for such HIV-1 activation are those that encode immediate early gene products [Nabel et al., 19881. Similarly, the immediate early gene region of CMV can also
stimulate HIV-1 gene expression [Davis et al., 19871.
Serological and immunological observations further
reinforce the potential role that herpes viruses might
play in HIV-1-infected individuals. One report demonstrated a close association between HSV-2 and HIV-1
infection [Holmberg et al., 19881. Another study demonstrated enhanced replication of HIV-1 following in-
Stimulation of HIV-1 Replication
fection by CMV of two chronically HIV-1-infected cell
lines [Skolnik et al., 19881.
The developments that lead in vivo to productive
HIV-1 replication from latent infection are still obscure. However, since reactivation and/or reinfection
by herpes viruses do take place, such events may explain why some HIV-1-infected individuals develop
symptoms and opportunistic diseases within a relatively short period. Indeed, the immunosuppressive
state induced by HIV-1 may facilitate reinfection and/
or reactivation by latent herpes viruses that may, as
shown here, have a n enhancing effect on HIV-1 replication. The fact that immunologically healthy individuals are commonly infected by a single strain of CMV
[Spector, 19821, whereas patients with AIDS are infected by multiple CMV strains, is of interest in this
regard [Drew et al., 19841.
ACKNOWLEDGMENTS
This work was carried out by M.T. in partial fulfillment of the requirements of the degree of Doctor of
Philosophy, Faculty of Graduate Studies, McGill University. We are grateful to Francine Busschaert for the
preparation of the manuscript. This research was supported by a grant to M.A.W. from Health and Welfare
Canada.
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