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Infection
May Contribute to Occult Hepatitis B Virus
and In Vitro Protein Prevent Its Excretion
Specific Amino Acid Substitutions in the S
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Specic Amino Acid Substitutions in the S Protein Prevent Its
Excretion In Vitro and May Contribute to Occult Hepatitis B Virus
Infection
Subhajit Biswas,
a
* Daniel Candotti,
a,b
Jean-Pierre Allain
a
Department of Haematology, University of Cambridge, Cambridge, United Kingdom
a
; National Health Service Blood & Transplant, Cambridge, United Kingdom
b
Occult hepatitis B virus (HBV) infection (OBI) is dened as low plasma level of HBV DNA with undetectable HBV surface
antigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown.
The potential role of specic amino acid substitutions in the S protein from OBI in HBsAg production and excretion was
examined in vitro. HBsAg was quantied in culture supernatants and cell extracts of HuH-7 cells transiently transfected
with plasmids containing the S gene of eight HBsAg
control samples
contained genotype B, four genotype C, and two genotype D strains. A
detailed description of OBI and HBsAg
blood
donors (95 sequences per genotype).
Site-directed mutagenesis (SDM) of pcDNA3.1-HBV clones was per-
formed using the QuikChange site-directed mutagenesis kit (Agilent
Technologies, La Jolla, CA) according to the manufacturers instructions.
The correct introduction of a mutation was veried by sequencing.
Cells and transfection. pcDNA3.1-HBV plasmids were transfected
into HuH-7 cells (3 g plasmid/3 10
5
cells) using FuGENE HD trans-
fection reagent (Promega, Madison, WI) as described previously (10). For
each sample, at least two independent transfection experiments were per-
formed in triplicate.
Analysis of extracellular and intracellular HBsAg production. Ex-
tracellular (EC) HBsAg production was tested in culture supernatants 72
h posttransfection using the Murex HBsAg v3 enzyme immunoassay
(DiaSorin, Dartford, United Kingdom). A standard curve was generated
by testing serial dilutions of a commercial calibrated recombinant HBsAg
(Source BioScience, Nottingham, United Kingdom) for quantication.
To evaluate intracellular (IC) HBsAg production, supernatant-free
cell monolayers were washed three times with 1 phosphate-buffered
saline (PBS) and lysed in 500 l of 1PBS, 1%Triton X-100, 10 units/ml
DNase I, and complete proteinase inhibitor/EDTA (Roche, Basel, Swit-
zerland). Cell lysates were centrifuged at 13,000 rpm for 15 min, and
supernatants were collected as cytosolic extracts. HBsAg in extracts was
quantied as described above.
The total amount of HBsAg in each transfected culture of 3 10
5
HuH-7 cells was calculated as the sumof HBsAg present in ICextract and
ECsupernatant. For each sample, the mean ICand ECHBsAg production
was calculated from 2 to 4 independent transfection experiments each,
including three replicates. For comparison, HBsAg production in each
culture was normalized according to the transfection efciency calculated
from the percentage of cells expressing -galactosidase activity.
Immunouorescence detection of intracellular HBsAg. HuH-7 cells
grown on glass coverslips and transfected with pcDNA3.1-HBV plasmids
were xed at 72 h posttransfectionusing 4%paraformaldehyde for 30 min
at room temperature. The xed cells were washed with 1PBS, blocked
with 1% bovine serum albumin (BSA)0.5% Triton X-100 in PBS for 30
min and incubated for 90 min in the presence of Alexa Fluor 488-labeled
mouse anti-HBsAg monoclonal IgG (Invitrogen). Cells were washed and
coverslips were mounted on slides using Prolong Gold antifade reagent
with DAPI (Invitrogen). Cells were examined by immunouorescence
microscopy (60oil immersion lens and 10objective) for detection of
intracellular HBsAg in cells at a magnication of 600.
Transmembrane structure predictionof HBsAg variants. The trans-
membrane distribution of S protein was predicted using the SOSUI server
(http://bp.nuap.nagoya-u.ac.jp/sosui/). Other programs were used to
predict the S protein transmembrane domains: DAS, TopPred, and
HMMTOP (topology prediction programs, Expasy Bioinformatics Re-
source Portal [http://www.expasy.org/tools/). Results were compared to
the generally used model by Persson and Argos (14).
Statistical analysis. IC and EC HBsAg production for M88 cl4 wild
type (wt) and its different mutants at position 178 were compared by
means of one-way analysis of variance (ANOVA) with Tukeys multiple
comparison (post hoc) test. Differences between HBsAg productions (to-
tal or EC) for OBI strains before and after SDM repair were compared
using Students t test (two-tailed for unpaired data), and the variances of
the data were measured by the F test. P values of 0.05 were considered
statistically signicant.
TABLE 1 Characteristics of HBsAg
and OBI clones were aligned with consensus sequences derived from 124 wild-type/HBsAg
se-
quences (Fig. 1). Substitutions at positions 3 to 5 were not consid-
ered, as they resulted fromthe use of the degenerated primer SPL3
in the initial genomic amplication procedure. The potential as-
sociation of 17 OBI-specic substitutions with pattern 2 was in-
vestigated by restoring the wild-type consensual residues using
SDM (Table 2). Eight of the selected substitutions were unique to
the pattern 2 sequences studied (M75T, P105R, K160N, W165R,
L176P, W182C, V184A, and I226N), whereas one (S167L) and six
(Y100S, P111S, G112E, G119E, S154P, and P178R) were also pres-
ent in OBI pattern 1 and pattern 3 sequences, respectively. In
addition, Q129R and A159V were found in sequences associated
with all three patterns.
The HBsAg production pattern was not modied when 14 se-
lected amino acids were restored to the wild-type residues by SDM
individually or in combinations (Table 2). In contrast, restoration
of methionine 75 in sample HK01556-cl2, tyrosine 100 in sample
HK6794-cl2, and proline 178 in samples HK3110-cl4 and
HK3475-cl6 resulted in a signicant increase of HBsAg produc-
tion (P 0.02 to 0.05, except HK01556-cl2) that was associated
with increased HBsAg excretion in culture supernatants, as re-
ected in the decrease of HBsAg IC/EC ratio observed (7 to 801
versus 0.125 to 4 after SDM). This nding was conrmed in all
four samples by immunouorescence analysis, showing a change
following SDM from the dense intracellular accumulation of
HBsAg associated with pattern 2 to a diffuse uorescence across
the cytoplasm typical of pattern 1 (Fig. 4A). These data were re-
produced in the hepatocyte cell line HepG2 (data not shown). The
P178Rsubstitution was present in the pattern 3 TW8964-cl1 sam-
ple, but the SDM restoration of proline did not produce any per-
ceptible change, possibly due to the apparently extremely low
HBsAg production that characterizes pattern 3 OBIs (Table 2).
The P178R substitution was not found in the sequences of 369
strains (124 genotype B, 95 genotype C, and 150 genotype D) from
HBsAg
sequence each.
To conrm the effect of M75T, Y100S, and P178R on HBsAg
excretion, these three substitutions were introduced into the ge-
FIG 3 Immunouorescence microscopy of HuH-7 cells expressing non-OBI
and OBI HBsAg. Cell nucleus were stained with DAPI (blue) and HBsAg was
detected with Alexa Fluor 488-labeled mouse anti-HBsAg monoclonal IgG
(green). Cells transfected with a reporter plasmid expressing LacZwere used as
negative controls.
TABLE 2 Impact of OBI-specic amino acid substitutions on HBsAg
production pattern in vitro
OBI clone
Substitution
repaired
a
Average total
HBsAg
(range) (ng)
b
IC/EC
ratio
IFA
pattern
c
Deduced
HBsAg
Production
pattern
HK01556-cl2 None 189 (93254) 800 R 2
T75 M 226 (127310) 0.125 WT 1
R105P 72 (47103) 41 R 2
P154S 214 (213229) 464 R 2
A184V 97 (83109) 73 R 2
HK6794-cl2 None 60 (5192) 8 R 2
S100Y 325 (307362) 3 WT 1
R165WL167S 102 (96107) 7 R 2
C182W 39 (2470) 9 R 2
TW9015-cl1 None 91 (56143) 12 R 2
S111PE112G 91 (8299) 13 R 2
E119G 93 (8999) 11 R 2
TW0498-cl2 None 18 (1323) 13 LF 3
R129Q 21 (1923) 22 LF 3
TW0498-cl3 None 135 (62217) 15 R 2
R129Q 63 14 R 2
HK3110-cl4 None 116 (88131) 20 R 2
V159AN160K 188 (184192) 20 R 2
P176SR178P 284 (267302) 2 WT 1
R178P 757 (692809) 4 WT 1
N226I 23 (1825) 22 R 2
HK3475-cl6 None 75 (49103) 7 R 2
R178P 464 (409541) 2 WT 1
TW8964-cl1 None 0.6 (0.30.8) NA
d
NF 3
R178P 2 (1.82.1) NA NF 3
a
Mutations in OBI sequences (in bold) were repaired by SDM. None, the native OBI
sequence was tested.
b
Cumulative amount of HBsAg (ng) in cytosol and culture supernatant.
c
IFA detection of intracellular S protein. WT, diffused granular pattern; R, retained
dense packed uorescence close to the nucleus; LF, low level uorescence; NF, no
uorescence detected.
d
NA, not applicable.
Biswas et al.
7886 jvi.asm.org Journal of Virology
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notype BHBsAg
blood donors
identied three patterns of HBsAg expression (Fig. 1). These pat-
terns were dened by the total HBsAg production estimated by
enzyme immunoassay (EIA), the IC/EC HBsAg ratio, and the in-
tracellular distribution of HBsAg detected by immunouores-
cence (Fig. 2 and 3). However, two genotype C controls (M92-cl2
and M95-cl8) presented an IC/ECratio and IFApattern similar to
OBI pattern 2, but total HBsAg was similar to pattern 1. In addi-
tion, pattern 2 TW9015-cl1 and pattern 3 TW6639-cl1 and
TW2256-cl3 IFA appeared to be intermediate between pattern 2
and 3. Nevertheless, there were clear differences in HBsAg expres-
sion properties between HBsAg
control (n
1/369; 0.27%). The Y100S mutation was observed in one genotype
A2, six genotype B, and four genotype D OBI strains (n 11/176;
6.25%), and in one genotype D HBsAg
sequences of genotypes A to
D and was considered OBI specic.
The rare M75T substitution was associated with HBsAg excre-
tion defect. This mutation is located in the C-terminus part of the
protein cytosolic loop that contains a putative core-envelope in-
teraction domain important for both virions and HBsAg secretion
(16). In previous studies, replacement of R73, R78, and R79 by
uncharged residues, and the naturally occurring mutation L77R
reduced HBsAg secretion (16, 18). The highly restricted perinu-
clear IFA staining pattern observed for mutant M75T in the pres-
ent study (Fig. 4A) was consistent with the HBsAg retention in the
ER-Golgi reported for mutant L77R (16). Mutant M75T provides
additional indirect support to a role of the cytosolic loop C termi-
nus in HBsAg secretion independently of its putative role in virion
morphogenesis.
Three genotype B OBI clonesHK01556-cl2, HK6794-cl2,
and TW6639-cl1with HBsAg excretion defect contained the
Y100S mutation(Fig. 1 andFig. 2). WhenrepairedinHK6794-cl2,
excretion of HBsAg was recovered and changed from pattern 2 to
pattern 1. However, Y100S alone did not cause HBsAg retention
when introduced in M88-cl4 control (Table 3) or when naturally
present in the T75M-repaired HK01556-cl2 clone (Table 2 and
Fig. 4), suggesting that the negative effect of mutant Y100S on
HBsAg excretion may be corrected by other, yet-uncharacterized,
S envelope mutation(s) in M88-cl4 and HK01556-cl2. Similarly,
the single mutation W74L has been reported to suppress the re-
tention phenotype of L77R (16). A Y100C substitution was pres-
ent in two pattern 3 OBI clones (HK8663-cl2 and TW3437-cl5) in
agreement with previous reports associating this substitution with
HBsAg-negative phenotype in OBI cases (12, 1921). However, it
was also found in all pattern 2/3 clones of the M92 control, indi-
cating that Y100C does not play a direct role in reducing total
HBsAg amounts or HBsAg reactivity with commercial assays, as
suggested by Mello and coauthors (12).
Previous studies have shown that both virions and HBsAg se-
cretion were affected by mutations within three of the putative
transmembrane (TM) alpha-helix domains TM1, TM2, and TM4
of the S protein (18, 22, 23). Mutations in TM2 and TM4 may
affect (i) possible intramolecular interactions between TM do-
mains withinthe S protein, resulting inalteredproteinfolding and
defective insertion into the ER membrane, or (ii) intermolecular
interactions with the peptide chains of other S proteins essential
for HBsAg morphogenesis (11, 23, 24). In the present study, the
P178R substitution in the N-terminal part of the putative TM3
prevented HBsAg protein excretion in the two distinct HK3110-
cl4 and HK3475-cl6 OBI clones and in the mutated M88-cl4 con-
trol. Introduction of a positively charged arginine residue in place
of a proline (a residue commonly found as the rst residue of an
alpha helix) may modify this transmembrane domain and affect
the excretion of the modied protein. The presence of a poten-
tially charged residue within the TM domain of a typical integral
membrane protein was shown to result in retention and rapid
degradation in the ER (25). This is in agreement with the reduced
total amount of HBsAg and its intracellular location characteriz-
ing OBI pattern 2. These effects of charged residues appeared to
correlate with the level of free energy required to partitioncharged
chains into a lipid bilayer (25), and it may explain the similar,
albeit lessened, effect of substitutions with other strongly polar
residues, including lysine and glutamine (Table 3). However, lim-
ited changes induced by nonpolar residues alanine and leucine
and the lack of signicant change with glutamate (Table 3) suggest
that these phenotypic changes may be also dependent on their
position within the transmembrane sequence and on the nature of
the amino acid side chains. Nevertheless, these data showthat, like
the three other transmembrane domains, TM3 plays a role in the
morphogenesis of HBsAg. However, the topology of the HBsAg
carboxy-terminal transmembrane domains is not precisely
known, and structural predictions rely on models that are contin-
uously rened.
Defective HBsAg secretion was also reported to be associated
with substitutions in the MHR of OBIs (5, 13, 17, 26, 27). Some of
these substitutions were observed in the OBI sequences studied
here, but their negative effect if any on HBsAg phenotype re-
mained unclear. For example, serine at position 126, previously
associated with a moderate decrease of HBsAg secretion (5), was
present in OBI pattern 1 TW5004-cl3, with no evidence of a secre-
tion defect. Similarly, Q129R was found in four OBI clones irre-
spective of their HBsAg excretion pattern (pattern 1 clone
TW4576-cl3, pattern 2 clone TW0498-cl3, and pattern 3 clones
TW6639-cl1 and TW8964-cl1). Moreover, the correction R129Q
did not restore efcient HBsAg excretion in TW0498-cl3 (Table
2), and the M88-cl4 excretion pattern was not modied by Q129R
(Table 3). The substitution G145A was reported to impair HBsAg
secretion in a genotype A OBI strain, but it showed no obvious
effect in an Asian strain in another study (5, 13). This substitution
was present in genotype B OBI TW0498-cl3 clone with pattern 2.
Similarly, D144E, previously reported to impair HBsAg detection/
secretion as mentioned above, was also observed in pattern 1
HK6921-cl3. Together, these data suggest that alterationof HBsAg
secretioncanbe due not only to a single substitutionbut also more
frequently to a combination of amino acid substitutions in differ-
ent regions of the protein. This is supported by reports showing
both positive and negative transcomplementation effect of S mu-
tations on HBsAg secretion inhibition (13, 16, 17).
It might be difcult to evaluate the exact importance of muta-
tions altering HBsAg secretion in the genesis of OBI, since such
Biswas et al.
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mutations appear to be rare and strain specic in most cases. In
addition, the HBsAg phenotype associated with these mutations
was generally examined from individual clones that may not be
representative of the variants constituting the quasispecies in in-
fected individuals, as observed for M75T carriage in HK01556
sample (data not shown). In contrast, the presence of P178R in
every closely related variant within HK3110 and HK3475 quasi-
species strongly supports anassociationbetweenHBsAg excretion
defect and OBI phenotype in these donors. A more complex situ-
ation was observed in HK6794 diverse quasispecies reected by
diversity in the patterns of HBsAg phenotype, suggesting that the
HBsAg phenotype of only few clones may not truly represent the
overall strain phenotype.
At present, the possible pathogenic role of intracellularly re-
tained HBsAg in the development of liver disease in individuals
with OBI can be only speculated on. However, impaired secretion
of mutated misfolded/unfolded proteins is known to induce ER
stress that can affect host cell physiology by activating intracellular
transductionpathways (28). Inhumanhepatocytes, accumulation
of HBV surface proteins in the ER has been reported to cause the
formation of ground glass hepatocytes and to induce oxidative
stress, cellular DNA damage, and mutations (29). In a transgenic
mouse model, the accumulation of nonsecretable HBsAg particles
within the hepatocyte ER appeared to cause severe and prolonged
liver dysplasia and damage accompanied by continual liver in-
ammation, regenerative hyperplasia, transcription deregulation,
aneuploidy, and the eventual development of hepatocellular car-
cinoma (HCC) (30). Altogether, these studies suggest that cytoso-
lic retention of the HBV surface proteins in hepatocytes may have
an oncogenic potential by inducing hepatocyte stress response
pathways that may stimulate transformation processes. In this
context, long-term OBI carriers infected with secretion-defective
HBVvariants might be at high risk of developing HCCif sufcient
time is allowed to elapse.
Inconclusion, thehighgeneticvariabilityobservedintheSgeneof
OBI strains is associated with heterogeneous patterns of intracellular
and extracellular HBsAg production in vitro. The identication of
three new OBI-specic mutationsM75T, Y100S, and P178R
associated with HBsAg intracellular retention supports further the
hypothesis that the lack of HBsAg secretion may signicantly con-
tribute to the multifactorial occurrence of OBI. M75T and P178R
mutants provide additional indirect support for a role of the cy-
tosolic loop and the third transmembrane domain in HBsAg mor-
phogenesis. Further studies are needed to investigate a potential
pathogenic role of intracellularly retained HBsAg in the develop-
ment of liver disease in OBI carriers.
ACKNOWLEDGMENTS
S. Linfromthe TaiwanBloodServices Foundation, Taipei, Taiwan, andC.
Kit Lin from Hong Kong Red Cross Blood Transfusion Centre, Hong
Kong, Peoples Republic of China, are thanked for providing OBI samples
studied.
S. Biswas was supported in part by a grant from NHSBT England and
by a grant from Novartis Diagnostics & Vaccines.
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July 2013 Volume 87 Number 14 jvi.asm.org 7891
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