Originally published online as doi:10.1189/jlb.0306151 on August 21, 2006
Published online before print August 21, 2006
(Journal of Leukocyte Biology. 2006;80:1013-1017.)
© 2006
by Society for Leukocyte Biology
Retroviral E-DNA: persistence and gene expression in nondividing immune cells
Andrea Cara* and
Mary E. Klotman
,1
* Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy; and
Division of Infectious Disease, Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA
1 Correspondence: Mt. Sinai School of Medicine, Box 1090, 1 Gustave L. Levy Place, New York, NY 10029, USA. E-mail: mary.klotman{at}mssm.edu
ABSTRACT
Following retroviral infection of cells, not only is the proviral
DNA integrated into the host genome, but there is also an accumulation
of unintegrated extrachromosomal DNA (E-DNA), both linear and
circular. Although the integrated DNA is responsible for the
production of viral proteins and new viral progeny, the role
of E-DNA has remained uncertain. Several reports have shown
that E-DNA is transcriptionally active producing both RNA, as
well as viral proteins and that circular E-DNA can persist in
nondividing cells, raising questions regarding the potential
consequences of this reservoir. Furthermore, integrase inhibitors,
presently in clinical trials, shifts the balance of proviral
DNA to the E-DNA form. This review is focused on recent work
in this field with an emphasis on exploring the potential role
of E-DNA in both pathogenesis of retroviral infections, especially
HIV-1, and as a tool to deliver and express genes.
Key Words: unintegrated DNA • HIV-1 • integrase inhibitors • vaccine • gene therapy
INTRODUCTION
Following retroviral fusion and entry into the host cell, viral
RNA is reverse transcribed and proviral DNA is transported into
the nucleus of the cell where it either integrates into the
host cell’s genome or remains unintegrated, forming characteristic
episomal forms [
1
]. The integration step is a process dependent
on the viral integrase (IN) enzyme, essential for the subsequent
steps of viral replication, occurring after both ends of linear
DNA have undergone loss of two nucleotides [
1
]. As a consequence,
integration of the viral genome into the cell’s chromosome
is a crucial step for completion of retroviral life cycle and
is an attractive target for the development of drugs that inhibit
integration [
2
].
In addition to the integrated provirus, unintegrated extrachromosomal forms of viral DNA (E-DNA) accumulate in the infected cells [3
4
5
] and are abundant during HIV-1 infection. The E-DNA consists of both fully reverse transcribed linear DNA, as well as closed circular forms of DNA. Circular E-DNA is considered a marker of nuclear entry since it is generally detected only in the nucleus of infected cells; however, a recent report showed that the E-DNA was detectable as early as two hours following virus entry in the cytoplasm of recombinant murine leukemia virus-infected cells [6
]. The circularized double-stranded linear DNA, contains either a single copy or a tandem double copy of the long terminal repeats (1-LTR and 2-LTR forms respectively) [1
, 7
, 8
]. In particular, 1-LTR circles can be produced by autointegration reaction, leading to rearranged circular forms [9
, 10
], reverse transcription intermediates failing to complete the reverse transcription process [11
], or homologous recombination between the two LTR [10
]. In addition, reverse transcribed linear DNA can also be a substrate for the host cell nonhomologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-stranded breaks by end-ligation, yielding the 2-LTR circular forms. A number of proteins have been reported to participate to this reaction, including Ku70/80 heterodimer, ligase 4, XRCC4 and RAD52 [12
, 13
]. Although the linear form of E-DNA is the precursor of the integrated proviral DNA, circular E-DNA is not a template for integration [1
, 14
]. For this reason, the 1-LTR and 2-LTR circular forms of E-DNA are considered terminal by-products of the reverse transcription process. To date, no essential viral or pathologic function has been associated with E-DNA. Although the presence of E-DNA has been reported in a number of animal retroviral systems [3
], this review focuses on the evaluation of the transcriptional potential and persistence of E-DNA during HIV-1 infection. These properties raise the question of whether E-DNA is more than just a side product of the viral replication cycle but could play an active role in viral pathogenesis.
E-DNA PERSISTENCE
Several reports have shown that E-DNA possesses a short half-life
[
7
,
14
15
16
17
18
19
]. Because of this apparent instability,
circular E-DNA, and in particular the 2-LTR episomal form, has
been proposed as a marker of ongoing viral replication in vitro
and in vivo. Studies focused on measuring circular E-DNA in
lentiviral infected human and nonhuman primates have shown the
persistence of 2-LTR circles in peripheral blood mononuclear
cells (PBMC) of the majority of HIV-infected individuals or
experimentally SHIV-infected monkeys in the absence of detectable
plasma viremia levels (<50 copies/ml of plasma). These data
have been used to support the presence of residual cryptic viral
replication in vivo [
18
,
20
21
22
23
24
25
]. However, the
persistence of 2-LTR circles could be due to either ongoing
replication if the circles are unstable or to their increased
stability in a particular viral reservoir. Consistent with the
view that these forms are unstable is the observation by several
groups that unintegrated circular DNA forms have short half-lives
in dividing cells. In fact, following in vitro infection of
replicating T-cells, E-DNA rapidly disappears, thus suggesting
that any persistence must be due to active viral replication
[
17
,
18
,
26
]. Because unintegrated DNA cannot replicate because
of the absence of an origin of replication, circular forms of
E-DNA would be diluted out with successive rounds of cell division.
However, recent work has demonstrated that unintegrated DNA
remains stable for up to 5-7 days in growth-arrested cells [
27
28
29
]
and for up to two months in nondividing macrophages [
30
]. Therefore,
persistence in vivo could represent either stability in a nondividing
cell or instability and turn over in a dividing cell population.
While clinical data clearly demonstrate persistence of detectable circular E-DNA in the absence of detectable plasma HIV RNA, it remains unclear what this persistence represents. In one longitudinal study, there was little correlation between the rapid drop in plasma viral RNA levels in the setting of antiretroviral therapy and the level of 2-LTR DNA, suggesting the latter was a poor marker of virus replication [22
]. The eventual clearing of 2-LTR DNA reported in one study after 7-8 years of therapy could either represent eventual suppression of replication or clearing of a long-lived reservoir [25
]. Importantly, the first in vivo evidence for instability of episomal HIV-1 circular DNA in PBMC from infected patients was recently reported [19
]. In patients on therapy, antiviral resistance mutations emerged in circular E-DNA, while proviral DNA retained wild-type sequences, providing direct evidence for the in vivo evolution and turnover of episomal viral cDNA.
However, the presence of a population of cells within PBMC where these forms were stable has not been excluded and if this were the case, the utility of the assay for monitoring ongoing viral infection would warrant further exploration.
E-DNA GENE EXPRESSION
An increasing number of reports indicate that E-DNA is transcriptionally
active, at least in cell culture model systems, albeit at lower
levels than the integrated counterpart [
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
].
These viral transcripts have been shown to produce viral proteins,
including Gag and Tat; however, fully infectious virions have
not been demonstrated in a number of systems [
34
,
36
,
38
,
45
]. Wu and Marsh [
46
,
47
] have demonstrated transcription
of the nef, env and tat encoding mRNA before integration with
production of the early viral protein, Nef. In addition, Poon
and Chen [
39
] have demonstrated that the presence of the viral
Vpr gene increases expression of viral proteins from unintegrated
templates. Moreover, we and others have shown that viral proteins
expressed from E-DNA result in phenotypic changes in the infected
cells, including down-regulation of the CD4 receptor when Nef
is expressed from an unintegrated template and enhanced T cell
activation and viral replication [
44
,
46
]. E-DNA, both in
the linear and circular configuration, contains promoter and
termination signals essential for synthesis of viral mRNA and,
consequently, viral proteins. In fact, transient transfection
of DNA molecules mimicking the extrachromosomal forms of the
virus produced infectious viral particles, although in a low
amount [
37
].
These findings suggest that circular E-DNA might serve as a reservoir for viral protein expression. This expression could result in subsequent antigen processing through the class I presentation pathway, similarly to the viral proteins produced by the integrated counterpart [48
]. In this setting, E-DNA persistence in infected individuals, although unable to support viral replication, could represent a reservoir of viral protein(s) expression and would have major implications for HIV-1 immune recognition and potentially for AIDS pathogenesis.
Integrase mutant or defective viruses are commonly used to evaluate and characterize the transcriptional activity of E-DNA although there is variability in transcriptional activity reported using IN defective viruses to infect cell lines, PBMC or terminally differentiated cells, including macrophages [31
, 33
, 34
, 36
, 38
, 43
, 45
, 49
50
51
]. This variability may, in part, be due to the mutation introduced, in that some mutations introduced in the catalytic center of the integrase gene specifically blocks the DNA cleaving and joining reactions of the integration step (Class I mutants), while others have more pleiotropic effects adversely affecting gag-pol precursor polyprotein processing or assembly, which may indirectly account for the lower transcriptional activity of some IN mutants (Class II mutants). In addition, it has been found that some cell lines are more promiscuous in supporting replication of class I IN mutants, leading to the definition of permissive, semipermissive, or nonpermissive cell lines [38
]. Although cell-type dependent nonspecific DNA recombination (i.e., nonintegrase-mediated E-DNA integration into the host cell’s chromosome) may contribute to class I IN mutant viral replication, other mechanisms, may be involved in the ability of IN mutant viruses to sustain multiple rounds of infection and expression from unintegrated templates. In this context, recent work has demonstrated that transcription from unintegrated viral templates is strong and persists in both growth-arrested cells or terminally differentiated cells such as primary macrophages and retinal ganglion cells [30
, 42
, 43
].
The low transcriptional capacity of E-DNA in certain cell types suggests that the structural conformation of this DNA does not make it an efficient template for viral expression. The transcriptional activity of E-DNA might be impeded by proteins of either cellular or viral origin, which may displace or block the attachment of transcriptional factors in the LTR [52
, 53
]. A recent report showed that the presence of the HIV-1 Vpr protein is essential for optimal expression from unintegrated viral templates [39
]. The proximity of the two LTR promoters or the use of the single LTR to both start and end transcription in the 2-LTR and 1-LTR circular forms, respectively, might contribute to the lower transcriptional ability of E-DNA compared with the integrated form [37
].
To enhance the transcriptional activity of E-DNA, one strategy is to enhance the stability of these forms in dividing cells. We and others recently reported that the insertion of the simian virus 40 (SV40) origin of replication in the backbone of either an integration-defective lentiviral vector or a HIV-1 molecular clone was associated with long-term expression and persistence of E-DNA [40
, 41
] in dividing cells in the presence of SV40 large T-antigen. Moreover, the persistence and episomal nature of circular E-DNA was confirmed by fluorescence in situ hybridization analysis up to 60 days after transduction [40
]. This approach provides a strategy for gene delivery based on nonintegrating lentiviral vectors, particularly for those diseases associated with episomally replicating viruses, including papilloma viruses, polyoma viruses, and herpes viruses. Importantly, a recent report provided the first evidence for effective delivery of a therapeutic gene using an IN-defective lentiviral vector [54
].
E-DNA AND INTEGRASE INHIBITORS
The evaluation of E-DNA may have implications in the setting
of antiretroviral therapy. One new class of drugs undergoing
preclinical and clinical evaluation targets the IN protein of
HIV-1 [
55
]. The development of IN inhibitors may prove to be
an important advance in antiretroviral therapy. By preventing
viral integration, these compounds have been shown to inhibit
subsequent viral production while increasing E-DNA with respect
to the integrated counterpart [
56
57
58
59
60
], thus recapitulating
the effects obtained in vitro after infection with IN defective
viruses. While IN-defective viruses can serve as a model for
studying E-DNA, the kinetics of HIV-1 infection in the presence
of IN inhibitors may be more clinically relevant than those
observed with IN-defective viruses. In this context, Wu and
Marsh recently demonstrated that HIV-1 Nef is expressed in the
presence of a diketo acid IN inhibitor [
47
]. Similarly, we
recently showed the ability of Nef expressed in the context
of an IN-competent virus but in the presence of an IN inhibitor
to down-regulate CD4 on primary CD4+ T lymphocytes [
44
]. Although
IN inhibitors would increase the amount of E-DNA, new rounds
of viral replication would be blocked, and E-DNA would be lost
over time; minimizing any clinical consequences from expression
of these forms.
E-DNA AS A VACCINE
Live attenuated SIV/SHIV expressing multiple viral proteins
prevents an AIDS-like disease after challenge with pathogenic
SIV/SHIV in monkeys, indicating that presentation of viral epitopes
in this context could be effective [
61
]. However, a major drawback
to the development of attenuated lentiviral vaccines was the
finding that a Nef-deleted SIV could give rise to an AIDS-like
disease in both neonatal and adult macaques [
62
and references
therein]. Additional concerns regarding the use of attenuated
lentiviruses arise from the recent finding that recombination
of live, attenuated SIV with challenge virus in some cases results
in an even more virulent strain [
63
]. One strategy to minimize
these unacceptable outcomes might be to exploit the coupling
of multiple gene products expressed from viral vectors in the
context of HIV-derived IN-defective vectors. This strategy would
take advantage of the presentation of viral antigens within
the context of a nonintegrating and nonreplicating whole virus,
therefore having a major potential safety advantage compared
with previously attenuated candidates. Interestingly, a recent
report showed persistence and expression from unintegrated templates
in an in vivo model of gene therapy for retinal degeneration
[
54
]. In addition, we have shown that episomal vectors based
on lentiviral E-DNA are maintained in culture and have sustained
expression of reporter genes eGFP and luciferase, in the absence
of cell division [
30
,
40
], and preliminary data indicate that
expression of viral proteins such as Env is sustained in the
absence of episomal replication (data not shown). Critical to
this approach is determining whether IN-defective HIV-1 results
in enough viral protein expression to elicit an immune response.
Thus, further exploration of the functional activity of viral
proteins expressed from E-DNA and their capability to stimulate
a long-lasting immune response is of interest.
CONCLUSIONS
HIV-1 E-DNA is normally produced/sustained during the course
of infection in infected patients, even in the absence of detectable
plasma viremia. In this setting, presence of E-DNA may indicate
either continuous cryptic viral replication and/or persistence
of this form in a nondividing long-lived reservoir. The latter
could come from tissue macrophages as well as nondividing memory
CD4+ T-lymphocytes as they can persist either in tissue or in
the periphery for prolonged periods of time. In either case,
this form of E-DNA might serve as a template for persistent
viral protein expression. Additionally, the demonstration that
viral proteins transcribed from E-DNA can modulate the pattern
of molecules expressed on the cell surface of infected cells
and that E-DNA expressed proteins might be recognized within
the context of MHC class I, suggests that E-DNA represents a
reservoir of biologically active proteins with implications
for AIDS pathogenesis, as well as vaccine and vector development
(
Fig. 1
). The full evaluation of E-DNA function and activities
has implications for antiviral development, particularly IN-inhibitors,
for the engineering of nonintegrating viral vectors for gene
therapy purposes and for the generation of nonintegrating lentiviral
vaccines.
ACKNOWLEDGEMENTS
We are grateful to Drs. V. Buffa, D.R. Negri, Z. Michelini,
L. Gillim-Ross, and J. Vargas, Jr., for critical insights.
Received March 4, 2006;
revised May 16, 2006;
accepted May 17, 2006.
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ABSTRACT
INTRODUCTION
, macrophages; DC, dendritic cells; MHC-1, and class-I major histocompatibility complex.
