Biochemical and Biophysical Research Communications 518 (2019) 26e31

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Different antiviral activities of natural APOBEC3C, APOBEC3G, and

APOBEC3H variants against hepatitis B virus
Arun Kanagaraj a, Naoya Sakamoto a, Lusheng Que b, Yingfang Li a, b, Md Mohiuddin a,
Miki Koura a, Kousho Wakae b, Makoto Kurachi a, Masamichi Muramatsu a, b, **,
Kouichi Kitamura a, *
a
Department of Molecular Genetics, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
b
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Some APOBEC3 family members have antiviral activity against retroviruses and DNA viruses. Hepatitis B
Received 11 July 2019 virus (HBV) is a DNA virus that is the major causative factor of severe liver diseases such as cirrhosis and
Accepted 1 August 2019 hepatocellular carcinoma. To determine whether APOBEC3 variants in humans have different anti-HBV
Available online 7 August 2019
activities, we evaluated natural variants of APOBEC3C, APOBEC3G, and APOBEC3H using an HBV-
replicating cell culture model. Our data demonstrate that the APOBEC3C variant S188I had increased
Keywords:
restriction activity and hypermutation frequency against HBV DNA. In contrast, the APOBEC3G variant
Hepatitis B virus
H186R did not alter the anti-HBV and hypermutation activities. Among APOBEC3H polymorphisms (hap
APOBEC
Cytidine deaminase
I-VII) and splicing variants (SV-200, SV-183, SV-182, and SV-154), hap II SV-183 showed the strongest
Antiviral protein restriction activity. These data suggest that the genetic variations in APOBEC3 genes may affect the ef-
ficiency of HBV elimination in humans.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction partially explained by the lack of proofreading activity during

Hepatitis B virus (HBV) is a DNA virus that causes severe liver
diseases such as cirrhosis and hepatocellular carcinoma [1]. Similar
to retroviral replication, the HBV genome is transcribed into a
pregenomic RNA (pgRNA) and other viral protein mRNAs in the
nucleus [2]. Two viral proteins, core and P protein, encapsidate
pgRNA to form a nucleocapsid, in which the P protein reverse-
transcribes pgRNA to produce relaxed circular DNA (rcDNA). The
nucleocapsid associates with surface proteins for secretion as an
infectious virion. It was suggested that the retrovirus-like replica-
tion cycle of HBV may produce a viral population with high genetic
heterogeneity, a quasispecies [3]. The high mutation rate of HBV is
Abbreviations: HBV, hepatitis B virus; pgRNA, pregenomic RNA; rcDNA, relaxed
circular DNA; 3D-PCR, differential DNA denaturation PCR.
* Corresponding author. Department of Molecular Genetics Kanazawa University
Graduate School of Medical Sciences 13-1 Takara-machi, Kanazawa, 920e8640,
Japan.
** Corresponding author. Department of Virology II National Institute of Infectious
Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
E-mail addresses: muramatsu@nih.go.jp (M. Muramatsu), kkita@med.
kanazawa-u.ac.jp (K. Kitamura).
reverse transcription. In addition, APOBEC-mediated hyper-
mutation in the HBV genome has been also described [4e10].
The APOBEC3 (A3) family of DNA/RNA cytidine deaminases,
clustered in human chromosome 22, comprises seven members
designated A3A-A3H. These enzymes have antiviral activity against
retroviruses and DNA viruses [11] including HBV [4e10,12,13] and
human papillomavirus type 16 [14e17]. A3 enzymatic activity re-
sults in obvious accumulation of C-to-T (opposite strand G-to-A)
mutations in the viral genome, which is called hypermutation. A3G
is well defined as a major restriction factor against HIV-1 via its G-
to-A hypermutation activity. A previous study found G-to-A mu-
tations in HBV DNA from patient's sera [18], indicating that the HBV
genome is also a target of A3 enzymes. Enzymes A3C, A3G, and A3H
have the ability to induce HBV DNA hypermutation and are highly
expressed in the human liver [6]. We also demonstrated that A3G
and AID induce hypermutation in the HBV genome [4,5,7]. Varta-
nian et al. showed that A3C, A3DE, A3G, and A3H are significantly
upregulated in HBV-related cirrhosis samples compared to healthy
liver, and HBV hypermutation was found in cirrhosis samples [10].
A3 variants and their antiviral activity have been extensively
studied in the HIV research field [19]. An ancient variant in primate
A3C, A3C I188 (Fig. 1A), is found in 9.6% of people of African descent,

https://doi.org/10.1016/j.bbrc.2019.08.003
0006-291X/© 2019 Elsevier Inc. All rights reserved.
 A. Kanagaraj et al. / Biochemical and Biophysical Research Communications 518 (2019) 26e31
Fig. 1. Schematic summary of A3C, A3G, and A3H variants. Human A3 members with polymorphisms and splicing variants (SV) tested in this study are shown. (A) A3C and A3G
variants. Positions of S188I and H186R are indicated. (B) A3H haplotypes at the amino acid positions 15, 18, 105, 121, and 178 based on SV-183. (C) A3H alternative splicing variants.
Gray boxes indicate the coding region in exons. Different usage of exons 4 and 5 results in varying C-terminal extensions. Diagram not to scale.
and restricts HIV-1 replication 5- to 10-fold more efficiently than
the common A3C S188 variant [19,20]. It has also been demon-
strated that A3C I188 confers dimerization capacity and a higher
mutagenic activity in comparison to the common A3C [21]. The A3G
H186R variant (Fig. 1A) was also found in African American in-
dividuals (37%) and is associated with AIDS progression, although
the biochemical difference between these variants was not deter-
mined [22]. A3H is the most polymorphic gene among the A3
family (Fig. 1B and C). Seven haplotypes (hap I-VII) and four mRNA
splicing variants (SV-154, SV-182, SV-183, and SV-200) have been
identified [23e25]. Previous studies revealed that these haplotypes
show different anti-HIV activities mainly owing to differences in
protein stability [23,25]. An epidemiological analysis of HBV
infection in the Moroccan population showed that the homozygous
A3G R186 allele increases the risk of developing chronic infection
compared with the H186 allele, but the frequency was not signifi-
cantly different [26].
In this study, to elucidate the differences in anti-HBV activities of
A3C, A3G, and A3H variants, we compared these variants using an
HBV-replicating cell culture system.
2. Materials and methods
2.1. Plasmids
The HBV replicon plasmid pPB was described previously [7,27].
All cDNA fragments of A3C, A3G, and A3H variants were subcloned
into the mammalian expression vector pcDNA3tag1A (Agilent
Technologies, PaloAlto, CA, USA). Plasmids of the most common
variants A3C S188, A3G H186, and A3H hap II SV-183 were
described previously [5,14]. DNA fragments containing A3C I188
and A3G R186 were generated from variants S188 and H186,
respectively, using PCR-based mutagenesis. A3H hap I cDNA was
amplified using RT-PCR from BL2 cells and subcloned. The hap III,
IV, and V cDNA fragments were generated using PCR to introduce
mutations at amino acid positions 15, 18, and 178 of hap II,
respectively. Hap VI was generated by replacement of the PmaCI/
BamHI fragment (containing G105 and K121) of hap I with the
counterpart of the hap IV vector. Hap VII was generated by
replacement of the AfaI fragment (containing K121 and E178) of hap
I with the counterpart of the hap II vector. A3H splicing variants
were amplified using RT-PCR with specific reverse primers. The
cloned inserts and the introduced mutations were confirmed by
sequencing. Primers are listed in Supplementary Table 1.
27
2.2. Cell culture and transfection
Huh7 cells were maintained in Dulbecco's modified Eagle's
medium (DMEM, Wako Chemicals, Osaka, Japan) supplemented
with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/
mL streptomycin at 37
C in a 5% CO2 atmosphere. Plasmids were
transfected using FuGENE6 (Promega, Madison, WI, USA), accord-
ing to the manufacturer's protocol.
2.3. Preparation of HBV DNA
HBV DNA was purified from culture supernatants. Secreted HBV
particles were precipitated with 50% PEG8000, resuspend in PBS,
and treated with DNase I (100 mg/mL) in 10 mM MgCl2. The pellet
was lysed in 50 mM Tris (pH 7.5), 20 mM EDTA, proteinase K
(500 mg/mL) and sodium dodecyl sulfate (SDS, 0.7%) at 50
C for 5 h.
DNA was purified with phenol:chloroform extraction and ethanol
precipitation. Purified DNAs were treated with the DpnI restriction
enzyme to avoid pPB plasmid contamination.
2.4. qPCR
qPCR analysis was performed as described previously [5] with
the primers 50 -GAATTGATGACTCTAGCTACCTG-30 and 50 -GAAAC-
CACAATAGTTGCCTGATC-30 using SYBR Premix ExTaq (Takara,
Kusatsu, Japan) and the MX3000 thermocycler (Stratagene, La Jolla,
CA, USA). HBV DNA copy numbers were determined using a HBV
plasmid standard curve.
2.5. Western blotting
Western blotting was performed as described previously [5,7].
Signals from western blots were detected using the LAS1000
Imager System (Fuji Film, Tokyo, Japan). The following antibodies
were used: rabbit anti-GAPDH (G9545, Sigma-Aldrich, St. Louis,
MO, USA), anti-FEN1 (GTX70185, GeneTex, Irvine, CA, USA), mouse
anti-FLAG (F3165, Sigma-Aldrich), anti-rabbit IgG-HRP (GE
Healthcare, Marlborough, MA, USA), and anti-mouse IgG-HRP (GE
Healthcare).
2.6. Mutation analysis
Hypermutation was analyzed with differential DNA denatur-
ation PCR (3D-PCR). The 3D-PCR procedure was performed as
described previously, with minor modifications [9,28]. The specific
28 A. Kanagaraj et al. / Biochemical and Biophysical Research Communications 518 (2019) 26e31

primers used for 3D-PCR targeting of the HBx region were as fol-
lows. First PCR: 50 - CGCGGAAATATACATYGTTTCYAT-30 and 50 -
GGTAAAGTTGYATGGTGYTGGTG-3'; second PCR: 50 -CATRGCTGC-
TARGCTRTACTGCCAA-30 and 50 - AAGTGCACACRGAYYGGCAGAT-3';
Y ¼ C or T, R ¼ G or A. The first PCR was performed as follows: 94
C
for 5 min, 38 cycles of 94
C for 20 s, 50
C for 30 s, and 72
C for
28 s, and a final elongation step at 72
C for 3 min. The nested PCR
was performed as follows: initial denaturation for 5 min at
94e81
C, followed by 35 cycles of 1 min at 94e81
C, 30 s at 47
C
and 25 s at 72
C, with a final elongation for 5 min at 72
C. In
addition to 3D-PCR as the hypermutation-enrich method, we also
utilized the sequence analysis from standard PCR fragments. For
this PCR, the primer set 50 - CTAAGTAAACAGTACATGAACCTT-30 and
50 -GGTAAAGTTGYATGGTGYTGGTG-30 was used and performed as
follows: 94
C for 5 min, 38 cycles of 94
C for 20 s, 50
C for 30 s,
and 72
C for 30 s, and a final elongation step at 72
C for 3 min. To
determine the mutation frequency, PCR fragments were cloned into
T vectors (Promega). Successfully recombined clones were
randomly selected and sequenced using ABI PRISM 3130 (Applied
Biosystems, Foster City, CA, USA).
2.7. Statistical analysis
Statistical analysis was performed using GraphPad Prism
(GraphPad Software, La Jolla, CA, USA). Significance between three
or more groups was determined using one-way ANOVA with
Tukey-Kramer post-hoc test. Significance between A3C S188 and
I188 in different dosage groups was determined using two-way
ANOVA (Fig. 2B). P-values of <0.05 were considered statistically
significant. Error bars indicate the standard error of the mean from
triplicate samples.
3. Results
3.1. Repression of HBV replication by A3C, 3G, and 3H variants
To investigate the differences in anti-HBV activities among A3C,
A3G, and A3H variants (Fig. 1AeC), a plasmid-based HBV replica-
tion system was utilized. Transfection of the HBV replicon plasmid
pPB [7,27] initiates transcription of viral RNA, which results in viral
replication including nucleocapsid formation, reverse transcrip-
tion, and release of virions to the culture supernatant. Three days
after transfection with pPB and A3 vectors, HBV DNA from culture
supernatants was quantified using qPCR assays (Fig. 2AeC). As
expected, all A3 vectors restricted HBV replication to various levels.
The replication-defective HBV plasmid, pPBDE, did not show any
production and secretion of HBV DNA in the culture supernatant,
ensuring that the HBV DNA detected in the supernatants was
derived from HBV replication and not from the transfected HBV
plasmid. HBV DNA levels from the A3C I188 transfectant were lower
than those from A3C S188, whereas no significant difference was
observed between A3G H186 and R186 (Fig. 2A). We further
examined the anti-HBV activity of A3C S188 and I188 with different
inputs of A3C plasmids, and confirmed the higher antiviral activity
of the I188 variant (Fig. 2B). Among A3H haplotypes and splicing
variants, hap II SV-183 caused the strongest reduction in HBV DNA
levels, whereas only a small reduction was observed in hap III, hap
IV, and SV-154 (with hap II) transfectants (Fig. 2C).
3.2. Expression levels of A3 proteins
Previous studies demonstrated that the protein stability of A3H
variants reflected anti-HIV activity [23,25]. Hence, we determined
the protein levels of A3 variants in Huh7 cells. Western blot ana-
lyses show that similar levels of protein expression were found in
samples transfected with A3C S188 and I188 plasmids (Fig. 2D).
Because A3C S188 and I188 showed different antiviral activities, we
determined the expression levels of both proteins with different
amounts of transfected A3C vectors (Fig. 2D). The results show that
the I188 variant has a higher anti-HBV activity in this HBV repli-
cation model. We also observed similar levels of protein expression
in A3G H186 and R186-transfected samples (Fig. 2E). A3H variants
revealed higher expression of A3H hap II, hap V, and hap VII than of
the other haplotypes (Fig. 2F, left). The highest protein levels of hap

Fig. 2. Overexpression of A3 variants in HBV-replicating Huh7 cells. (AeD) Quantification of HBV DNA in culture supernatants of A3 transfectants. HBV replicon plasmid (pPB)
and A3 expression vectors were transfected into Huh7 cells. Three days after transfection, the culture supernatant was collected and HBV DNA was purified and detected using qPCR
analysis. pPB and GFP control vector transfectants were used and defined as one. Replication-incompetent HBV plasmid (pPBDE) was used as a negative control. (A) Comparison of
A3C and A3G variants. (B) Further analysis of A3C variants. Huh7 cells were transfected with pPB and different amounts of A3C S188 and I188 vector DNA (0.5, 1.0, and 1.5 mg per
well; GFP control vector was used for normalization of total DNA amount). (C) Left: A3H haplotypes on SV-183. Right: A3H alternative splicing variants based on hap II. Asterisks
indicate statistically significant differences compared with control GFP group; ***P < 0.001. Data are representative of three independent experiments, and error bars represent
standard errors of the mean. (DeF) Western blotting analyses of A3 proteins overexpressed in Huh7 cells. FLAG-tagged A3 expression vectors were transfected into Huh7 cells.
Variants of A3C (D), A3G (E), and A3H (F) were detected with anti-FLAG antibody. GAPDH or FEN1 protein were used as a loading control.
 A. Kanagaraj et al. / Biochemical and Biophysical Research Communications 518 (2019) 26e31 29

II and hap VII proteins were correlated with their anti-HBV activ- 3.4. A3C I188 induced massive hypermutation into HBV DNA
ities in Fig. 2C. The hap V protein also showed similar levels with
those of hap II and VII, but the anti-HBV activity of hap V was Because A3C I188 showed higher anti-HBV activity than the
weaker than that of hap II and VII. Protein levels of splice variants in S188 variant even with similar protein expression levels (Fig. 2), we
hap II of SV-200, SV-183, and SV-182 were comparable, whereas further investigated the mutation frequency of HBV DNA (Fig. 4).
SV-154 showed much lower levels than other variants (Fig. 2F, Cytoplasmic nucleocapsid-associated HBV DNA was purified from
right). Interestingly, the single amino acid addition of SV-183 from A3C-expressing hepatocytes. The HBV X gene region, which is a
SV-182 resulted in higher anti-HBV activity but with similar protein hotspot for APOBEC3-mediated hypermutation [9,28], was ampli-
levels (Fig. 2C and F). fied using a standard PCR protocol (denaturation temperature at
94
C). The PCR fragment was cloned into a T vector, and the
3.3. Hypermutation inducted by A3 variants sequence of the X gene in 20 different clones was determined.
Sequencing analysis shows that a 1.8-fold higher mutation fre-
We investigated the hypermutation activity of A3 variants using quency in HBV DNA was detected from A3C I188 compared to S188.
3D-PCR analysis of HBV DNA collected from the Huh7 transfectants. An average of 8.4 G-to-A mutations per clone was identified within
3D-PCR is a method that selectively amplifies AT-rich DNA by the small X gene region (694 base pairs) of A3C I188 transfectants.
lowering the denaturation temperature in the PCR condition,
thereby detecting hypermutated DNA [9,28]. The 3D-PCR results
show that all tested A3 variants exhibited hypermutation activity,
although the hypermutation activity of A3H hap VI appeared to be
lower than that of the other variants (Fig. 3A). The hypermutation
detected in the 3D-PCR was confirmed by sequencing analyses of
DNA amplified from denaturation temperatures lower than 83.7
C.
The analyses revealed extensive G-to-A and C-to-T mutations in the
3D-PCR amplicon, which is typical of APOBEC3-induced mutations
(Fig. 3B). The A3C-induced hypermutation was strongly biased to
G-to-A, as no C-to-T mutation was detected. Variant I188 slightly
increased the mutation frequency (171 G-to-A) compared with
S188 (138 G-to-A). In A3G and A3H hap II, V, and VII, clear C-to-T as
well as G-to-A hypermutations were detected. The dinucleotide Fig. 4. Mutation matrix of HBV sequence from standard PCR products. Standard
PCR products (94
C denaturation temperature) from A3C S188 and I188 transfectants
preference of this hypermutation was not altered between tested
were excised from the gel, cloned into a T vector, and sequenced. Mutation frequency
variants (Supplementary Fig. 1). in amplified 694 bp (except primer sequence) was analyzed.

Fig. 3. 3D-PCR analysis of HBV DNA from transfectants of A3 variants. (A) HBV replicon plasmid (pPB) and A3 expression vectors were transfected into Huh7 cells. Three days
after transfection, the culture supernatant was collected and HBV DNA was purified and analyzed using 3D-PCR with a denaturation temperature (Td) gradient of 94.0
C, 86.0
C,
83.7
C, 82.1
C, and 81.0
C. PCR products (83.7
C and 81
C) were excised from the gel, cloned into a T vector, and sequenced. pPB and GFP control vector transfectants were used
as control. (B) Mutation matrix of 3D-PCR products (167 bp, except primer sequence) from A.
30 A. Kanagaraj et al. / Biochemical and Biophysical Research Communications 518 (2019) 26e31
These results suggest that the higher hypermutation activity of this
A3C variant reflected its higher anti-HBV activity.
4. Discussion
In this study, we compared variants of A3C, A3G, and A3H
regarding their anti-HBV activities, protein levels, and hyper-
mutation activities in HBV plasmid-transfected Huh7 cells. In A3C,
the I188 variant showed a higher anti-HBV activity than the common
type S188 with similar protein levels. The stronger antiviral activity
of I188 was also reported in HIV-1 [20]. Our sequence analysis
revealed extensive G-to-A hypermutation by the I188 variant. The
mutation frequency induced by I188 was approximately 1.8-fold
higher than that of S188 (Fig. 4). Recently, it was reported that the
dimerization of A3C through I188 increased processivity on DNA
deamination activity [21]. The higher mutagenic activity of this A3C
variant may reflect its higher anti-HBV activity.
The A3G H186R variant is abundant in African Americans (37%)
but rare in Caucasians. It was reported that this variant is associated
with rapid progression in AIDS [22], but not with chronic HBV
infection [26]. Our results also show no significant difference be-
tween A3G H186 and R186 on anti-HBV activity and protein sta-
bility. In the HIV-1 genome, A3G deaminates entirely the 1st-strand
HIV-1 DNA, inducing only G-to-A hypermutations. C-to-T hyper-
mutations in the HIV-1 genome seem to be rare [29]. As mentioned
above, the HBV replication cycle partially resembles that of retro-
viruses. The G-to-A HBV hypermutation induced by A3s may be
caused by a mechanism similar to that of retrovirus such as HIV-1.
However, the biased C-to-T hypermutation induced by A3G and
some A3H variants implies that the deamination occurred at the
viral RNA level or after the double-strand synthesis of the viral
genome.
Differences in restriction activity and protein levels in the A3H
polymorphisms and splicing variants were more pronounced.
Deletion of N15 common to hap III, IV, and VI, and G105 in hap I and
VI were associated with low protein levels in transfected Huh7 cells
(Fig. 2F), consistent with HIV-1 studies showing that low activity
results from protein instability [23,25]. Hap II, V, and VII have re-
striction activity against Vif-deficient HIV-1 [25]. Our data also
show the higher protein levels in hap II, V, and VII, and the highest
anti-HBV activity in hap II (Fig. 2). Unexpectedly, we observed that
the low protein levels of hap VI showed relatively high anti-HBV
activity, whereas the high protein levels of hap V showed moder-
ate anti-HBV activity (Fig. 2). Among the four alternative splicing
variants of A3H [24], SV-154 corresponds to skipped exon 4 and is
predicted to be structurally disrupted. As expected, we detected
only small amounts of SV-154 protein in transfected Huh7 cells
(Fig. 2F). The protein levels of SV-200, SV-183, and SV-182 were
comparable in our experiments, but only SV-183 showed a strong
anti-HBV activity; the other two showed moderate activity (Fig. 2).
These three variants share exons 1 to 4 but have different C-ter-
minal ends. The biochemical properties of these C-terminal differ-
ences regarding anti-HBV activity should be investigated in the
future.
In this study, we demonstrated the different restriction and
mutagenic activities of A3C, A3G, and A3H variants against HBV
nucleocapsid DNA in overexpression experiments in cell culture.
We have previously demonstrated that endogenous A3C is highly
expressed in a hepatoma cell line, and endogenous A3G can be
induced by IFNg [5]. These results suggest that the genetic varia-
tions in endogenous A3 genes may modulate the persistence of
HBV infection in humans. It has been suggested that several A3
members can also deaminate HBV cccDNA in the nucleus [4,5,8].
Comparison of anti-HBV cccDNA activities of A3 variants will be
performed in further studies.
Author contributions
Study conception and design: MaM and KK. Data acquisition:
AK, NS, MoM, and MiK. Data analysis and interpretation: AK, NS, LQ,
YL, MoM, and KK. Material supports: MiK. Manuscript preparation:
AK, KW, MaK, MaM, and KK. Study supervision: MaM.
Acknowledgements
We thank Ms. Shimadzu for technical support. This study was
supported by the Japan Society for the Promotion of Science [grant
numbers 26460993and 19K07583 to KK and MM, respectively], the
Ministry of Health, Labour and Welfare of Japan [grant number
H26-kanen jitsuyouka-wakate008 to KK], the Japan Agency for
Medical Research and Development, AMED [grant number
18fk0310103j0302 to MM], the Takeda Science Foundation to AK,
MM, and KK, the Miyakawa Memorial Research Foundation to MM,
and the GSK Japan Research Grant 2016 to KK. The funders had no
role in study design, data collection and analysis, or decision to
publish.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.08.003.
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2019.08.003
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