Reassessing the role of APOBEC3G in human immunodeficiency virus type 1 infection of quiescent CD4+ T-cells.

Abstract

HIV-1 is restricted for infection of primary quiescent T-cells. After viral entry, reverse transcription is initiated but is not completed. Various hypotheses have been proposed for this cellular restriction including insufficient nucleotide pools and cellular factors, but none have been confirmed as the primary mechanism for restriction. A recent study by Chiu et al. implicates APOBEC3G, an anti-retroviral cytidine deaminase, as the cellular restriction factor. Here, we attempted to confirm these findings using the same strategy as reported by Chiu et al. of siRNA targeting knock-down of APOBEC3G expression. In contrast to the published study, our results do not support a role for APOBEC3G in restriction of HIV-1 in quiescent CD4+ T-cells. In our study, we tested the same siRNA as reported by Chiu et al. as well as two additional siRNAs targeting APOBEC3G, one of which showed 2-fold greater knock-down of APOBEC3G mRNA. However, none of the three siRNAs tested had a discernable effect on enhancing infection by HIV-1 in quiescent CD4+ T-cells. Therefore, we conclude that the primary mechanism of HIV-1 restriction in quiescent CD4+ T-cells remains to be elucidated.

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Reassessing the Role of APOBEC3G in Human
Immunodeficiency Virus Type 1 Infection of Quiescent
CD4+ T-Cells
Masakazu Kamata1, Yoshiko Nagaoka2, Irvin S. Y. Chen1*
1Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United
States of America, 2Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California,
United States of America
Abstract
HIV-1 is restricted for infection of primary quiescent T-cells. After viral entry, reverse transcription is initiated but is not
completed. Various hypotheses have been proposed for this cellular restriction including insufficient nucleotide pools and
cellular factors, but none have been confirmed as the primary mechanism for restriction. A recent study by Chiu et al.
implicates APOBEC3G, an anti-retroviral cytidine deaminase, as the cellular restriction factor. Here, we attempted to confirm
these findings using the same strategy as reported by Chiu et al. of siRNA targeting knock-down of APOBEC3G expression.
In contrast to the published study, our results do not support a role for APOBEC3G in restriction of HIV-1 in quiescent CD4+
T-cells. In our study, we tested the same siRNA as reported by Chiu et al. as well as two additional siRNAs targeting
APOBEC3G, one of which showed 2-fold greater knock-down of APOBEC3G mRNA. However, none of the three siRNAs
tested had a discernable effect on enhancing infection by HIV-1 in quiescent CD4+ T-cells. Therefore, we conclude that the
primary mechanism of HIV-1 restriction in quiescent CD4+ T-cells remains to be elucidated.
Citation: Kamata M, Nagaoka Y, Chen ISY (2009) Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-
Cells. PLoS Pathog 5(3): e1000342. doi:10.1371/journal.ppat.1000342
Editor: Thomas J. Hope, Northwestern University, United States of America
Received December 4, 2008; Accepted February 18, 2009; Published March 20, 2009
Copyright: ? 2009 Kamata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the NIH grant A1028697 (UCLA CFAR, http://grants.nih.gov/grants/oer.htm). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: syuchen@mednet.ucla.edu
Introduction
Nonproliferating quiescent CD4+ T-cells are resistant to the
infection with human immunodeficiency virus type 1 (HIV-1)
unless they are activated by mitogenic stimulation [1–5] or by
cytokine stimulation [6]. Other studies demonstrated that
quiescent T-cells at the stage of G0or G1aof the cell cycle are
nonpermissive to HIV-1 infection, but cells in the G1bphase of the
cell cycle which show high levels of RNA synthesis, but no DNA
synthesis [7] are permissive, and the induction of cell cycle
progression per se is not needed to render cell permissive [8,9].
Earlier studies showed that HIV-1 infection in quiescent CD4+ T-
cells results in 4-fold lower levels of viral entry, and incomplete
reverse transcription and minimum levels of integration are
observed [5,10,11]. Although the precise mechanisms involved in
this blockage remain unidentified, a number of cellular factors
have been reported as restriction factors to HIV-1 infection in
quiescent CD4+ T-cells (reviewed in more detail [12,13]),
including Murr1 [14] and most recently APOBEC3G [15].
APOBEC3G is a cytidine deaminase and has well characterized
potent anti-retroviral activity, including against HIV-1 [16–20]. In
the case of HIV-1, it acts through incorporation into virions where
it edits newly synthesized viral DNA in the next infection cycle by
deaminating dC to dU, resulting in lethal G-to-A hypermutations
in the single stranded viral DNA intermediate (reviewed in
[16,21]). The HIV-1 encoded viral infectivity factor (Vif)
counteracts the effects of virion incorporated APOBEC3G by
mediating its degradation [22–24]. A deaminase-independent anti-
viral activity has also been identified [15,25,26] but the detailed
mechanism of action is poorly understood.
Using siRNA mediated knock-down, Chiu et al. concluded that
APOBEC3G plays role in HIV-1 restriction in quiescent resting
CD4+ T-cells. In those cells, APOBEC3G exists as a low molecular-
mass (LMM) ribonucleoprotein complex that inhibits HIV-1
infection prior to reverse transcription probably through its RNA
binding activity. In this report, we attempted to reproduce the
findings using the identical strategy of siRNA mediated knock-down
of APOBEC3G expression as reported by Chiu et al. By
nucleofection of the same siRNA as reported by Chiu et al
(siA3G240WT) as well as two additional siRNAs targeting APO-
BEC3G, we confirm reduction in APOBEC3G mRNA and protein
in quiescent CD4+ T-cells. However, none of the siRNAs resulted in
a significant enhancement of HIV-1 infection in those cells.
Therefore, we conclude that the role of APOBEC3G in the
mechanismofHIV-1restrictioninquiescentCD4+T-cellsisunclear.
Results
Synthesized siRNAs can transduce into quiescent CD4+ T-
cells with high efficiency by nucleofection
Chiu et al. reported a 37-fold enhancement of HIV-1 infection
by an HSA bearing reporter virus in unstimulated quiescent CD4+
T-cells following nucleofection of siRNA directed to APOBEC3G
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- a level that is nearly comparable to that observed in PHA/IL-2
stimulated cells [15]. We mimicked the experimental conditions of
Chiu et al. using the same siRNA (siA3G240 WT) and VSV-G
pseudotyped HSA reporter virus (NL4-3 HSA R-E- [27]).
We first tested the efficiency of nucleofection using fluorescein
isothiocyanate (FITC) conjugated siRNA. Quiescent CD4+ T-cells
(56106) were nucleofected with 2 mg of siRNA following the
manufacturer’s protocol and the efficiency of siRNA transduction
monitored by flow cytometry. We consistently observed 5–20%
higher mortality between nucleofected cells compared to untreated
control cells 48 hr after nucleofection. This level was similar or
slightly lower than that reported by Chiu et al. [15]. As shown in
Figure 1A, FITC-conjugated siRNA was nucleofected into nearly
100% of the cells from two independent blood donors. We further
tested whether the RNAi mechanism is functional in quiescent
CD4+ T-cells using siRNA specific to the CD4 molecule. After
nucleofection, the cell surface expression of CD4 molecules on
quiescent CD4+ T-cells decreased 3-fold as measured by mean
fluorescent intensity (MFI) (Figure 1B, siCD4) compared to control
siRNA nucleofected cells (Figure 1B, siControl). Identical results
were also observed using cells from another independent donor
(data not shown).
The expression of APOBEC3G effectively knock-down by
nucleofection of siRNA directed to the sequence of
APOBEC3G in quiescent CD4+ T-cells
We next examined that the levels of knocking-down of
APOBEC3G by siRNA reported by Chiu et al., that targeted
APOBEC3G (siA3G240WT). We also tested two additional siRNAs,
one previously published siRNA [28] and another identified by
ourselves directed to distinct sequences of APOBEC3G (siA3G726
and siA3G883, respectively). These siRNAs gave greater downreg-
ulation of APOBEC3G mRNA than that reported by Chiu et al.;
6-fold reduction was observed in the case of siA3G883(Figure 2A).
We further examined the protein levels of APOBEC3G in siRNA
nucleofected cells by Western blotting (Figure 2B). All three
siRNAs targeting APOBEC3G showed comparable levels of
reduction of APOBEC3G protein at 48 hr after nucleofection;
the band intensities of the cells nucleofected with siRNA targeting
APOBEC3G compared to that of control siRNA nucleofected cells
were 35%, 33%, and 31% by siA3G240 WT, siA3G726, and
siA3G883, respectively, a decrease in levels similar to that reported
by Chiu et al [15].
APOBEC3G knock-down in quiescent CD4+ T-cells does
not affect HSA reporter virus infection
Using above described protocol, we nucleofected siRNAs
targeted to APOBEC3G [siA3G240 WT, siA3G726 (see below),
and siA3G883] or the mutant (siA3G240 MT) and non-specific
siRNA (siControl) into quiescent CD4+ T-cells (Figure 3). The
cells were then infected by an NL4-3 HSA reporter virus at 2 day
post-nucleofection. As expected, CD4+ T-cells stimulated with
PHA/IL-2 were highly susceptible to infection by the HSA
reporter virus and the virus infection was strongly diminished in
the presence of AZT. In striking contrast to Chiu et al., we did not
observe any enhancement of HSA expression in quiescent CD4+
T-cells in the presence of siRNA to APOBEC3G reported by Chiu
et al. (Figure 3, siA3G240 WT). We further tested two additional
siRNAs that were more efficient at decreasing APOBEC3G
expression as shown in Figure 2. However, there was no
discernable effect by these new siRNAs targeting APOBEC3G
on enhancing reporter virus infection (Figures 3 and 4, siA3G883
are shown; Figure 5, siA3G726 and siA3G883 are shown). We
repeated the experiment using quiescent CD4+ T-cells from 6
different donors and in all cases, we did not observe enhancement
of the levels of the HSA expression after nucleofection of
APOBEC3G specific siRNA (data not shown). We further tested
infection with three different amounts of NL4-3 virus carrying the
EGFP reporter rather than the HSA reporter. The infection
efficiency reached maximal level with 125 ng of p24 per
16105cells (Figure 4). However, we did not observe any
significant differences in EGFP expression between the cells
nucleofected with siRNAs targeting APOBEC3G (Figure 4,
siA3G240 WTand siA3G883) or control siRNA (Figure 4, siControl)
even with the higher amounts of virus (250 ng and 500 ng of p24).
Chiu et al. also reported that the knock-down of APOBEC3G by
siRNA in quiescent CD4+ T-cells continued for at least 88 hr after
nucleofection [15]. Therefore, we examined infection with the
same EGFP reporter virus at 72 and 96 hr after nucleofection
(Figures S1A and S1B, respectively). However, we did not observe
any effect of APOBEC3G siRNA transduction on EGFP
expression at those time points. Furthermore, we tested the same
culture medium used by Chiu et al.[15] that contained 10% fetal
calf serum (FCS) instead of 10% human serum, but neither culture
condition showed any enhancement of reporter virus infection in
quiescent CD4+ T-cells (data not shown). Thus, our results
obtained under similar conditions were discrepant with those of
Chiu et al.
Knock-down of APOBEC3G does not interfere with HIV-1
entry into quiescent CD4+ T-cells
To ensure that the reporter viruses successfully entered
quiescent CD4+ T-cells and to exclude the possibility that the
nucleofection prevented the infection of the reporter virus,
quiescent CD4+ T-cells were infected with virus and immediately
stimulated with PHA/IL2. The marker gene expression was
monitored by flow cytometry at 48 hr after stimulation. The
stimulation status was monitored by the expression levels of two
activation markers, CD25 and CD69. Nucleofection did not affect
the expression levels of the activation markers (Figure S2). Similar
to the results seen in Figures 3 and 4, we did not detect significant
gene expression from the reporter virus in APOBEC3G specific
siRNAs nucleofected cells (Figure 5, siA3G240 WT/-, siA3G726/-
and siA3G883/-). Upon stimulation by PHA/IL-2, EGFP
Author Summary
In 1990, we demonstrated that unstimulated quiescent T-
lymphocytes are resistant to HIV-1 infection. Viruses can
get into the cell, and reverse transcription is started, but it
is not completed. Various hypotheses have been proposed
for this blockage including insufficient free nucleotides
and inhibiting cellular factors, but none have been
confirmed as the primary mechanism for this blockage. A
recent study by Chiu et al. provided one possible
mechanism for the blockage in quiescent T-lymphocytes:
APOBEC3G, an anti-retroviral cytidine deaminase that was
implicated as the responsible factor for this blockage. We
have attempted to confirm these findings using published
methods. Chiu et al. reported a 37-fold enhancement of
HIV-1 infection in quiescent T-lymphocytes by decreasing
the levels of APOBEC3G. In contrast to the published study,
our results do not support a role for APOBEC3G in the
inhibition of HIV-1 infection in quiescent T-lymphocytes.
We believe the block of HIV-1 infection of quiescent T-cells
remains unknown.
Reassessing of the Role of APOBEC3G
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expression was observed in all nucleofected cells (Figure 5,
siA3G240 WT/Stim, siA3G726/Stim and siA3G883/Stim). There
was a 2–3 fold difference in EGFP expression between
unstimulated control cells (Figure 5, Unstimulated/Stim) and
siRNA nucleofected cells (Figure 5, siControl/Stim, siA3G240 WT/
Stim, siA3G726/Stim, and siA3G883/Stim). However, there were
no obvious differences between the cells nucleofected with siRNAs
targeting APOBEC3G (Figure 5, siA3G240
siA3G726/Stim, 3.67%; and siA3G883/Stim, 5.47%) and the
mutant (Figure 5, siA3G240 MT/Stim, 5.70%) or control siRNA
(siControl/Stim, 4.91%). This result indicated that similar
WT/Stim, 3.16%;
amounts of reporter viruses entered into the quiescent CD4+ T-
cells under the five experimental conditions, and none of siRNAs
targeting APOBEC3G interfered with the entry of the reporter
viruses into quiescent CD4+ T-cells. As described above, the
difference in EGFP expression between siRNA nucleofected cells
and unstimulated control cells after the stimulation is likely to be
caused by the cytotoxic effect of nucleofection. The mortality rate
increased when using the combination of nucleofection and post-
stimulation (45–50% mortality of siRNA nucleofected cells,
compared to 25% in unstimulated control cells). Upon infection
with heat inactivated reporter virus (Figure 5, HI), no EGFP
Figure 1. siRNAs can transduce into quiescent CD4+ T-cells with high efficiency by nucleofection. (A) Quiescent CD4+ T-cells were
purified from freshly isolated PBMCs of two independent donors with CD4 magnetic beads and nucleofected with siRNA targeting APOBEC3G
(siA3G883), conjugated with (FITC) or without FITC (None). Cells were analyzed by flow cytometry 3 hr after nucleofection. (B) To monitor the integrity
of the RNAi machinery, quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNA targeting CD4 (siCD4), control siRNA (siControl), or
no siRNA (None). The levels of cell surface CD4 expression were monitored by flow cytometry using PE-conjugated anti-CD4 antibody or isotype-
matched control 48 hr after nucleofection and represented by mean fluorescent intensity (MFI) in each panel. The solid line represents PE-CD4
antibody stained cells, whereas the shaded area represents isotype control staining.
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expression was observed upon stimulation, indicating that the
EGFP signals derived from the infected vectors and were not due
to pseudo-infection or auto-fluorescence from the cells. Thus, the
knock-down of APOBEC3G by siRNA does not affect the
efficiency of HIV-1 entry in quiescent CD4+ T-cells.
Knock-down of APOBEC3G does not affect the levels of
reverse transcription in HIV-1–infected quiescent CD4+ T-
cells
HIV-1 can enter into quiescent CD4+ T-cells, but it remains in
an inactive state before completion of reverse transcription. Upon
subsequent mitogentic stimulation, HIV-1 completes reverse
transcription and p24 Gag expression is observable after
approximately a 48 hr delay [5,10]. Chiu et al. reported that this
restriction on reverse transcription in quiescent CD4+ T-cells was
alleviated by knock-down of APOBEC3G. We lastly examined the
status of HIV-1 reverse transcription following downregulation of
APOBEC3G. As reported by Vatakis et al.[10], the levels of early
reverse transcripts decreased in stimulated cells over time, whereas
they hardly changed in unstimulated cells until 48 hr after the
reporter virus infection. Similar levels of early reverse transcripts
were detected 12 hr after infection and there were no significant
differences between siRNAs targeting APOBEC3G (Figure 6,
siA3G240WT, siA3G726, and siA3G883) and control siRNA
(Figure 6, siControl) nucleofected cells. Heat inactivation of the
reporter viruses for 15 min at 60uC decreased the amount of both
early and late reverse transcripts to background levels (Figure 6,
HI). The levels of reverse transcription in cells nucleofected with
either siRNA targeting APOBEC3G, the mutant, or control
siRNA were 2-fold lower than that in the unstimulated control
cells (Figure 6, unstimulated). This difference may be caused by
the cytotoxic effect of nucleofection as described above; the
mortality rate in this experiment was 26–32% in siRNA
nucleofected cells 48 hr after nucleofection. In contrast, it was
19% in untreated control cells at the same time point. Comparable
results were also observed when we measured late reverse
transcripts. The levels of late reverse transcripts increased over
time, but the levels were similar or slightly lower than those in
unstimulated control cells and, in contrast to the results of Chiu et
al. [15], never reached the levels observed in stimulated cells.
Equivalent results were obtained when replication competent
HIV-1 NL4-3 was used for the infection instead of the reporter
virus (data not shown). The above results clearly indicated that the
knock-down of APOBEC3G by siRNA affected neither the
efficiency of initiation of reverse transcription nor the subsequent
elongation of cDNA in quiescent CD4+ T-cells.
Discussion
The mechanisms involved in the restriction of HIV-1 infection
in quiescent CD4+ T-cells remain unidentified. Some potential
mechanisms for this blockage may be the presence of cellular
inhibitors in quiescent cells [14,15], the lack of cellular factors
required for completion of HIV-1 infection [9] or both. Chiu et al.
provided one possible mechanism for the restriction in quiescent T
cells [15]. In contrast to their study, our results do not support a
role for APOBEC3G in restricting HIV-1 infection of quiescent
unstimulated human CD4+ T-cells. We also tested two additional
siRNAs that were more efficient at decreasing APOBEC3G, both
at the level of mRNA and protein. None of the siRNAs tested
resulted in efficient HIV-1 reporter virus infection of quiescent
CD4+ T-cells.
It is unclear to us why our results are discrepant with those of
Chiu et al. Our experimental conditions for maintaining quiescent
T-cells are that which we used previously including in our first
description of the block to HIV-1 infection [5]. From our past
experience in working with quiescent T-cells, several factors may
influence their behavior, including the source of cells and culture
conditions. Our cells are obtained from fresh leukopaks from the
UCLA blood bank and processed for peripheral blood mononu-
clear cell (PBMC) by a Ficoll-Hypaque density gradient within
24 hours. The cells are then used within 0.5 hours. In the past we
used selected lots of human AB serum monitored for absence of
growth stimulating activity rather than FCS. However, if carefully
screened, FCS is also satisfactory and we observed no difference
between use of 10% FCS and 10% human AB serum in our results
(data not shown). Chiu et al. used 10% FCS for the culture of
quiescent CD4+ T-cells. We also considered whether the
nucleofection method might affect the results, in particular, the
cytotoxicity associated with the procedure. Even under optimized
conditions, the nucleofected cells consistently contained around
20% dead cells monitored by forward and side scatter compared
to untreated control cells 48 hr after nucleofection. Those dead
Figure 2. siRNAs directed to the sequence of APOBEC3G
effectively knock-down the expression of APOBEC3G in
quiescent CD4+ T-cells. (A) Quiescent CD4+ T-cells derived from
PBMCs were nucleofected with siRNAs and cultured for two days. Total
RNA was isolated, and the levels of APOBEC3G mRNA were monitored
by quantitative real time RT-PCR using b-actin as an internal control
[31]. P values (asterisks) versus control siRNA were 0.00022 (siA3G240
WT), 0.00013 (siA3G726), 0.00005 (siA3G883), respectively. (B) Quiescent
CD4+ T-cells derived from PBMCs were nucleofected with siRNAs and
cultured for two days. Cells were lysed in 0.5% SDS, and the levels of
APOBEC3G protein were monitored by Western blotting. b-actin was
used as a loading control.
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Figure 3. siRNA mediated knock-down of APOBEC3G does not affect the infectivity of HIV-1 reporter virus. Quiescent CD4+ T-cells
derived from PBMCs were nucleofected with siRNAs and cultured for two days. Cells were then infected with VSV-G pseudotyped NL4-3 HSA reporter
virus (125 ng of p24 per 16105cells) for 3 hr and subsequently cultured in the absence or presence of 25 mM AZT. Expression of HSA was monitored
48 hr after the reporter virus infection by flow cytometry using PE-conjugated anti-HSA antibody. Unstimulated non-nucleofected cells
(Unstimulated) and unstimulated cells nucleofected with control siRNA (siControl) served as negative controls. Cells stimulated with PHA (5 mg/ml)
and IL-2 (20 U/ml) served as positive controls (Stimulated). Comparable results were obtained using cells from six different donors.
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