Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 297304
doi:10.1111/j.1745-7270.2007.00275.x
Cell-specific Regulation of
APOBEC3F by Interferons
Songcheng YING1#, Xuzhao
ZHANG1#, Phuong Thi Nguyen SARKIS2#,
Rongzhen XU1, and Xiaofang YU1,2*
1
Second Affiliated Hospital, Cancer Institute, School of Medicine, Zhejiang
University, Hangzhou 310009, China;
2
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg
School of Public Health, Baltimore, Maryland 21205, USA
Received: December
3, 2006
Accepted: January
24, 2007
This work was
supported by the grants from the NIH (AI062644), the National Natural Science
Foundation of China (30425012) and the Cheung Kong Scholars Program Foundation
of the Chinese Ministry of Education
# These authors
contributed equally to this work
*Corresponding
author: Tel, 410-955-3768; Fax, 410-614-8263; E-mail, [email protected]
Abstract Human cytidine deaminase
APOBEC3F (A3F) has broad anti-viral activity against hepatitis B virus and
retroviruses including human immunodeficiency virus type 1. However, its regulation
in viral natural target cells such CD4+ T lymphocytes,
macrophages, and primary liver cells has not been well studied. Here we
showed that A3F was up-regulated by interferon (IFN)-a in primary hepatocytes and
multiple liver cell lines as well as macrophages. Although the IFN-a signaling
pathway was active in T lymphoid cells and induction of other IFN stimulated
genes such as PKR was detected, A3F and APOBEC3G (A3G) were not induced by IFN-a in these
cells. Thus, additional factors other than known IFN-stimulated genes also
regulated IFN-a-induced A3F expression distinctly. A3F and A3G expression levels in
primary hepatocytes, especially after IFN-a stimulation, were
comparable to those in CD4+ T lymphocytes in some
individuals. Significant variations of A3F and A3G expression in primary
hepatocytes from various subjects were observed. Individual variations in A3F
and/or A3G regulation and expression might influence the clinical outcomes of
hepatitis B infection.
Key words APOBEC3F; interferon; human immunodeficiency virus type 1; hepatitis
B virus; cytidine deaminase
APOBEC3G (A3G) and APOBEC3F (A3F) are members of the APOBEC family
proteins with homologous cytidine deaminase domains [1]. Although their
cellular targets are not known, A3G and A3F are potent antiviral proteins that
can suppress human immunodeficiency virus type 1 (HIV-1), hepatitis B virus
(HBV), and endogenous retroelements [2–8]. The antiviral mechanism of A3G against
HIV-1 has been well studied. In the absence of the viral Vif protein, the A3G
cytidine deaminase converts cytidines to uridines in single-stranded viral cDNA
during reverse transcription resulting in lethal hypermutation of the virus
genome. The HIV-1 Vif protein, however, can degrade A3G through a
proteasomal-dependent pathway involving cullin 5 containing E3 ubiquitin ligase
[9]. By targeting A3G for degradation in the virus-producing cell, Vif is able
to prevent A3G molecules from incorporating into virions where they would
otherwise be carried into the newly infected cell to inhibit productive
infection. Although HBV is a DNA virus, it replicates through reverse
transcription and can likewise be targeted by A3G and A3F [10]. Many studies have focused on the antiviral effects and
post-translational regulation of A3G and A3F. However, little is known about
how they are transcriptionally regulated. It is not known if A3G or A3F could
be induced in vivo in response to inflammation or cytokines, or whether
A3G or A3F participate in interferon (IFN)-mediated host defenses is unclear.
One previous study found that A3G was induced by phorbol myristate acetate but
not by IFNs in an immortalized CD4+ T cell line H9 [11].
However, a recent report suggested that A3G could be induced by IFNs in
macrophages [12]. The effects of IFNs on A3G transcription in primary CD4+ T cells have not been reported.
Regulation of A3F by IFNs in relevant cells such as hepatocytes,
macrophages, and primary CD4+ T cells has not been well
studied. In the present study, we observed that IFN-a can up-regulate A3F mRNA
in macrophages, primary hepatocytes, and liver cell lines but not in primary
CD4+ T cells. We also observed a wide variation in the level of A3F
induction by IFN in primary hepatocytes from different subjects, suggesting
that individual variation in IFN responsiveness might account for differences
in viral pathogenesis and clinical outcomes of HBV infections.
Materials and Methods
Cell culture and IFN treatment
Freshly isolated primary hepatocytes from anonymous donors were
obtained from BD Biosciences (San Jose, USA). Hepatocytes were received within
24–48
h of isolation as adherent cultures in 6-well plates in Hepato-STIM medium (BD
Biosciences) and used immediately for induction studies. Hep3B, HepG2, and
QSG7701 are hepatocellular carcinoma cell lines maintained in DMEM (Invitrogen,
Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS). Primary CD4+ T cells were purified from freshly isolated PBMC by incubation with
CD4-conjugated magnetic microbeads (Miltenyi Biotech, Gladbach, Germany)
according to the manufacturer’s instructions and cultured in RPMI 1640 with 10%
FBS. To obtain macrophages, freshly isolated PBMC were plated in 6-well plates
overnight at 2?107 cells/ml
in RPMI 1640 with 10% FBS, after which non-adherent cells were removed and the
medium replaced every 2 d. Macrophages, differentiated by adherence to the
plastic, were used on day 12 after isolation for induction studies. All
cytokines were obtained from EMD Biosciences. IFN-a and IFN-g were dissolved
in phosphate-buffered saline with 0.5% bovine serum albumin (control medium)
and stored in single-use aliquots at –70 ?C. Unless otherwise stated, IFN-a was used at
1000 IU/ml and IFN-g at 10 IU/ml. In IFN induction experiments, cells were treated with
equal volumes of IFN or control medium.
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR (qRT-PCR) was carried out according to
standard protocols [13]. Briefly, total RNA from cells was isolated using the
RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s
instructions, including an on-column DNase digestion step using the RNase-free
DNase set (Qiagen). One-fifth of the RNA was reverse transcribed using random
primers and the Multiscribe reverse transcriptase (Applied Biosystems, Foster
City, USA). The cDNA was amplified using TaqMan universal PCR master mix
(Applied Biosystems) and an ABI Prism 7000 sequence detection system (Applied
Biosystems). The primer/probe sets were pre-designed TaqMan gene expression
assays specific for A3G, A3F, PKR and IRF-1 (Hs00222415_m1, Hs00736570_m1,
Hs00169345_m1 and Hs00233698_m1, respectively). Amplification of target genes
was normalized using amplification levels of b-actin as an endogenous
control (human ACTB endogenous control FAM/MGB probe; Applied Biosystems). The
efficiency of the PCR was tested by amplification of the target from serially
diluted cDNA generated from reverse transcription of a stock set of human RNA.
Data analysis and calculations were carried out using the 2-DDCT comparative method as previously described [13]. Gene expression is
expressed as a fold induction of a gene measured in IFN-treated samples
relative to samples treated with control media (phosphate-buffered saline plus
0.5% bovine serum albumin).
Results
Results
A3F is induced by IFN-a in liver cell lines and primary
hepatocytes
A3F was reported to have potent anti-HBV activity [10]. However,
whether A3F expression is regulated by IFNs in liver cells is not known [14].
To examine the effect of IFNs on A3F transcription, we treated the various cell
types with IFN-a, IFN-g, or control media for various times. Cells were then collected to
isolate RNA for measuring A3F mRNA by qRT-PCR. PKR and IRF-1 mRNA, inducible
primarily by IFN-a and IFN-g, respectively, were also measured as positive controls for IFN
treatment. mRNA expression in IFN treated cells were expressed as a fold induction
over control treated cells after normalizing to b-actin. We observed that A3F mRNA was expressed in liver cell line HepG2
and, more importantly, its expression in these cells was up-regulated by IFN-a in a
dose-dependent fashion [Fig. 1(A)]. As positive controls for IFN
stimulation, we detected other known IFN stimulated genes (ISG) such as PKR,
which was mainly induced by IFN-a [Fig. 1(B)], and IRF-1, which was
primarily induced by IFN-g [Fig. 1(C)]. The time courses of IFN-a and IFN-g induced A3F
expression was also examined. IFN-a induced A3F expression peaked 4–12 h after
treatment in HepG2 cells [Fig. 1(D)]. IFN-g induced A3F expression
peaked 8–24 h after treatment in these cells [Fig. 1(D)]. A similar
time-dependent pattern of IFN-a induced PKR expression [Fig. 1(E)] or IFN-g induced IRF-1
expression [Fig. 1(F)] was observed in HepG2 cells.Our data differed from a recent report which did not detect any IFN
induced A3F expression in HepG2 cells [14]. To determine if IFN induced A3F
expression in liver cells is a common feature, we studied A3F expression in
another liver cell line, Hep3B. Similar to HepG2 cells, IFN-a induced A3F
expression in Hep3B cells in a dose- [Fig. 2(A)] and time-dependent
fashion [Fig. 2(D)]. A3F induction was more sustained by IFN-g than IFN-a in Hep3B cells
[Fig. 2(D)]. PKR [Fig. 2(B,E)] as well as IRF-1 [Fig. 2(C,F)]
mRNA were induced, indicating that the IFN doses used were effective for
inducing known ISG. We also observed A3F up-regulation by IFN-a in another
liver cell line, QSG7701 (Fig. 3).
IFN-a
only induced A3F expression moderately (3- to 4-fold) in HepG2 and Hep3B cell
lines
It has been reported that A3F could be detected in liver tissues
[15,16]. However, little is known about its regulation by IFNs in primary human
hepatocytes. Therefore, we determined the effect of IFN treatment on A3F
expression in cell-cultured primary hepatocytes from several healthy donors. We
observed that A3F was detectable in primary hepatocytes from multiple donors
and, more importantly, IFN-a induced A3F mRNA expression in these primary human hepatocytes [Fig.
4(A)]. However, A3F induction by IFN-a varied widely between 2-
to 9-fold among various donors [Fig. 4(A)]. A previous study detected
A3G induction by IFN-a in primary hepatocytes from a single donor [14]. Therefore, it is
not known whether IFN-a induced A3G expression also varies among various donors. A3G
induction by IFN-a in primary hepatocytes varied among various donors ranging from
approximately 5- to 19-fold [Fig. 4(A)]. IFN-g was less effective than
IFN-a in up-regulating A3F in the primary hepatocytes [Fig. 4(B)]
although IRF-1 was efficiently induced by IFN-g in these primary cells [Fig.
4(B)].
A3F is not induced by IFN-a in primary CD4+ T lymphocytes
Phorbol myristate acetate activates A3G in CD4+ H9 T cells through protein kinase C activation [11]. However, IFN-a did not induce A3G
expression in H9 cells [11]. We have also observed that IFN-a did not induce
the expression of A3F in H9 cells (data not shown). The ability of IFN-a to induce A3F
expression in primary CD4+ T lymphocytes was evaluated. In
contrast to liver cells, we observed that A3F was not induced by IFN-a or IFN-g in primary CD4+ T cells over a time course of 16 h [Fig. 5(A)] or in primary
CD4+ T cells from several other individuals stimulated with IFNs for 8 h
[Fig. 5(E,F)]. However, other ISG, including PKR and IRF-1, were readily
induced by IFN-a and IFN-g, respectively, in all primary CD4+ T cells
[Fig. 5(B,C,E,F)]. These data indicate that IFN
mediated signaling pathway is functional in primary CD4+ T
lymphocytes. Therefore, the transcriptional regulation of A3F by IFNs appears
to be cell type-dependent and might be distinct from those of PKR and IRF-1. In
addition to A3F, we have also observed that the related cytidine deaminase A3G
was also not induced by IFN-a or IFN-g in primary CD4+ T cells [Fig. 5(D)], an
extension from a previous report [11].
A3F is induced by IFN-a in macrophages
A3G was not induced by IFN-a in H9 cells (data not shown and previous
report [11]) or primary CD4+ T lymphocytes [Fig. 5(D)],
but it was efficiently induced by IFN in macrophages from the same donor [Fig.
6(A)]. Similarly, IFN-a also induced A3F expression in macrophages from the same donor [Fig.
6(B)] from which A3F was not induced by IFN-a in primary CD4+ T lymphocytes [Fig. 5(A)]. Thus, A3F and A3G were not
induced by IFN-a in primary CD4+ T lymphocytes but both were
efficiently induced by IFN-a in macrophages. IFN-a induced consistent A3F expression in
macrophages from various donors [Fig. 6(B–D)]. Interestingly, IFN-g induced A3F expression in macrophages was
more donor-dependent (Fig. 6). In one individual IFN-g induced A3F
expression in macrophages as efficiently as IFN-a [Fig. 6(C)],
whereas in others IFN-g induced no appreciable A3F expression [Fig. 6(B,D)].
Relative A3F and A3G
expression in untreated or IFN-a treated primary liver cells
versus CD+
T lymphocytes
Using qRT-PCR, we compared the relative levels of A3F and A3G mRNA
in various liver cell lines, primary liver cells, and T cells that were either
untreated (control) or treated with IFN-a (1000 IU/ml for 16 h). The
relative expression of the target genes was normalized to the endogenous
control gene b-actin. The expression levels of A3F and A3G, both lowest in
the QSG liver cell line, were set as unitless values of 1 and A3F or A3G
expression in all other cells were calculated as a relative value above
1. The A3G expression varied over four logs of magnitude between these
cell types, and the order of expression in control (untreated) cells from lowest
to highest were: liver cell lines (QSG, Huh7, Hep3B, HepG2), primary
hepatocytes, primary CD4+ T cells (Fig. 7). IFN-a up-regulated
A3G in the liver cell lines and primary liver cells, but not in primary CD4+ T cells. Although A3G was not up-regulated by IFN-a in T cells, A3G
expression was relatively high in these cells. Interestingly, A3G expression in
some of the primary liver cells after IFN induction approached the levels seen
in T cells. Although IFN could induce A3G significantly in liver cell lines,
the basal levels of A3G in those cells were low and remained relatively low
even after IFN treatment as compared to the other cells tested. In contrast to A3G expression, A3F expression between untreated
cells was less variable. Except for the QSG cell line, where A3F expression was
just above detection, A3F expression varied over just one log between the other
cells tested. In general, A3F expression in untreated CD4+ T cells was higher than in primary liver cells or liver cell lines,
with the exception of Hep3B liver cells which had relatively high A3F
expression at baseline. In all primary liver cells, IFN-a significantly
up-regulated A3F. IFN-a also significantly up-regulated A3F in the QSG and HepG2 cell lines
but less significantly in Huh7 or Hep3B cells at the same time point.
Discussion
Like A3G, A3F has potent antiviral activity against diverse
retroviruses [3–8] and HBV [10]. A3F and the related cytidine deaminase A3G can now
be classified among other well-known ISG, such as PKR, ISG15 and MX1 that
mediate a variety of antiviral effects. Macrophages and liver cells are primary
targets of lentiviruses and HBV, respectively. It is therefore plausible that
up-regulation of A3F by IFNs could contribute to innate antiviral defenses
against HBV in liver cells and lentiviruses in macrophages. Our results differed considerably from a recent report which did not
observe IFN-a induced A3F expression in HepG2 cells [14]. Plausible explanations
for this divergence might be the differences in cell culture conditions,
differences in the sources of IFN-a used in these studies, or differences in A3F
quantifications. Induction of A3F in macrophages by IFN-a observed in
this study was consistent with another recent report [12]. In addition to HepG2 cells, we have observed that A3F could also be
induced by IFN-a in other liver cell lines Hep3B (Fig. 2) and QSG7701 (Fig.
3). More importantly, IFN-a efficiently induced A3F expression in primary hepatocytes from
multiple healthy donors [Fig. 4(B)]. This is the first report showing
that A3F was induced by IFN-a in primary hepatocytes and macrophages but not CD4+ T cells. Although A3G could also be induced by IFN-a in primary
hepatocytes (Fig. 4 and previous report [14]), our data suggests that
the IFN-a inducible anti-HBV A3F could contribute to HBV suppression in
vivo. This argument would be consistent with the in vivo observation
of HBV G-to-A mutation patterns characteristics of APOBEC3 cytidine deaminases
[16–18].
A3G primarily mediates GG-to-GA mutations, whereas A3F mostly generates
GA-to-AA mutations [3–8]. GA-to-AA mutations are highly represented in viral sequences
recovered from HBV-infected individuals [17,18]. Therefore, A3F or other
related APOBEC3 proteins in addition to A3G might represent a major selection
force against HBV in vivo. The abilities of spontaneous clearance of viral infection after HBV
exposure differ significantly among various subjects [19–21]. Variations
in the magnitudes of A3F induction by IFN-a differed among various
healthy donors. We noted significant variations in IFN-induced A3F expression (Fig.
4) in primary hepatocytes from various subjects. Using primary hepatocytes
from multiple donors, we also observed that expression of IFN induction of A3G
in primary hepatocytes also varied significantly among individual donors (Fig.
4). This finding raises the interesting possibility that individual
differences in APOBEC3 protein expression and interferon inducibility in liver
cells might contribute to different courses of viral pathogenesis in infected
individuals. A3F was regulated differently than other IFN-a-responsive
genes, such as PKR. PKR was induced by IFN-a in all cells tested: T
cells, macrophages, and liver cells. A3F was induced in macrophages and liver
cells but not H9 T cells or primary CD4+ T
lymphocytes. Similarly, A3G was also not induced by IFN-a in CD4+ T lymphocytes (Fig. 5) or H9 cells [11]. H9 cells are
relatively resistant to certain IFN induced responses including cell growth
properties and down-regulation of c-myc [22–25]. However, we observed
normal IFN-a-induced PKR expression in H9 cells as well as primary CD4+ T lymphocytes, suggesting at least some IFN-a-mediated
signaling pathways are functional in these T cells. It is conceivable that IFN-a-induced A3F or
A3G expression requires an additional factor/s which is/are absent in T cells.
Therefore, A3F and A3G appeared to be uniquely regulated by IFNs in a cell type-dependent
manner. Consistent with previous reports [15,16], our data in multiple liver
cell lines, primary hepatocytes, and macrophages also indicate that A3F and A3G
expressions are coordinately regulated.
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