Vol. 65, No. 5
JOURNAL OF VIROLOGY, May 1991, p. 2695-2698
0022-538X/91/052695-04$02.00/0
Copyright ©) 1991, American Society for Microbiology
Unintegrated Human Immunodeficiency Virus Type 1 DNA in
Chronically Infected Cell Lines Is Not Correlated with
Surface CD4 Expression
N. J. BESANSKY,* S. T. BUTERA, S. SINHA, AND T. M. FOLKS
Retrovirus Diseases Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases,
Centers for Disease Control, Atlanta, Georgia 30333
Using a polymerase chain reaction-based assay on total cell Ijsates, we have detected unintegrated human
immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and
promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4
inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted
recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in
these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels
of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction.
ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, Jl cells, with
surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar
to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.
lysates by polymerase chain reaction (PCR) analysis, eliminating the need for isolation of unintegrated DNA by
procedures such as Hirt fractionation. The two-LTR circular
form was chosen as a marker for total unintegrated viral
DNA because of our ability to uniquely and specifically
detect this form in the presence of other forms of unintegrated and integrated viral DNAs. PCR primers binding the
U5 and U3 regions of the LTR were oriented so that
amplification would proceed efficiently only across the LTRLTR junction of two-LTR circular HIV-1 DNA to produce a
173-bp product. Other forms of unintegrated and integrated
HIV-1 are not detected because the distance separating the
primers is too great (-10 kb) for efficient amplification.
In the first experiment, these primer pairs were used to
test for the presence of unintegrated HIV-1 DNA in lysates
from acutely infected cultures and persistently infected cell
lines, including the T-lymphocytic 8E5 (10), ACH-2 (4), and
Jl (23) cell lines and the promyelocytic OM-10.1 cell line (3).
By using HIV-specific primer pairs (19), a gag region was
amplified in parallel as a total HIV-1 DNA reference. A gag
signal was observed in all HIV-1-infected cultures (Fig. 1).
With the two-LTR primer pairs, amplified products of the
expected molecular size were obtained from the acutely
infected cultures as well as from three of four chronically
infected cultures. Interestingly, the signal intensities varied
among the different cultures. Given equivalent amounts of
cellular DNA in the PCR reactions, the stronger two-LTR
signals indicate a much higher copy number of unintegrated
HIV-1 DNA in the acutely infected, OM-10.1, and Jl cultures than in ACH-2 cultures. In addition, OM-10.1 and Jl
lysates showed a ladder of bands above the expected 173-bp
band. The additional bands appear to be HIV-1 specific,
since the conditions employed in the PCR assay were
stringent and no signals were observed from uninfected
cultures. Furthermore, the ladder of bands is not an artifact
of long-term cell culture, since the banding pattern was
similar in lysates from acutely infected peripheral blood
lymphocytes (PBLs) and A3.01 cells (overexposed in Fig. 1
to enhance the signals from the persistently infected cells).
Human immunodeficiency virus type 1 (HIV-1) preferentially infects cells expressing CD4 on their surfaces (5). Early
after primary infection of host cells, three forms of unintegrated HIV-1 DNA accumulate transiently. These include a
linear copy of the viral genome with long terminal repeats
(LTRs) and two covalently closed circular forms with one or
two tandem LTRs. The linear form of unintegrated DNA
appears to be the precursor to integration into the host
chromosome (2).
Infection with HIV-1 may lead either to destruction of the
host cell or to a persistent, noncytopathic infection. Like the
outcome of infection with certain avian (14, 29) and feline
(17) retroviruses, the outcome of infection with HIV-1 has
been correlated with the amount of unintegrated viral DNA
present in the host cell (22). In vitro, the accumulation of
large quantities of unintegrated viral DNA is a hallmark of an
acute, cytopathic infection. Such DNA accumulates to high
levels in the cell as a result of second-round superinfection
(22, 25) and may kill the cell directly or indirectly through
unknown mechanisms. In contrast, significant amounts of
unintegrated viral DNA are not generally observed in persistent HIV-1 infections. This has been attributed to protection from reinfection due to the loss or down-modulation of
the CD4 receptor, a feature of most chronically infected cell
lines (5, 10, 28).
In this paper we report the presence of unintegrated HIV-1
DNA in chronically infected T-lymphocytic and promyelocytic cell lines. Our results indicate that unintegrated HIV-1
DNA is unstable in these cell lines and originates from a
continuous process of reinfection. Although the accumulation of unintegrated viral DNA was inhibited by 3'-azido-3'deoxythymidine (AZT), soluble CD4 (sCD4), and anti-CD4
monoclonal antibody (anti-CD4 MAb), it was not correlated
with degree of surface CD4 expression in chronically infected cell lines.
Unintegrated HIV-1 DNA was detected in whole-cell
*
Corresponding author.
2695
Downloaded from http://jvi.asm.org/ on February 20, 2016 by PENN STATE UNIV
Received 27 November 1990/Accepted 28 January 1991
2696
J. VIROL.
NOTES
_
__
"j
_-
'T _~
40-
VW
_
a
FIG. 1. PCR analysis of two-LTR circular HIV-1 DNA in chronically infected cell lines. Cells were washed twice with phosphatebuffered saline and suspended in PCR lysis buffer (3) at 6 x 106 cells
per ml. After lysis, PCR was performed with 25 ,ul of lysate and 100
ng of each primer for 35 cycles at 94°C for 1 min, 61°C for 1 min, and
72°C for 30 s, as described elsewhere (26). The 5' two-LTR primer
binds positions 566 to 585 of HXB2 (24), the 3' primer binds
positions 9170 to 9151, and the two-LTR probe binds positions 591
to 620. A region of gag was amplified in parallel by using primers
SK38 and SK39 (19). Amplification products were analyzed by
agarose gel electrophoresis and Southern hybridization to a 5 _32p_
labeled two-LTR probe or to gag probe SK19 (19). The predicted
size of the two-LTR amplification product is 173 bp; that of the gag
product is 116 bp (19). Uninfected A3.01 cells (9) served as a
negative control, and acute infections of A3.01 cells and normal
human PBLs with lymphadenopathy-associated virus stock served
as positive controls. Continuously infected T-lymphocytic cell lines
were derived from acute infections of A3.01 cells (8E5 and ACH-2)
or Jurkat cells (Ji) with lymphadenopathy-associated virus stock.
Continuously infected promyelocytic cell line OM-10.1 was derived
from lymphadenopathy-associated virus-infected HL60 cells (3).
The additional bands may correspond to variant forms of
unintegrated HIV-1 DNA resulting from insertion and recombination events (21). No two-LTR products were detected from 8E5 cells, although a strong gag signal was
present. That the virions produced by 8E5 cells are defective
in reverse transcriptase function (10, 11) suggests a role for
that enzyme in maintaining extrachromosomal viral DNA.
A
24h
Oh
M
M A
C
48h
M A C
72h
M A
C
.99t
'i:;"'.,
2-LTR
173 bp
48h
M PA
B
2-T
..w
OM
173 bp
72h
M PA
OM
10.1
10.1
gag
116 bp
uEhIIIIEmhIirn. ?--I'lImImhmhmEhPMWh
173
b
pp
2-LTR
2-LTR
173 bp
*
Ji
Ji1
gag
116 bp
I
..Ii
EIm I I.
FIG. 2. Effect of AZT or sCD4 on accumulation of two-LTR circles. (A) Cells were treated with AZT (Burroughs Wellcome) or
recombinant CD4 (Genentech) at 10 or 20 ,ug/ml of medium, respectively. Untreated (lanes M), AZT-treated (lanes A), and sCD4-treated
(lanes C) cells were sampled upon setup (0 h) and at 24-h intervals, and then equal numbers were subjected to PCR with either two-LTR
primers or gag primers. (B) Removal of AZT from OM-10. 1 and Jl cultures permitted accumulation of two-LTR circles. Cultures from which
AZT was removed (lanes PA) and untreated cultures (lanes M) were sampled upon setup and at 24-h intervals and then PCR amplified with
two-LTR primers.
Downloaded from http://jvi.asm.org/ on February 20, 2016 by PENN STATE UNIV
To distinguish reinfection from reverse transcription of
intracellular viral transcripts as possible sources of unintegrated viral DNA, cultures of OM-10.1 and Ji were maintained in the presence or absence of either AZT, a viral
replication inhibitor (15), or sCD4, which blocks reinfection
(8). PCR analysis was performed on samples removed at
setup and at 24-h intervals. Within 72 h, both treatments
reduced the level of two-LTR circles in both cell lines, with
no discernible effects on cell viability, efficiency of amplification, or total detectable HIV-1 DNA (Fig. 2A). Thus, the
presence of unintegrated HIV-1 DNA in these cultures
reflects reinfection by progeny virions. In addition, since
AZT and sCD4 inhibit prospective rather than preexisting
unintegrated viral DNA, the rate at which the two-LTR
signal was diminished indicates that this form is unstable
within the cell and persists no longer than 72 h.
To control for variations in total cellular DNA among PCR
reactions, the PCR method was modified by reducing the
number of amplification cycles and including primers homologous to the gamma interferon gene, a single-copy cellular
gene, as an internal DNA standard. To determine the twoLTR copy number, known amounts of a plasmid containing
the two-LTR circle junction were subjected to PCR in
parallel (data not shown). This quantitative procedure, carried out on lysates from OM-10.1 grown in the presence or
absence of AZT or sCD4 as described above, confirmed
previous results (Table 1). Three days of treatment reduced
the abundance of two-LTR circles by 10-fold, from approximately 1 copy per 50 cells in control cultures to 1 copy per
500 cells after treatment.
The length of time necessary to restore unintegrated viral
DNA to pretreatment levels was determined by washing
AZT-treated OM-10.1 and JI cells and suspending them in
medium alone. Samples removed at zero hour and at 24-h
intervals were subjected to PCR analysis and compared with
untreated cultures (Fig. 2B). By 72 h post-AZT treatment,
two-LTR circles had returned to pretreatment levels, implying that reinfection is a continuous process.
Acute, cytopathic HIV-1 infection has been associated
with the massive accumulation of unintegrated viral DNA
through reinfection of cells bearing relatively high levels of
surface CD4 (22, 25). In contrast, persistent HIV-1 infection
has been associated with little or no unintegrated viral DNA
NOTES
VOL. 65, 1991
TABLE 1. Quantitative PCR analysis of two-LTR circular HIV-1
DNA in OM-10.1 cells treated with AZT or sCD4a
CD4 *
a
-
CD8
Two-LTR circle production atb:
Treatment
None
AZT
sCD4
-m
2697
0h
24 h
48 h
72 h
100.0
100.0
73.8
108.0
100.0
25.6
0
100.0
12.1
3.1
in cells bearing little or no detectable surface CD4. One
model to explain the different courses of infection proposes
that the rate and extent of reinfection are directly related to
the level of surface CD4 expression by the target cell (27). To
test this relationship, we determined the level of surface CD4
on three chronically HIV-1-infected cell lines in which we
had detected various levels of unintegrated viral DNA (Fig.
3). By flow cytometry, very little surface CD4 was detected
on ACH-2 cells, and, as expected, they had a small amount
of unintegrated viral DNA (Fig. 1). Similarly, a correlation
was observed with the OM-10.1 cell line, in which relatively
high levels of surface CD4 were associated with a large
accumulation of unintegrated viral DNA. However, surface
CD4 expression by the Jl cell line was not detected at all,
and yet the level of unintegrated viral DNA and rate of
reinfection (Fig. 2B) were similar to those of OM-10.1 cells.
Thus, the rate of reinfection appears to be independent of the
number of CD4 receptors.
The possibility remained that Jl cells lack any CD4 on
their surface, implying that HIV-1 superinfection can occur
via a CD4-independent pathway. In this case, the blocking of
reinfection by recombinant sCD4 could be explained by the
ability of sCD4 to neutralize HIV-1 by causing the shedding
of gpl20 from virions (16) rather than by competitive inhibition of the virus-cell interaction. To distinguish CD4-dependent from CD4-independent superinfection, Ji cells were
first cleared of unintegrated HIV-1 DNA by culture in the
presence of AZT for 7 days. Then, these AZT-treated Jl
cells were cultured with (per milliliter) 1.25 ,i.g of either the
anti-CD4 MAb OKT4A (Ortho), which prevents acute
HIV-1 infection by specifically blocking the gpl20-binding
site of CD4, or, as a negative control, the anti-CD4 MAb
OKT4 (Ortho), which binds a CD4 epitope not involved with
gpl20 interactions (13). After 72 h, equal numbers of cells
were removed from these cultures and compared by PCR
analysis with AZT-treated Ji cells that were maintained in
medium (AZT free) over the same period. Values corresponding to the PCR band for the two-LTR circle were
generated by densitometry and expressed as a percentage of
the value for the medium culture. After AZT treatment,
culture of Jl cells in the presence of OKT4 allowed for the
reaccumulation of circular HIV-1 DNA and resulted in a
level 146.1% that of the medium culture. However, under
identical conditions, culture of Jl cells in the presence of
OKT4A prevented the accumulation of circular HIV-1 DNA
Downloaded from http://jvi.asm.org/ on February 20, 2016 by PENN STATE UNIV
a Equal numbers of untreated, AZT-treated, and sCD4-treated cells were
sampled at setup and at 24-h intervals. For quantitative PCR, each reaction
contained gamma interferon-specific primers (18) as an internal cellular DNA
reference, and the number of cycles was reduced from 35 to 25. After
amplification, liquid hybridization was performed (6) with 250,000 cpm each of
a two-LTR and a gamma interferon gene 5'-32P-labeled probe (positions 4669
to 4688 in reference 12). Following autoradiography, densitometry was
performed on an LKB UltroScan XL laser densitometer.
b The densitometric measurements of bands from the two-LTR product
were first normalized by dividing by the measurements from the corresponding cellular reference product (gamma interferon) for each PCR reaction.
Results were then expressed as a percentage of the medium control for each
time point.
I
(a)
4-
C')
C)
E3
z
Relative Log Fluorescence
) by chroniFIG. 3. Expression of surface CD4 antigen (
cally HIV-1-infected OM-10.1, ACH-2, and Jl cell lines. Surface
CD8 expression (... .) was used as a negative control in all cases.
Surface CD4 expression was detected by an indirect immunofluorescence assay, as described elsewhere (3). Briefly, cells were first
reacted with anti-CD4 (OKT4; Ortho) and anti-CD8 (OKT8; Ortho)
MAbs followed by a phycoerythrin-tagged goat anti-mouse immunoglobulin G secondary antibody (Tago, Inc.). After paraformaldehyde fixation, cells were analyzed on a Becton Dickinson FACScan
system. In each case, 104 total cells were analyzed, and the data are
presented as histogram profiles comparing relative log fluorescence
with total cell numbers.
2698
NOTES
REFERENCES
1. Besansky, N. J. Unpublished data.
2. Brown, P. 0. 1990. Integration of retroviral DNA. Curr. Top.
Microbiol. Immunol. 157:19-47.
3. Butera, S. T., V. L. Perez, N. J. Besansky, W. C. Chan, B.-Y.
Wu, G. J. Nabel, and T. M. Folks. J. Cell. Biochem., in press.
4. Clouse, K. A., D. Powell, I. Washington, G. Poli, K. Strebel, W.
Farrar, P. Barstad, J. Kovacs, A. S. Fauci, and T. M. Folks.
1989. Monokine regulation of human immunodeficiency virus-1
expression in a chronically infected human T cell clone. J.
Immunol. 142:431-438.
5. Dalgleish, A., P. Beverley, P. Clapham, D. Crawford, M.
Greaves, and R. Weiss. 1984. The CD4 (T4) antigen is an
essential component of the receptor for the AIDS retrovirus.
Nature (London) 312:763-766.
6. Ehrlich, G. D., S. Greenberg, and M. A. Abbott. 1990. Detection
of human T-cell lymphoma/leukemia viruses, p. 325-336. In
M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.),
PCR protocols: a guide to methods and applications. Academic
Press, Inc., San Diego.
7. Fauci, A. S. 1988. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239:617-622.
8. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S.
Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch,
R. T. Schooley, and R. A. Flavell. 1988. HIV infection is blocked
in vitro by recombinant soluble CD4. Nature (London) 331:7678.
9. Folks, T. M., S. Benn, A. Rabson, T. Theodore, M. D. Hoggan,
M. Martin, M. Lightfoote, and K. Sell. 1985. Characterization of
a continuous T-cell line susceptible to the cytopathic effects of
the acquired immunodeficiency syndrome (AIDS)-associated
retrovirus. Proc. Natl. Acad. Sci. USA 82:4539-4543.
10. Folks, T. M., D. Powell, M. Lightfoote, S. Koenig, A. S. Fauci, S.
Benn, A. Rabson, D. Daugherty, H. E. Gendelman, M. D.
Hoggan, S. Venkatesan, and M. A. Martin. 1986. Biological and
biochemical characterization of a cloned Leu-3- cell surviving
infection with the acquired immune deficiency syndrome retrovirus. J. Exp. Med. 164:280-290.
11. Gendelman, H. E., T. S. Theodore, R. Willey, J. McCoy, A.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Adachi, R. J. Mervis, S. Venkatesan, and M. A. Martin. 1987.
Molecular characterization of a polymerase mutant immunodeficiency virus. Virology 160:323-329.
Gray, P. W., and D. V. Goeddel. 1982. Structure of the human
immune interferon gene. Nature (London) 298:859-863.
Hoxie, J. A., J. D. Alpers, J. L. Rackowski, K. Huebner, B. S.
Haggarty, A. J. Cedarbaum, and J. C. Reed. 1986. Alterations in
T4 (CD4) protein and mRNA synthesis in cells infected with
HIV. Science 234:1123-1127.
Keshet, E., and H. M. Temin. 1979. Cell killing by spleen
necrosis virus is correlated with a transient accumulation of
spleen necrosis virus DNA. J. Virol. 31:376-388.
Mitsuya, H., K. J. Weinhold, P. A. Furman, M. H. St. Clair,
S. N. Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, and S.
Broder. 1985. 3'-Azido-3'-deoxythymidine (BW A509U): an
antiviral agent that inhibits the infectivity and cytopathic effect
of human T-lymphotrophic virus type III/lymphadenopathyassociated virus in vitro. Proc. Natl. Acad. Sci. USA 82:70967100.
Moore, J. P., J. A. McKeating, R. A. Weiss, and Q. J. Sattentau.
1990. Dissociation of gpl20 from HIV-1 virions induced by
soluble CD4. Science 250:1139-1142.
Mullins, J. I., C. S. Chen, and E. A. Hoover. 1986. Diseasespecific and tissue-specific production of unintegrated feline
leukemia virus variant DNA in feline AIDS. Nature (London)
319:333-336.
Neubauer, A., B. Neubauer, and E. Liu. 1990. Polymerase chain
reaction based assay to detect allelic loss in human DNA: loss of
P-interferon gene in chronic myelogeneous leukemia. Nucleic
Acids Res. 18:993-998.
Ou, C.-Y., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky,
J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman.
1988. DNA amplification for direct detection of HIV-1 in DNA
of peripheral blood mononuclear cells. Science 239:295-297.
Pang, S., Y. Koyanagi, S. Miles, C. Wiley, H. V. Vinters, and
I. S. Y. Chen. 1990. High levels of unintegrated HIV-1 DNA in
brain tissue of AIDS dementia patients. Nature (London) 343:
85-89.
Pauza, C. D., and J. Galindo. 1989. Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to
accumulation of self-integrated viral DNA and to production of
defective virions. J. Virol. 63:3700-3707.
Pauza, C. D., J. E. Galindo, and D. D. Richman. 1990. Reinfection results in accumulation of unintegrated viral DNA in
cytopathic and persistent human immunodeficiency virus type 1
infection of CEM cells. J. Exp. Med. 172:1035-1042.
Perez, V. L., T. Rowe, S. Justement, S. T. Butera, C. H. June,
and T. M. Folks. Submitted for publication.
Ratner, L., W. Haseltine, R. Patarca, K. J. Livak, B. Starcich,
S. J. Josephs, E. R. Doran, J. A. Rafalski, E. A. Whitehorn, K.
Baumeister, L. Ivanoff, S. R. Petteway, Jr., M. L. Pearson, J. A.
Lautenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C.
Gallo, and F. Wong-Staal. 1985. Complete nucleotide sequence
of the AIDS virus, HTLV-III. Nature (London) 313:277-284.
Robinson, H. L., and D. M. Zinkus. 1990. Accumulation of
human immunodeficiency virus type 1 DNA in T cells: result of
multiple infection events. J. Virol. 64:4836-4841.
Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi,
G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermolabile
DNA polymerase. Science 239:487-491.
Stevenson, M., C. Meier, A. M. Mann, N. Chapman, and A.
Wasiak. 1988. Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for
persistence in AIDS. Cell 53:483-496.
Stevenson, M., X. Zhang, and D. J. Volsky. 1987. Downregulation of cell surface molecules during noncytopathic infection of
T cells with human immunodeficiency virus. J. Virol. 61:37413748.
Weller, S. K., A. E. Joy, and H. M. Temin. 1980. Correlation
between cell killing and massive second round superinfection by
members of some subgroups of avian leukosis virus. J. Virol.
33:494-506.
Downloaded from http://jvi.asm.org/ on February 20, 2016 by PENN STATE UNIV
and resulted in a level only 19.7% that of the medium
culture. Therefore, while OKT4 had no inhibitory effect on
reinfection, treatment with OKT4A reduced by fivefold the
two-LTR circular HIV-1 DNA accumulation, showing that
CD4 is present on the Jl cell surface and that reinfection is
CD4 dependent.
Our results demonstrate that the occurrence of extrachromosomal HIV-1 DNA depends on viral replication and not
on the degree of CD4 surface expression. Furthermore, little
or no cytopathicity is observed during unintegrated HIV-1
DNA accumulation in our chronically infected cell models in
contrast to observations in previous studies with other in
vitro retroviral systems involving acute infections (14, 29).
However, the difference may be due to the much higher
levels of unintegrated viral DNA found in acutely infected
cells.
As the level of unintegrated retroviral DNA in vitro has
been correlated with cytopathicity, so the level of unintegrated retroviral DNA in vivo has been correlated with
disease (17, 20). Although clinical HIV-1 infection has a long
period of latency, for which the persistently infected cell
lines serve as models, it ultimately leads to AIDS (7). We
have been able to detect the two-LTR form of HIV-1 DNA
in PBLs from HIV-1-infected asymptomatic individuals as
well as in PBLs from AIDS patients (1) and anticipate that
this form can be used to monitor viral activity in such
individuals, given the ease with which PCR can be applied to
small amounts of clinical samples. Clinical studies are under
way in our laboratory to establish a correlation between
two-LTR circles and disease progression.
J. VIROL.