Negative regulation of human immune deficiency virus replication in monocytes: Distinctions between restricted and latent expression in THP-1 cells

Abstract

In THP-1 monocytoid cells infected with HIV, viral expression can be regulated in several ways: (a) latency (no viral expression); (b) restricted expression (chronic low-level viral expression with little or no detectable virus released); and (c) continuous production. In cells with restricted HIV expression, nuclear factor(s) were found that blocked tat-associated DNA binding complex formation, suggesting that initiation of transcription was negatively regulated. Also, viral particles were seen budding into and accumulating within intracytoplasmic vacuoles with little virus released, suggesting multiple levels of regulation. These cells with restricted expression had no detectable viral antigens on the cell surface and were not lysed by IL-2-activated large granular lymphocytes. However, they could cause viral-mediated T cell cytolysis in cell-cell assays, suggesting viral transmission through cell contact. In addition, cells with latent HIV were identified and could still produce infectious virus after 5-azacytidine exposure 10 mo later. LPS and other treatments could increase viral production in cells with restricted but not latent expression, suggesting they occur by distinct mechanisms. These infected cells provide a reservoir for viral transmission to uninfected T cells that itself is not detected by immune surveillance mechanisms.

Full text

NEGATIVE
REGULATION
OF
HUMAN
IMMUNE
DEFICIENCY
VIRUS REPLICATION
IN
MONOCYTES
Distinctions
between
Restricted
and
Latent
Expression
in
THP-1
Cells
BY
JUDY
A
.
MIKOVITS,'
RAZIUDDIN,`
MATTHEW
GONDA,T
MARTIN
RUTA,
1
NANCY
C
.
LOHREYS
HSIANG-FU
KUNG,N
AND
FRANCIS
W
.
RUSCETTIS
From
the
'Department
of
Biological Carcinogenesis
Development
Program
and
the
TLaboratory
of
Cell
and
Molecular
Structure,
Program
Resources,
Inc
.
;
the
SLaboratory
of
Molecular
Immunoregulation
and
the
Il
Laboratory
of
Biochemical
Physiology,
National
Cancer
Institute,
Frederick
Cancer
Research
Facility,
Frederick,
Maryland
21701
;
and
the
Division
of Blood
and
Blood
Products,
Centers
for
Biologic
Evaluation
and
Research,
Bethesda,
Maryland
20892
Human
immunodeficiency
virus
1
(HIV
1) is
a
retrovirus
that
has
many
similari-
ties
with
members
of the
nontransforming
and
cytopathic
animal
lentivirus
family
(1)
.
These
viruses,
including
HIV
1,
cause
slowly
progressive, chronic,
and,
in
some
instances,
fatal
diseases
in
their
hosts
.
The
time
from
initial
viral
infection
to
clini-
cally
observed
symptoms
of
disease
is
usually
measured
in
years
(2)
.
Elucidation
of the
viral
life
cycle
during
the
subclinical
phase
of
infection
is
critical
in
gaining
a
clearer
understanding
of
the
pathophysiology
of
AIDS
.
Several
studies
have
sug-
gested
that
a
persistent
state
of
latent
or
chronic
low
level
productive
infection
exists
in
fresh
and
cultured
cells
(3-7)
.
These
data
imply
that
viral
latency
can
be
a
compo-
nent
of
HIV
infectivity
.
The
mechanisms
involved
in
developing
these
latent
or
re-
stricted
states
of
HIV
expression are
not
well
understood
.
Animal
lentiviruses
show
a
tropism
for
cells
of
the
monocyte/macrophage
lineage
during
viral
latency
and
persistence (8)
.
There
is
now
substantial
evidence
suggesting
that
the
monocyte/macrophage
also
serves
as
a
reservoir
for
HIV
infection
(9)
.
Fresh
monocytes
can
be
infected
in
vitro
with
HIV
.
Also,
HIV
can
be
cultured
from
mono-
cytes
obtained
from
blood
and
organs
of
patients infected
with
HIV
(9)
.
In the
brain,
the
macrophage
is
the
major
infected
cell
type
and
is
associated
with the develop-
ment
of
neurologic
symptoms
seen
in
AIDS
patients
(10,
11)
.
This
study
was
initiated
to
develop
HIV-infected
monocyte
cell
lines
that
could
be
used
to
understand
mechanisms
of
viral
latency
and
restricted
low
level
chronic
expression
.
THP-1,
derived
from
a
patient
with acute
monocytic
leukemia,
possesses
morphologic,
histochemical,
phenotypic,
and
functional properties
of
monocytes
(12,
13)
.
Analysis of
THP-1
cultures
after
an
initial
productive
HIV
infection
revealed
THP-1
cultures
with
either latent
or
restricted
HIV
expression
as well as
cultures
This
project
has
been funded
at least in
part
with
Federal
funds
from
the
Department
of
Health
and
Human
Services
under
contract
no
.
NOI-CO-74102
.
The
content
of
this
publication
does
not
neces-
sarily
reflect
the
views
or
policies
of
the
Department
ofHealth and
Human
Services,
nor
does
mention
of
trade
names,
commercial
products,
or
organizations
imply
endorsement
by
the
U
.
S
.
government
.
Address correspondence
to
Dr
.
Frank
Ruscetti,
Laboratory
of
Molecular Immunoregulation,
Building
560,
Room
21-89A,
Frederick
Cancer
Research
Facility,
Frederick,
MD,
21701
.
The
Journal of Experimental
Medicine
-
Volume
171

May
1990

1705-1720

1705
on December 15, 2016Downloaded from
Published May 1, 1990

1706

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
remaining
productively
infected
.
In
cells
with
restricted
HIV
expression,
viral
ex-
pression
is
negatively
regulated
in
such
a
manner
as to
escape
immune
surveillance
and
still
be
capable
of transmitting virus
to
T
cells
.
In
cells
with
latent
virus,
no
viral
expression
is
seen
but
infectious
virus
can
be
activated
by
a
mechanism
distinct
from
the
upregulation
of
viral
expression
in
cells
with
restricted
expression
.
Materials
and
Methods
Cell
Lines
.

THP-1
cells
were
maintained
in
RPM
I
with
10'/o
FCS,
penicillin
(100
hg/ml),
streptomycin
(100
kg/ml),
and
glutamine
(300
t~g/ml)
;
cells
were
subcultured
1
:5
every
4-5
d
.
HELA
and
murine
CB2MX3
cells
producing
HIV-1
tat,
obtained
from
G
.
Pavlakis
(BRI,
Frederick,
MD),
were
grown
as
monolayer
cultures
in
DMEM
as previously
described
(14)
.
Viruses
and
Infections
.

All
viruses
were
isolated
from
PBMCs
.
HIV
-1
strain
BP-1
was
grown
in
HUT
78
(15),
strain
ADA
was
grown
in
U-937
(16),
and
HIV-2
Rod
was
grown
in
CEM
(17)
.
10
7
THP-1
cells
in
log
phase growth were
infected
with
5
x
10
5
tissue
culture
infec-
tious
doses
at
a
50%
endpoint
(TCID
5

)'
harvested
from
cell-free
supernatants
of
each
virus
after
5
d
of
growth
.
Infections
were
done
in
1
ml
serum-free
RPMI
with
2
Ag/ml
of
polybrene
for
1-2
h
at
37°C
in
a
shaking
water
bath
.
Cells
were washed
twice
to
remove
unabsorbed
virus
and
subcultured
for
growth
.
For
the
multiplicity
of
infection
(MOI
;
number
of
TCID
5o
U/cell)
study,
concentrated
viral stocks,
made
using
a
tangential
flow
Millitan
Apparatus
(Mil-
lipore
Continental
Water
Systems,
Bedford,
MA),
were used
to
give
the
indicated
MOI
.
Virus Detection
.

Viral
p24
antigen
was
determined
on
tissue
culture
supernatants
or
cell
pellets
lysed
with
1%
Triton
X-100by
ELISA
(Cellular
Products, Inc
.,
Buffalo,
NY)
.
As
pre-
viously
shown,
HIV
p24
ELISA
kits
do not
discriminate
between
HIV-1
and
HIV-2
p24
(18)
.
TCID5o
was
determined
using
microtiter
wells
of
HUT-102B2
with
serial
dilutions
of
cell-free
virus
.
Electron
micrographs
of
HIV
infected
cells
were prepared
by
OS04-fixed,
rap-
idly
dehydrated
THP-1
or
HUT-102B2
cells
and
embedded
in
epoxy
resin
using
standard
procedures
.
Thin
sections
were mounted,
stained
with
uranyl
acetate
and
lead
citrate,
and
viewed
in
a
microscope
(H-7000
;
Hitachi,
Tokyo,
Japan)
as previously
described
(1)
.
For
syn-
cytia
formation,
200
infected
THP-1
cells
were
incubated
with
10
6
HUT-102132
cells
.
HIV-1-
induced
syncytia
were
recorded
by
microscopic
analysis
.
Reverse
transcriptase
(RT)
activity
was
measured
in
cell
supernates
pelleted
by
high-speed
centrifugation
using
poly(rA)-oligo
(dT12-18)
template
primer,
20
mM
Mg
2
'
as
cofactor,
and
appropriate
deoxynucleotide
triphosphates
as
previously
described
(19)
.
Results
were
adjusted
to
cpm
of
[
3
H]TTP-in-
corporated
[3
H]/ml
.
Phenotypic Analysis
of
HIVInfected
THRL

Cytofluorometric
analysis
was
performed
as
de-
scribed
(15)
.
Cells
washed
in
PBS
were
fixed
in
-10°C
absolute
methanol
for
intracellular
p24
antigen
determination
.
The
cells
were
not
fixed
for
all
other
assays
.
mAbs
used
were
directed
against
HIV
p24,
HIV
gp
160
:41,
and
HIV
-1
gp
120
(Cellular
Products,
Inc
.)
.
Other
monoclonals
against
Leu-3A
(CD4), Leu-2
(CD8),
Leu-M3
(CD14),
and
HLA-DR
were
pur-
chased
from
Becton
Dickinson
&
Co
.
(Sunnyvale,
CA)
.
Functional
Assays
.

Forviral-mediated
cell
cytotoxicity,
HIV-infected
cultures
are
tested
ei-
ther
by
titering
out
5-d supernatants
against
10,000
MT-2
cells
per
well
in
a
round-bottomed
96-well
plate
or
by
coculturing
with
infected
cells
.
After
various
days
of
incubation,
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT)
is
used
to
measure
in
vitro
growth by
cell-mediated
reduction
of
tetrazolium
(20)
.
The
OD
at
540
nm
is
directly
propor-
tional
to
the
number
of
viable
cells
.
Macrophage
functional
assays
were performed
plus
or
minus
PMA
(50 ng/ml)
treatment
for
24 h
.
Phagocytosis
was
performed
by
incubating
10
6
cells
with
100
lcl
zymosan
for
1
h
.
Cell
smears were
then
stained
with
Jenners,
and
1,000
cells
were
examined
microscopically
.
For
accessory
cell
function,
monocyte-free
lymphocytes
obtained
by
elutriation
were
separated
into
T4
and
T8
populations
as
previously
described
(15)
.
THP-1- and
HIV-infected
THP-1
irradiated
at
8,000
rad
were
added
to 10
5
T8
cells
in
a
culture
volume
of
0
.2
ml
at
serial
dilutions
of
THP-1
cells
with or
without
Con
A
(5
ttg/ml)
.
'
Abbreviations
used
in
this
paper
:
LGL,
large
granular
lymphocyte
;
LTR,
long
terminal
repeat
;
MOI,
multiplicity
of
infection
;
PCR,
polymerase
chain
reaction
;
RT,
reverse
transcriptase
;
TCID5o,
tissue
culture
infectious
dose
50%
endpoint
.
on December 15, 2016Downloaded from
Published May 1, 1990

MIKOVITS ET AL
.

1707
Stimulated
cultures
were
incubated
72 h
at
37'C
.
MTT
assay
was
performed
as
previously
described (20)
.
NK
Cytotoxicity
Assay
.

For
cytotoxicity
assays,
large
granular
lymphocytes
(LGL)
were
purified
on
Percoll
gradients
and
activated
overnight
at
10
5
cells/ml
with 100
U
of
rIL-2
(Bio-
gen,
Cambridge,
MA)
and
cytotoxicity
assays
performed
as
previously described
(15)
.
A
6%
increase
in
isotope
release,
above
baseline,
was
consistently
statistically
significant
at
p<0
.05
(student's
t
test)
.
Analysis
of
Viral
Nucleic
Acids
in
THP1
Cells
.

Preparation
of
total
cellular
RNA
for
Northern
transfer
experiments
was
done
by
the
guanidine
thiocyanate
CsCl
gradient
method
.
Poly-
adenylated
RNA
was
prepared
by
oligo(dT)-cellulose
column
chromatography
.
RNA
pellets
were
twice
precipitated
with
ethanol
and
quantitated
by
absorbance
at
260
nM
.
Ethidium
bromide
staining
was
used
to
equalize
amounts
of
nucleic
acids
.
After
equilibration,
the
RNAs
were
separated
on
0
.9%
agarose/formaldehyde
gels
and
blotted
onto
nitrocellulose
.
Northern
blots
were
probed by primer
extension
of
DNA
fragments
of
the
HIV
-1
strain
HXB2
(21)
.
The
probes
were used
at
a
concentration
of 4 x
10''
cpm/ml
of
dCTP['
2
]-labeled
pHXBA11
(a
gift
of
George
Pavalakis,
Bionetics
Research
Inc
.,
Frederick
Cancer
Research
Facility,
Frederick,
MD)
.
Blots
were
prehybridized
and
hybridized
at
42°C
for
24 h
each
.
Hybridiza-
tion
and
washings
were
done
as previously
described
(22,
23)
.
Long
Terminal
Repeat
(LTR)-directed
Nuclear
Run-on
Competition
Experiments
.

Analysis
of
RNA
transcripts
was
carried
out
by
nuclear
transcription
run-on
assay
.
Adaptions
of the
method
of
Greenberg and
Ziff
(22)
were
made
as
previously
described
(23)
.
The
; `
2
P-labeled
RNA
was
recovered
by
treating
with
a
final
concentration
of 0
.2
M
NaOH
for
10
minon
ice,
neu-
tralized
by
acid-free
Hepes
to a
final
concentration
of
0
.24
M
.
This
purified
labeled
RNA
was
hybridized
at
65°C
for
30
h
to
purified
HIV-1
LTR
fragment
immobilized
on
nitrocellu-
lose
.
A
recombinant
construct
pL3CAT
consisting
of
a
Barn
HI-Hind
III
fragment
of
HIV
-1
(nucleotides
-1068
to
+83),
which
contains
the
HIV-1
LTR
promoter
as
well
as
downstream
TAR
sequences
(14),
was
used
to
isolate
template
DNA
.
pL3CAT
was
digested
with
Kpn
1-Hind
III,
purified
on
a
1%
low
melting agarose
gel
;
phenol
was
extracted
and
washed
with
70%
ethanol,
and
then the dried
template
was
used
in
the assay
.
Hybridizations
and
washings
were done
as previously
described
(22,
23)
.
Mobility
Shaft
Assay
.

For
binding
assays,
the
plasmid
pL3-CAT
(14)
was
digested
with
Hind
III,
dephosphorylated
with
CIAP,
ethanol
precipitated,
and
then
5'-labeled
with
7-["PIATP
and
T4
polynucleotide
kinase
.
The
labeled
fragment
was
digested
again
with
Eco
RV
.
The
199-bp
fragment
was
gel
purified
and
recovered
as
described
above
.
The
assay
used
was
a
slight
modification
of
the
procedure
of
Kadonaga
et al
.
(24)
.
The
assay
was
done
in 20-pl
reaction
volume
containing
20
mM
Hepes
(pH
7
.6),
60
mM
KC1,
10%
glycerol,
0
.5
mM
EDTA,
0
.5
mM
DTT,
1
wg
poly
dl-poly
c1C,
0
.05%
NP-40,50,000
cpm
of
end-labeled
DNA
probe
(Eco
RV-Hind
III
of
HIV
-1
LTR),
and
nuclear
extracts
at
5 or 10
pg
protein
.
Nuclear
extracts
were
prepared
by
the
method
of
Parker
and
Topol
(25)
.
For
competition
studies,
a
25-fold
excess
of
the
same
unlabeled
DNA
fragment
was
added
to
the
reaction
mixture,
In
mixing
experiments,
a
ratio
of
one
part of
extract
for
inhibition
to
four
parts
extract
to
be
inhibited
was
used with
the
protein
content
held
constant
at
either
5 or
10
ug
.
The
reaction
mixture
was
incubated
at
30°C
for
1
h
and
then
subjected
to
electrophoresis
at 10
V/cm
through
a
4%
polyacrylamide
(nondenaturing)
gel
in
Tris-EDTA-Borate
buffer
.
Polymerase
Chain
Reaction
(PCR)
Analysis
of
RNA
Products
in
HIV-1-infected
THP-1
.

1
kg
of
DNA
or
RNA
from
infected
or
uninfected
cells
was
amplified as
previously described
(26)
in
reaction
mixtures
containing
primer
pairs
specific to
HIV-1
gag
region of the
HIV-1
viral
genome
.
50
pmol
of
each
primer
was
used
.
For detection of
RNA,
an
additional
step
of
re-
verse
transcription
of
RNA
to
DNA
by
avian
myeloblastosis
virus
RT
was
incorporated
where
the
amplification
primer
initiated
the
reverse
transcription
(26)
.
PCR
products
were
ana-
lyzed
by
hybridization
with
specific
probes
spanning
the
region
between
primer
pairs
fol-
lowed
by
analysis
on
polyacrylamide
gels
where
the
expected
radiolabeled
band
for
gag
is
114
by
(26)
.
Results
Differential
Expression
of
HIV
after
Infection
of
THP-1
Cells
.

An
acute
infection
of
THP-1
cells
was
established
by
incubating
HIV
-1
(strain
BP-1)
at
an
MOI
of
0
.05
on December 15, 2016Downloaded from
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170
8

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
with
cells
in
the log
phase
of
growth
.
In
each
experiment,
productive
infection
as
measured
by
extracellular
virus
was
not detected
at
day
7,
butwas
detected
between
day
14
and
17
.
Extracellular
virus
was
detected
in
media
by
four
assays
:
presence
of
p24
core
antigen,
RT,
viral-mediated
T
cell
cytolysis,
and
syncytia
formation
(Table
I)
.
Phenotypic
analysis
of the
cells
showed
that
monocyte
surface
antigens,
such
as
CD14
(Leu-M3),
were
unaffected
by
HIV
1,
while
CD4
antigen
(Leu-3A)
could
no
longer
be
detected
on
the
cell
surface,
presumably
blocked
by
the
binding
of
HIV
virions
(Fig
.
1
b)
.
Also,
30-40%
of the
cells
contained
surface
antigens
recognized
by
anti-gp 160
:41
(Fig
.
1
b)
.
In
contrast,
45-60
d
after
the onset of acute
infection,
entire
cultures
of
these pre-
viously
productively
infected
cells
produced
little
or
no
extracellular
virus (Table
I,
lines
3,
5,
and
8-10)
.
Cell
surface
analysis
showed
that
CD4
antigen
could
be
recognized again while
the
presence
of
viral
antigens
could
not
be detected (Fig
.
1,
c)
.
Presence
of virus
as
measured
by
RT,
infectivity,
T
cell
cytopathology,
and
syncytia
formation
was
not
detected in the
media
ofthese
cells
.
However,
the
media
of
some
of
these
cultures
contained
p24
core antigen
at
a
concentration of
<20
ng/ml,
as
compared
with
200-500 ng/ml
for
productive
cultures
.
These
cultures
remained
HIV
infected
as
shown
by
increased
expression
after
treatment
ofthe
cells
with
LPS,
(Table
I,
line
4)
or
irradiation
(Fig
.
1
d)
.
72
h
after
activation
of the
cells,
extracel-
lular
virus,
as
measured
by
all
the
criteria
used,
was
present
and
viral
antigens
could
again
be
detected
on
the
cells
.
These
data
suggested
that
viral
expression
was
re-
stricted
in
these
cells
.
In
addition,
several
nonproducer
THP-1
cultures
remained
nonproducers
after
activation
(Table
1)
.
This
absence
or
low
level
of
viral
production
in
infected
THP-1
cells
was
reproducibly
seen
with
two
other
isolates,
HIV
-1
ADA,
a
monocytoid
isolate,
and HIV-2
Rod
(Table
I)
.
Conditions
for
Establishing
Restricted
Viral
Production
in
THR1
Cells
.

To
better
un-
derstand
the
conditions
necessary
for
the
establishment
of
restricted
expression,
ex-
periments
were
done
to
askthe
effect
of
MOI
on
subsequent
viral
expression
in
THP-1
TABLE
I
Analysis of
HIV-1-infected
THP-1
Cells
THP-1
cells
were
grown and
infected
with
HIV
strains at
an
MOI
of
0
.05,
as
described
in
Materials
and Methods
.
Analysis
of
the
viral
expression
was
made
60
d
post-infection,
as
described
in
Materials
and
Methods
.
Values
are
for at
least
three
separate
infections
.
Cells
were
incubated
for
48
h
with
10
hg
of
LPS, and
viral
assays
were
performed
.
Viral
strain
Viral
expression
Syncytia
(percent
positive)
p24
antigen Viral
RT
ng/ml
cpm/ml
1
.
None None
0
0
0
2
.
BP-1
Producer
2-5
200-500
30-60,000
3
.
BP-1
Restricted
0
0
.5-20
0
4
.
BP-1
Restricted
(LPS)'
1-2
200-500
15-20,000
5
.
BP-1
Nonproducer
0
0 0
6
.
BP-1
Nonproducer
(LPS)
0
0
0
7
.
ADA
Producer 5-10
1,500
50-100,000
8
.
ADA
Nonproducer
0
0
0
9
.
ROD
Restricted
>1
0
.1-100
5-10,000
10
.
ROD
Nonproducer 0 0
0
on December 15, 2016Downloaded from
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100
1000
1
100
I
%POS=9
MEAN=157

100
100
MEAN=215
100
1000
100
MIKOVITS
ET
AL
.

170
9
%POS=66
MEAN=600
100
1000
1
100
.
100
%POS=O MEAN=O
%POS=37
MEAN=285
%POS=1
MEAN=493
%POS=34
MEAN=458
1000
FIGURE
1
.

Phenotypic
characterization of
HIV
infection
of
HIV
.
FACS
analysis
using
Leu-3a
(CD4),
Leu-m3
(CD14),
and
HIV
-1
gp
160
:41
was
performed
as
described
in
Materials
and
Methods
.
(a)
Uninfected
THP-1
;
(b)
productively
infected
THP-1
;
(c)
infected
THP-1
with
no
expression
;
(d)
infected
THP-1
with
no
expression
48
h
after
irradiation
.
Antibodies
used
were
A-CD4,
B-CD14,
and
C-HIV
1
gp
160
:41
.
Analysis
was
performed
as
described
in
Materials
and Methods
.
cells
(Table
II)
.
At
high
MOI,
productive
infection
with
cell
cytotoxicity
was
evident
with
no
restricted
expression
of
HIV
seen
.
As
the
MOI
was
lowered,
initial
produc-
tive
infection
was
followed
by
development
of
several
cultures
with
restricted or
no
viral
expression
.
Infection
of
THP-1
with
HIV
-2
Rod
resulted
in
only
restricted
ex-
pression
.
This
was
probably
due
to
not
being
able
to
obtain
a
viral
preparation
with
a
higher
titer
.
Kinetics ofexpression of
p24
in
THP-1
cells
was
also
followed
after
HIV
infection
.
Until
day
21,
both
productive
and
restricted
HIV
cultures
contained
the
same
number
of
viral
p24-positive
cells
.
However,
by
day
60,
>95
To
of the
cells
were
positive
in
the
productive
cultures,
whereas
only
32%
were
positive
in
the
cultures
with
re-
stricted
expression
.
It
seems
likely
that
restricted
HIV-1
expression
also
leads
to
de-
creased
viral
spread
.
At
no
time
after
the
development
of
nonproducer
cultures
were
any
cells
in
these cultures
positive
for
HIV
-1
p24
antigen
.
Characterization
of
THR1
Cells
with
Restricted
HIV
Expression
.

Since
little
or
no
de-
tectable
extracellular
virus
was
found
in
some
HIV-infected
THP-1
cultures,
the
status
of
viral
expression
was
examined
in
these
cells
.
High
levels
of
viral
RNA
were
found
in
the productively
infected
THP-1
(Fig
.
2
A, lane
2)
with
prominent
peaks
at
the
genomic
9
.1-kb
size,
along
with
subgenomic
4
.4-
and
2
.0-kb
mRNAs
.
However,
THP-1
cultures
with
restricted
expression
contained
markedly
less
viral
RNA
(Fig
.
2
A,
lane
4)
than
the productively
infected
cultures
(Fig
.
2
A, lane
3)
with
subgenomic
RNAs
of
7
.5
and 4
.4
kb
preferentially
accumulating
over
full-length
9
.1-kb
RNA
(Fig
.
2
B, lane 5)
.
A
7
.5-kb
RNA
species
has not
been
previously
reported
and
its
Z
C
7
O
1
U
V
100
5
`
J
P~OS=95
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171
0

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
TABLE
II
Effect
of
MOI
on
HIV
Infection
of
THP-1
Cells
*
MOI
is
the
number
of
TCID5o
U/cells
.
THP-1
cells
were
grown and
infected
with
HIV
strains
as
described
in
Materials
and
Methods
.
1
Number
of
days
post-infection
that
detectable
virus
was
present
in
extracellular
media
as
assayed
by
viral
p24
antigen
.
Cytolysis
and
cell
death
were
observed
microscopically
.
Infected cultures
of
ADA
at
high
MOI
showed
complete death
.
II
Analysis of
the restricted
viral
expression
was
made
60
d
post-infection
using
the
criteria
of
no
syncytia
formation
and
low
or
absent
extracellular
p24,
as in
Table
I
.
Restricted
and
nonproducer
cultures
are
included
in
total
.
presence
may
be
related
to
the
mechanism
of
restricted
expression
in
these
cells
.
To
our
surprise,
the
nonproducer
cultures
produced
no
detectable
viral
RNA
(Fig
.
2
A,
lane
5)
even
if
60
1~g
of
poly(A)
+
-selected
RNA
(Fig
.
2
B,
lane
2)
was
analyzed,
suggesting
that
HIV
expression
in
these
cells
was
truly
latent
(i
.e
.,
complete
absence
of
viral
expression)
.
Since
some
ofthe
nonproductive
cultures
possessed
viral
RNA,
the
status
of
intra-
cellular
viral
particles
was
studied
by
EM
.
In those
cultures
with
restricted
viral
ex-
pression,
intracellular
assembly
of
numerous
viral
particles
was
observed
.
Most
of
the
virions
were
seen
within
intracytoplasmic
vacuoles
.
Many
immature
and
ma-
ture
forms,
as well as
virions
budding
into
the
vacuoles,
could
be
seen
.
The
viral
particles
seen
on
ultrastructural
analysis
were
identical
to
previous
descriptions
of
HIV
(1)
.
As
seen
in
freshly
infected
macrophages
(27),
these
vacuoles
were
predomi-
nantly
found
in
the perinuclear
golgi
region
.
Surprisingly,
few
if
any
extracellular
virions
were
seen
.
Approximately
30%
.
of the
cell
sections
were
associated
with
viral
particles
.
In
cultures
with
latent infection,
no
intracellular
viral
particles
or
viral
RNA
could
be
detected
.
Negative
Regulation of
HIV
Expression
in
THP-1
with
Restricted
Expression
.

Since
the
accumulation
of
viral
RNA
in
the
HIV
restricted
cells
is
many
fold
lower
than
in
the
HIV-producing
cells
(Fig
.
2
A),
the
rate of
viral
transcription
wasmeasured
using
in
vitro
transcription
directed
by
the
LTR
in
a
nuclear
run-on
assay(Fig
.
3)
.
Exoge-
nous
LTR
was
added
to
the
in
vitro
transcription
mixture
so
that
initiation
and
elongation of
viral
RNA
could
be
measured
.
Nuclei
from
HELA
and
uninfected
THP-1
showed
a
small
basal
level
of
transcription
.
Nuclei
from
the
THP-1
cells
with
restricted
expression
showed
a
small
increase
in
the
rate
of
transcription
over
control
HELA
and
uninfected
THP-1
(Fig
.
3,
lane
3),
but
the
transcription
was
much
re-
Viral
strain
MOP
Days
to
infection)
Cytolysis'
No
.
productive/
no
.
total
No
.
restricted))/
no
.
total
BP-1 10
4
+
10/10
0/10
1
7
+/-
10/10
0/10
0
.1
14
-
9/10
1/10
0
.01
14-18
-
6/10 4/10
ADA
10
4 +
+
+
3/3
0/3
1
4
+
+
3/3
0/3
0
.1
7-10
+3/3
0/3
0
.01
14
+/-
1/3 1/3
ROD
1
14
-
0/2 2/2
0
.1
21-24 -
0/2 2/2
on December 15, 2016Downloaded from
Published May 1, 1990

MIKOVITS ET
AL
.
1711
FIGURE
2
.

Analysis
of
viral
nucleic acids
in in-
fected
THP-1
cells
.
Northern
transfer
and
hybrid-
izations
on
total
RNA
were
performed
as
de-
scribed
in
Materials
and
Methods
.
(a)
Lane
1,
uninfected
THP-1
;
lane
2,
THP-1
productively
infected
with
HIV
-1
BP-1
from a 30-min
exposure
;
lane
3,
THP-1
productively
infected
with
HIV-1
from
a
2-h
exposure
;
lane
4,
THP-1
with
restricted
HIV
-1
expression
from a
2-h
exposure
;
lane
5,
THP-1
.
with
latent
HIV
virus
from a
2-h
exposure
.
(b)
Lane
1,
THP-1
productively
infected
with
HIV
1,
poly(A)'
RNA
20
gg
;
lane
2,
THP-1
with
latent
HIV
virus,
poly(A)'
RNA
60 Ag
;
lane
3,
THP-1
with
latent
HIV
virus
72 h
after
5-azacytidine
(10
AM)
treatment
;
lane
4,
THP-1
with
latent
HIV
-1
virus
72
h
after
5-azacytidine
treatment
cocultured
with
HUT
102132 for
10
d
;
lane
5,
THP-1
with
restricted
HIV-1
expression
.
FIGURE
3
.

HIV
-1
LTR-directed
nuclear
run-on
competition
experiments
.
Analysis
of
RNA
tran-
scripts
was
performed by
nuclear
transcription
run-on
assay
as
described
in
Materials
and
Meth-
ods
.
Unless
indicated,
reactions
contained
75
At
nuclei
and
25
lxl
buffer
.
Lane
1,
THP-1
nuclei
;
lane
2,
HELA
nuclei
;
lane
3,
nuclei
from
THP-1
cells
with
restricted
HIV
-1
(BP-1)
expression
;
lane
4,
nuclei
from
THP-1
cells
productively
infected
with
HIV
1
;
lane 5,
nuclei
from
productively
in-
fected
THP-1
(75
Al)
and
nuclei
from
THP-1
with
restricted
HIV
-1
expression
(251x1)
;
lane
6,
nuclei
from
productively
infected
THP-1
(75
1x1)
and
nuclei
from
THP-1
(251x1)
;
lane
7,
nuclei
from
productively
infected
THP-1
(75
Al)
and
nuclei
from
THP-1
with
latent
virus
(25
1x1)
.
on December 15, 2016Downloaded from
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171
2

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
duced
from
levels
of
transcription
observed
using
nuclei
from
productively
infected
cells
(Fig
.
3,
lane
4)
.
To
ascertain
whether
nuclear
factors
in restricted
nuclei
were
affecting
LTR-directed
transcription, nuclei
from
productively
infected
cells
were
incubated
with
various
mixtures
of
nuclei
(Fig
.
3)
.
Mixing
nuclei
in
a
1
:4
ratio
from
either
uninfected
THP-1
or
THP-1
with
latent
virus
with
nuclei
from
productively
infected
THP-1
cells
did not
affect
the
rate
of
transcription
(Fig
.
3,
lanes
6
and
7)
.
However,
there
was
a
marked
decrease
(5-10-fold)
in
the
transcriptional
level
of
nuclei
from
HIV
producing
THP-1
cells
when
the
same
ratio
(1
:4)
of
competing
nuclei
from
THP-1
cells
with
restricted
expression
was
added
(Fig
.
3,
lane
5)
.
This
nuclear
material
would
also
inhibit
LTR-directed
transcription
of
nuclei
from
HIVinfected
T
cells
but
not
HTLV
1
LTR
directed transcription
in
HTLV
1-infected
T
cells
(data
not
shown)
.
To
determine
whether
this
negative
regulation
of
viral
transcription
in
these
cells
with
restricted
expression
was
at
the
level
of
DNA
binding
complex
formation,
gel
mobility
shift
assays
were
performed
using
the
enhancer-TAR
region
(-117
to
+82)
of the
HIV-1
LTR
(Fig
.
4)
.
Two
concentrations
of
each
nuclear
extract
were
used
.
Extracts
from
HIV-1-producing
THP-1
cells
(Fig
.
4,
lanes
4
and
5)
and
a tat-producing
mouse
cell
line
(lanes
8 and
9)
give
the
same
complex
formation,
while
the
extract
from
the
restricted
cell
line (lanes
6
and
7)
did
not
have
any
DNA
binding
in
the
area
where
extracts
from
both the productively
infected
THP-1
cells
or
the
tat-
producing
cells
bound
the
DNA
.
A
second
(lower)
complex
was
found
from
both
extracts
of
productive
and
restricted
cells,
but not
the
tat-producing
cells
.
Mixing
FIGURE 4
.

Gel
mobility
shift
analysis
ofprotein
binding
from
infected
THP-1
cells
to
HIV
LTR
.
A
"P-labeled
oligonucle-
otide
spanning
the
enhancer-
TAR
region (-117
to
+82)
of
the
HIV
-1
LTR
was
incubated
with
two
concentrations
(5
and
10
wg)
of
nuclear
extracts
pre-
pared
from
various
cells
as
de-
scribed
in
Materials
and
Meth-
ods
.
Lane
1,
probe
alone
;
lanes
2
and
3,
uninfected
THP-1
;
lanes
4
and
5,
productively
in-
fected
(BP-1)
THP-l
;
lanes
6and
7,
THP-1
with
restricted
HIV
-
1
expression
;
lanes
8
and
9,
tat-
producing
murine
cell
line
;
lanes
10
and
11,
productively
infected
(BP-1)
THP-1
plus
uninfected
THP-1
(4
:1
ratio)
;
lanes 12
and
13,
productively
infected
THP-1
plus
THP-1
with
restricted
HIV
-1
expression
(4
:1
ratio)
;
lanes 14
and
15,
productively
infected
THP-1
plus
tat-producing
murine
cell
line
(4
:1
ratio)
;
and
lane
16,
productively
infected
THP-1
with
excess
cold
probe
.
on December 15, 2016Downloaded from
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MIKOVITS ET AL
.

171
3
an
extract
from
uninfected
THP-1
with
an
extract
from
productively
infected
cells
did not
affect
binding
(Fig
.
4,
lanes
10
and
11)
.
In
contrast,
when
an
extract
from
cells
with
restricted
expression
was
mixed
with
an
extract
from
productively
infected
cells,
the
binding
of
productive
cell
extract
to
the
LTR
(Fig
.
4,
lanes
12
and
13)
was
eliminated,
suggesting
one
mechanism
of
restricted
HIV
-1
expression
was
at
the
level
of
initiation
of
transcription
by
blocking
DNA
binding
complex
formation
with the
HIV-1
LTR
.
Thus,
HIV
expression
in
cells
with
restricted
expression
may
be
actively
suppressed
by
some
DNA
binding
factor
.
Such
expression
is
not
detect-
able
in
cells
with
latent
viral
infection
(data
not
shown)
.
Reactivation
of
Virus
from
Cultures
with
Latent
Infection
.

Since
THP-1
cells
with
la-
tent
virus exhibited
no
detectable
viral
expression
and
did
not
negatively
regulate
viral
transcription,
we
asked
if,
using
PCR,
we
could
detect
viral
expression
in
the
cells
with
latent
virus (Fig
.
5)
.
Using
primer
pairs
to
the
gag
region,
which
has
been
shown
to
be
the
most
sensitive
region
for
HIV
detection
(26),
two
latently
infected
cultures
(Fig
.
5,
lanes
3 and
4)
showed
no
detectable
viral
RNA
after
2
h
(Fig
.
5
A)
or
24
h
(Fig
.
5
B)
of
gel
exposure
.
For
comparison,
productively
infected
T
cells
FIGURE
5
.

PCR
analysis
of
RNA
from
infected
THP-1
cells
under
various
conditions
of
RNA
isolated,
as
described
in
Materials
and
Methods,
was
amplified
using
gag
primers
.
At
the
end
of 35
cycles,
aliquots
were
hybridized
to
a
32
P-end-labeled
oligonucleotide
probe
spanning
the
region
between
the
primer
pairs
.
PCR
products
were
analyzed
on
a
20%
polyacrylamide
gel
using
the
gag
probe
.
(a)
Lane
1,
uninfected
H9
;
lane
2,
HIV
IIIB
H9
;
lane 3,
latent
THP-1
ADA
;
lane
4,
latent
THP-1
BP-1
;
lane
5,
latent
THP-1
BP-1
;
lane
6,
THP-1
;
lane
7,
BP-1
HUT
78
;
lane
8,
BP-1
THP-1
;
lane
9,
restricted
THP-1
BP-1
;
lane
10,
THP-1
;
lane
11,
restricted
THP-1
LPS
treated
;
lane
12,
restricted
THP-1
PMA
treated
;
lane
13,
productive
THP-1
;
lane
14,
latent
THP-1
ADA
;
lane
15,
latent
THP-1
ADA
with
5-azacytidine
;
lane
16,
latent
THP-1
BP-1
LPS
;
lane
17,
latent
THP-1
BP-1 with
5-azacytidine
;
lane
18,
latent
THP-1
BP-1 with
5-azacytidine
plus
LPS
.
Results
from
2-h
exposure
.
(b)
Same
lanes
24
h
after
exposure
.
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171
4

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
(lane
7),
productively
infected
THP-1
(lane
8),
and
THP-1
with
restricted
expres-
sion (lane
9)
are
shown
.
We
next studied
under
what
conditions the
virus
could
be
reactivated
.
Neither
LPS
(Fig
.
5,
lane
16)
nor
IUdR
(Fig
.
5,
lane
17)
could
induce
viral
expression
from
cells
with
latent
virus
.
Since
previous
work
has
shown
that
the
HIVLTR
stably
transfected
in
fibroblasts
was
methylated
(28),
5-azacytidine
was
used
and
found
to
be an
inducer
of
virus
expression
(Fig
.
5
B,
lane
18,
or
Fig
.
2
B,
lane
3),
even
after
the
virus
was
latent
for
10
mo
.
We
cannot
be sure
whether
the
level
of
RNA
is
due
to
activation
of
a
small
population
of
cells
or
to
a
general
lower
level
of
transcription
.
However,
sufficient
amounts
of
infectious
virus
were
pro-
duced
to
get
a
massive
infection
ofthe
T
cells line,
HUT
102B2, within
10
d
of
trans-
mission
(Fig
.
2
B,
lane
4)
.
Electron
micrographs
of
reactivated
latent
virus
trans-
mitted
into
HUT-102B2
show
typical
morphological
characteristics
of
HIV
assembly
and
budding
(Fig
.
6)
.
Viral
and
Cellular
Biology
of
Restrictedly
and
Latently
HIV-infected THP-1
.

The
mor-
phological,
phenotypic,
and
functional
characteristics
of
THP-1
cells
with
restricted
and
productive
expression
of
HIV
-1
were
essentially
the
same
as
uninfected
cells
.
Phagocytosis
of
yeast
particles,
accessory
cell
function
for
T
cell
activation,
and
de-
velopment
of
anchorage
dependence
were
all
normal
before
and
after
PMA-induced
differentiation
.
Since
these
cells
were
functionally
normal,
we
next
asked
whether
they
were
im-
munologically
normal
.
As we had
previously
shown
(15),
IL-2-activated
LGL
can
recognize
and
lyse
HIV-infected
cells
.
Specific
LGL-mediated
cytotoxicity
was
ob-
served
only
on
the
HIV-1-producing
THP-1
cells
.
IL-2
stimulation increased the
magnitude
of
LGL-mediated
cytotoxicity
.
No
cytotoxicity
was
seen
on
the
cells
with
restricted
HIV
expression,
suggesting
that
such
cells
can
avoid
recognition
by
specific
immune
mechanisms
.
The
biological
characteristics
of the
virus residing
in
these
functionally
and
im-
munologically
normal
THP-1
cells
were
determined
.
T
cell
cytopathology
was
mea-
sured
using
an
assay
where
virus-producing
cells
cause
cytolysis
of
MT-2
target
cells
(Fig
.
7
A)
.
Using
HIV-1-infected
HUT-78
cells,
50°Io
of the
target
cells
are
killed
by
100
cells,
while
all
the
cells
are
killed
by
250
cells
.
Using
the
THP-1
cells
with
restricted
expression,
50
0
/c
of
the target
cells
are
killed
by
200
cells
.
This
cell
cytotox-
icity
is
blocked
when
azidothymidine
(AZT)
is
added
to
the
cultures,
showing
that
it
is
HIV
mediated
.
The
supernatant
of
these
cells
with
restricted
expression
cannot
induce
MT-2
cytotoxicity
(Fig
.
7
B),
while those
from
cells
productively
infected
kill
very
efficiently
.
Supernatant
from
irradiated
cells
with
restricted
expression
will
now
kill
MT-2
.
Consistent with
this
is
the
observation
that
THP-1
cells
with
restricted
expression
will
not
kill
MT
-2
cells
when
they are
separated
by
a
permeable
barrier
(data
not
shown)
.
Virus
reactivated
from
latently
infected
THP-1
cells
after
10
mo
of
culture
was
able
to
kill
T
cells
as
efficiently
as
virus released
from
continuously
productive
cultures
.
Discussion
An
acute productive
infection
of
THP-1
cells
was
established
using
HIV
-1
and
HIV-2
.
Between
days
14
and
21,
20-4070
of the
cells
contain
antigens
recognized
by
anti-p24
and
anti-gp 160/41
.
These
data are
consistent
with
published
reports
using
CD34'
hematopoietic
stem
cells
(29),
fresh
monocytes
(9),
and
U937
(6,
7)
.
on December 15, 2016Downloaded from
Published May 1, 1990

MIKOVITS ET AL
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on December 15, 2016Downloaded from
Published May 1, 1990

171
6)

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
A
,
.2~
FIGURE
7
.

Viral
mediated
T
cell
cytotoxicity
assays
.
HIV
infected
cultures
were
tested
ei-
ther
by
titering
out
5-d
super-
nates against
10,000
MT-2
cells
per
well in
a
round-bottomed
96-well
plates
or
by
coculturing
with
infected
cells at
several
concentrations
.
After
5-7
d
of
incubation,
MTT
is
used
to
measure
in
vitro
growth
by
cell-
0 .2
k

'\-

____

mediated
reduction
of
tetrazo-
lium
(20)
.
The
OD
at
540
nm
is
directly
proportional
to
the
0

25

50

75

,00

number
of viable
cells
.
(A)
Volume
(NI)

Ability
of
extracellular
virus
to
B

induce
cytolysis
of
MT-2
.
5-d
, .0

old
culture
supernatants
from
THP-1
cells
with
restricted
HIV
I
expression
(/),
THP-1
cells
with
restricted
expression
after
irradiation
(A),
control
THP-1
(O),
and
HIV-1-infected
HUT
78
(0)
are
shown
.
(B)
Ability
of
HIV-infected
cells
to
induce
cy-
tolysis
of
MT
2
.
THP-1
cells
with
restricted
HIV
expression
(A),
control
THP-1
(N),
and
HIV-1-infected
HUT-78
(0)
are
0

100 200 300

400 500
shown
.
Cell
Number
E
c
m
a
e
Q
0
0
.8
$
0
.6
A
e
2
0
.4
a
Several
weeks
after
infection
of
THP-1
cells
by
HIV,
entire
cultures
spontaneously
became
restricted
in
viral
expression,
while
in
the
producer
cultures,
>90%
of the
cells
were
viral
antigen
positive
.
In
some
cases,
spontaneous
nonproducers
have
been
reported
for
T
cells
(3,
4)
but not
for
monocytes
.
Two
distinct
types of
infected
cul-
tures
with
altered
expression
were
identified
in
THP-1
:
(a)
cells
with
restricted
HIV
expression
;
and
(b)
cells
with
latent
competent
virus
.
It
is
unlikely
that these cultures arose
due
to
selection
of
clonal variants,
since
with
infection
at
low
MOI,
no
THP-1
cytotoxicity
or
loss
of
viability,
growth,
or
function
was
seen
in
infected cultures
.
However,
it is
not
possible
to
rule
out
that
the
cells
with
restricted
or
latent
infection
were
present
from
the
first
day
of
infection
and
eventually
overgrew
the other
cells
in
the
culture
.
While
it is
not
possible
to
determine
how
these cultures
arose,
their
existence
has
important
implications
for
HIV
viral
persistence
and
pathology
.
Furthermore,
in
both
types of
restricted cul-
tures,
cells
could
be
induced
to
produce
virus
after
10
mo
in
culture
.
Thus,
the
pheno-
type
of
these
viral
cultures
was
stable
.
Characterization
of
these
two
types of
infected
monocytoid
cultures
has
shown
that
they are
clearly
different
at
the
molecular
and
cellular
level
.
In
cells
with
re-
stricted
HIV
expression,
there
is
a
greatly
reduced
rate
of
transcription
and
slower
accumulation
of
viral
RNA,
as
shown
by
nuclearrun-on,
PCR,
and
Northern
anal-
ysis
.
This
is
due
at
least
in
part
to
HIV
specific
factor(s)
present
in
the
nucleus
of
on December 15, 2016Downloaded from
Published May 1, 1990

MIKOVITS
ET
AL
.

1717
restrictedly
infected
cells
that
can
negatively
regulate
transcription
of
productively
infected
cells
.
Gel
mobility
shift
analysis
showed
that
formation
of
DNA
binding
complexes
associated
with
tat
is
eliminated
by
nuclear
extracts
of
restrictedly
in-
fected
cells,
suggesting
that
initiation
of
transcription
is
being
regulated
.
In
addi-
tion,
production
of
genomic
viral
RNA
is
being
reduced
in
these
restricted
cells
with
a
concomitant
appearance
of
a
novel
subgenomic
7
.5-kb
RNA
(Fig
.
2)
.
This
RNA
is
not
seen
when
the virus
is
then
reinfected
into
T
cells
.
In
cells
with
latent virus,
viral
expression
and
the
ability
to
negatively
regulate
transcription
of productively
infected
cells
were
not
observed
.
By
PCR
analysis,
no
viral
RNA
was
found
including
nef
RNA
(data
not
shown),
suggesting
that
nef
does not
serve
as
a
negative
regulator
in
these
cells
.
This
is
the
first
demonstration
that
HIV
infection
of
monocytoid
cells
can
lead
to
latency
at
the
molecular
level
.
In
addition,
the
ability
of various agents
to
activate
viral
expression
in
these
two
infected
cell
types
is
distinct
.
In
agreement
with
several
others
(30-33),
we
find var-
ious
cytokine treatments,
LPS,
PMA,
TNF
a,
and
GM-CSF,
can
stimulate
expres-
sion
in
restricted
cells
such
that
viral
levels
approach
that
of
a
productive
cell
.
How-
ever,
none
of
these
treatments
stimulate
detectable
production
from
cells
with
latent
virus
.
The
ability
of
5-azacytidine
to
reactivate
virus
production
suggests
that
meth-
ylation
is
involved
in
the
regulation
of
HIV
expression
in
these
latently
infected
cells
.
Using
a
stably
transfected
LTR
into
fibroblasts,
it
has
been
previously
shown
that
methylation
of
HIV LTR
sequences could
occur
(28)
.
Treatments
that positively
regulate
HIV
transcription
can
be mediated by
cel-
lular
factors
such
as
NFKB
(34,
35),
and
AP-1
(36)
that
bind
to
specific
sequences
in
the
HIVLTR
.
In
addition, that
has
been
postulated
to
mediate
its
effects
through
cellular
factors
that
bind
to
the
sequences
responsive
to
the
transactivating
response
region
(37)
.
Viral
factors
such
as
the
nef
gene product,
which
can
repress
HIV
tran-
scription
(38),
and
vpu,
which
affects
viral
release
(39),
could
be
important
in
estab-
lishing
these
states
of
viral
suppression
.
In addition
to
the
cytokines
that
upregulate
viral
expression,
IFN-a
has
recently
been
shown
to
restrict
viral
production
in
human
monocytes
(40)
and
U-937
cells
(7)
.
It
is
clear that
multiple
pathways
regulate
HIV
transcription,
and
that
negative
regulation
of
viral
expression
in
THP-1
cells
prob-
ably
involves
viral
and
cellular
factors
.
The
biological
consequences
of
these types
of
restricted
HIV
expression
may
be
important
in
the
pathogenesis
of the
disease
.
In the
cells
with
restricted
expression,
most
if
not
all
infectious
virus
produced
is
sequestered
intracellularly
.
The
cells
even-
tually
store
sufficient
virus
to
kill
T
cells
as
efficiently
as
productively
infected
T
cells,
probably
through
cell-cell
contact
with
the
uninfected
target
.
Suppression
of
extracellular
virus
production
and
cell
surface
viral
antigen
expression
allows
the
monocyte
with
restricted
expression
to
escape recognition
and
subsequent
lysis
by
the
immune
system,
which
is
the
fate
of productively
infected
cells
(15)
.
In the
cells
with
latent infection,
no
virus
is
seen
.
Infectious
virus
can
be
activated
and can
efficiently
kill
T
cells
even
after
being
quiescent
for
long
periods
of
time
.
From
the
molecular
and
biological
aspects,
these
two
states
are
mechanistically
and
function-
ally
different
.
Chronic
low
level
expression
is
not
a
model
for
viral
latency
at
the
molecular
level,
and
viral
latency
has
no
low
level
expression
(33)
.
However,
each
of
these
states
provides
at
least
one
mechanism
of
the
establishment
of
HIV
viral
persistence
.
It
would
be
important
to
determine
the
status
of
viral
latency
in
HIV
on December 15, 2016Downloaded from
Published May 1, 1990

171
8

HUMAN
IMMUNE
DEFICIENCY
VIRUS
INFECTION
IN
THP-1
infected
patients
.
Understanding
these
diverse
interactions
between
HIV
and
mono-
cytes
is
important
in
understanding
the
nature
of
viral
persistence
and
its
relation-
ship
to
disease
.
Summary
In
THP-1
monocytoid
cells
infected
with
HIV,
viral
expression
can be
regulated
in several
ways
:
(a)
latency
(no
viral
expression)
;
(b)
restricted
expression
(chronic
low-level
viral
expression
with
little
or
no
detectable
virus released)
;
and
(c)
con-
tinuous
production
.
In
cells
with
restricted
HIV
expression,
nuclear
factor(s)
were
found
that
blocked
tat-associated
DNA
binding
complex
formation,
suggesting
that
initiation
of
transcription
was
negatively regulated
.
Also,
viral
particles
were
seen
budding
into
and
accumulating
within
intracytoplasmic
vacuoles with
little
virus
released,
suggesting multiple
levels
of
regulation
.
These
cells
with
restricted
expres-
sion
had
no
detectable
viral
antigens
on
the
cell
surface
and
were
not
lysed
by
IL-2-
activated
large
granular
lymphocytes
.
However,
they
could
cause
viral-mediated
T
cell
cytolysis
in
cell-cell
assays,
suggesting
viral
transmission
through
cell
contact
.
In
addition,
cells
with
latent
HIV
were
identified
and
could
still
produce
infectious
virus
after
5-azacytidine
exposure
10
mo
later
.
LPS
and
other
treatments
could
in-
crease
viral
production
in
cells
with
restricted
but
not
latent
expression,
suggesting
they
occur
by
distinct
mechanisms
.
These
infected
cells
provide
a
reservoir
for
viral
transmission
to
uninfected
T
cells
that
itself
is
not
detected
by
immune
surveillance
mechanisms
.
We
thank
Mike
Baseler
and
Louise
Finch
for
FAGS
analysis
;
Kunio
Nagashima
for
EM
anal-
ysis
;
Owen
Weislow
for
MTT
assay
;
John
Ortaldo
and
Robin
Winkler
for
LGLs
;
George
Pavalakis
and
Barbara
Felber
for
molecular
constructs
;
Scott
Koenig
for
viral
isolates
;
and
Joost
Oppenheim,
Dan
Longo,
and
Jeff
Rossio
for
reviewing
this
manuscript
.
Received
for
publication
28
November
1989
and
in
revised
form
1
February
1990
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