Published Ahead of Print 8 May 2013. 2013, 87(14):7882. DOI: 10.1128/JVI.00710-13. J. Virol. 

Subhajit Biswas, Daniel Candotti and Jean-Pierre Allain InfectionMay Contribute to Occult Hepatitis B Virus

andIn VitroProtein Prevent Its Excretion Specific Amino Acid Substitutions in the S

http://jvi.asm.org/content/87/14/7882Updated information and services can be found at:

These include:

REFERENCEShttp://jvi.asm.org/content/87/14/7882#ref-list-1This article cites 30 articles, 8 of which can be accessed free at:

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

Specific Amino Acid Substitutions in the S Protein Prevent ItsExcretion In Vitro and May Contribute to Occult Hepatitis B VirusInfection

Subhajit Biswas,a* Daniel Candotti,a,b Jean-Pierre Allaina

Department of Haematology, University of Cambridge, Cambridge, United Kingdoma; National Health Service Blood & Transplant, Cambridge, United Kingdomb

Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surfaceantigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown.The potential role of specific amino acid substitutions in the S protein from OBI in HBsAg production and excretion wasexamined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfectedwith plasmids containing the S gene of eight HBsAg� controls and 18 OBI clones. The intracellular (IC)/extracellular (EC)HBsAg production ratio was �1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strainsclones: pattern 1, an IC/EC ratio of 1.0, was found in 5/18 OBI clones, pattern 2, detectable IC but low or undetectable ECHBsAg (IC/EC, 7.0 to 800), was found in 6/18 OBIs, and pattern 3, low or undetectable IC and EC HBsAg, was found in 7/18clones. Intracellular immunofluorescence staining showed that in pattern 2, HBsAg was concentrated around the nucleus,suggesting retention in the endoplasmic reticulum. The substitution M75T, Y100S, or P178R was present in 4/6 pattern 2OBI clones. Site-directed-mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introducedinto a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies ex-pressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of twoOBI strains may contribute significantly to the OBI phenotype.

Occult hepatitis B virus (HBV) infection/carriage (OBI) ischaracterized by the presence of very low levels of HBV DNA

in plasma and/or in liver with hepatitis B surface antigen (HBsAg)being undetectable using the most sensitive commercial assays,with or without antibodies to hepatitis core antigen (anti-HBc) orhepatitis B surface antigen (anti-HBs), outside the preseroconver-sion window period (1). OBI represents a particular form of per-sistent or chronic infection that is encountered globally, albeit atdifferent frequencies depending on endemicity and genotype (2).OBI is a potential source of HBV transmission by transfusion andorgan transplantation, can reactivate in association with immu-nodeficiency or immunosuppressive treatments, and might be arisk factor for liver cancer (2). Several mechanisms have beenproposed as responsible for OBI, such as imperfect control bythe host immune system (3, 4), multiple amino acid substitu-tions in the S protein affecting HBsAg detection with commer-cial immunoassays (5, 6), mutations in regulatory elementsnegatively affecting virus replication (7, 8), and mutations af-fecting posttranscriptional mechanisms regulating S proteinexpression (9, 10).

A high frequency of mutations has been observed especiallywithin the major hydrophilic region (MHR) of S protein fromOBI strains, and these may alter antigenicity, HBV infectivity, celltropism, and virion morphogenesis (4–6, 11). In addition, recentstudies reported amino acid substitutions in the MHR of OBIstrains that impaired virion and/or S protein secretion in in vitro-transfected hepatocyte cell lines and infected mice that might con-tribute to the OBI phenotype (5, 12, 13).

In the present study, the potential role of OBI-specific aminoacid substitutions in the S protein, within and outside the MHR,on HBsAg production and excretion was examined in vitro.

MATERIALS AND METHODSCloning and sequencing of the HBV S protein coding region, HBsAgexpression plasmid, and site-directed mutagenesis. HBV DNA was iso-lated from randomly selected plasma samples from 18 previously charac-terized blood donors with OBI and eight HBsAg� blood donors as con-trols (10). Fifteen, two, and one OBI donors were infected with HBVgenotype B, C, and D, respectively. Two HBsAg� control samplescontained genotype B, four genotype C, and two genotype D strains. Adetailed description of OBI and HBsAg� control donors is provided inTable 1.

The whole HBV genome was initially PCR amplified, and a consensusviral sequence was obtained by direct sequencing of the PCR products aspreviously described (4, 10). A subgenomic fragment (positions 156 to1803) containing the S coding region and the HBV posttranscriptionalregulatory element (PRE) was further amplified by using primers SPL3(5=-gcgcgcgctagcacCATGGGGARCAYCRYATCRGGA-3=; nucleotides[nt] 156 to 177) and SPL2 (5=-gcctttgcaagcttCASACCAATTTATGCCTAC-3=; nt 1803 to 1785) (lowercase indicates NheI and HindIII restrictionsites introduced for cloning purpose into SPL3 and SPL2 primers, respec-tively). Amplicons were cloned into the pcDNA3.1� expression vector(Invitrogen, Paisley, United Kingdom) under the control of the humancytomegalovirus (HCMV) immediate early (IE) promoter. The HCMV IE

Received 12 March 2013 Accepted 30 April 2013

Published ahead of print 8 May 2013

Address correspondence to Daniel Candotti, dc241@cam.ac.uk, or Jean-PierreAllain, jpa1000@cam.ac.uk.

* Present address: School of Life Sciences, University of Lincoln, Lincoln, UnitedKingdom.

S.B. and D.C. contributed equally to this work.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00710-13

7882 jvi.asm.org Journal of Virology p. 7882–7892 July 2013 Volume 87 Number 14

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

gene promoter replaced the natural pre-S2/S promoter, with the tran-scriptional initiation site being located 75 nucleotides upstream of the 5=end of the S mRNA in order to limit variation in mRNA transcription andconsequently examine S protein expression through HBsAg quantifica-tion. The HBV DNA insert and ligation sites of selected clones (1 to 19clones/sample) were sequenced, and S amino acid sequences were de-rived. Amino acid substitutions in S protein sequences of OBI and controlclones were identified by comparison with a consensus sequence of therespective genotype obtained by aligning sequences from HBsAg� blooddonors (�95 sequences per genotype).

Site-directed mutagenesis (SDM) of pcDNA3.1-HBV clones was per-formed using the QuikChange site-directed mutagenesis kit (AgilentTechnologies, La Jolla, CA) according to the manufacturer’s instructions.The correct introduction of a mutation was verified by sequencing.

Cells and transfection. pcDNA3.1-HBV plasmids were transfectedinto HuH-7 cells (3 �g plasmid/3 � 105 cells) using FuGENE HD trans-fection reagent (Promega, Madison, WI) as described previously (10). Foreach 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 72h posttransfection using the Murex HBsAg v3 enzyme immunoassay(DiaSorin, Dartford, United Kingdom). A standard curve was generatedby testing serial dilutions of a commercial calibrated recombinant HBsAg(Source BioScience, Nottingham, United Kingdom) for quantification.

To evaluate intracellular (IC) HBsAg production, supernatant-freecell monolayers were washed three times with 1� phosphate-bufferedsaline (PBS) and lysed in 500 �l of 1� PBS, 1% Triton X-100, 10 units/mlDNase I, and complete proteinase inhibitor/EDTA (Roche, Basel, Swit-zerland). Cell lysates were centrifuged at 13,000 rpm for 15 min, andsupernatants were collected as cytosolic extracts. HBsAg in extracts wasquantified as described above.

The total amount of HBsAg in each transfected culture of 3 � 105

HuH-7 cells was calculated as the sum of HBsAg present in IC extract andEC supernatant. For each sample, the mean IC and EC HBsAg productionwas calculated from 2 to 4 independent transfection experiments each,including three replicates. For comparison, HBsAg production in eachculture was normalized according to the transfection efficiency calculatedfrom the percentage of cells expressing �-galactosidase activity.

Immunofluorescence detection of intracellular HBsAg. HuH-7 cellsgrown on glass coverslips and transfected with pcDNA3.1-HBV plasmidswere fixed at 72 h posttransfection using 4% paraformaldehyde for 30 minat room temperature. The fixed cells were washed with 1� PBS, blockedwith 1% bovine serum albumin (BSA)– 0.5% Triton X-100 in PBS for 30min and incubated for 90 min in the presence of Alexa Fluor 488-labeledmouse anti-HBsAg monoclonal IgG (Invitrogen). Cells were washed andcoverslips were mounted on slides using Prolong Gold antifade reagentwith DAPI (Invitrogen). Cells were examined by immunofluorescencemicroscopy (60� oil immersion lens and 10� objective) for detection ofintracellular HBsAg in cells at a magnification of �600.

Transmembrane structure prediction of 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 topredict the S protein transmembrane domains: DAS, TopPred, andHMMTOP (topology prediction programs, Expasy Bioinformatics Re-source Portal [http://www.expasy.org/tools/). Results were compared tothe generally used model by Persson and Argos (14).

Statistical analysis. IC and EC HBsAg production for M88 cl4 wildtype (wt) and its different mutants at position 178 were compared bymeans of one-way analysis of variance (ANOVA) with Tukey’s multiplecomparison (post hoc) test. Differences between HBsAg productions (to-tal or EC) for OBI strains before and after SDM repair were comparedusing Student’s t test (two-tailed for unpaired data), and the variances ofthe data were measured by the F test. P values of �0.05 were consideredstatistically significant.

TABLE 1 Characteristics of HBsAg� control and OBI donorsa

Donor Status Origin GenderAge(years)

HBV DNAload (IU/ml)

HBsAg(IU/ml) level Anti-HBc

Anti-HBslevel (IU/L)

HBVgenotype

M38 Control Malaysia M 30 1.6 � 107 8.3 � 102 Pos Neg CM86 Control Malaysia F 19 3.1 � 108 2.1 � 104 Pos Neg CM88 Control Malaysia M 23 1.1 � 108 3.9 � 104 Pos Neg BM90 Control Malaysia F 22 1.6 � 108 1.2 � 105 Pos Neg BM92 Control Malaysia M 49 1.0 � 107 1.1 � 103 Pos Neg CM95 Control Malaysia M 26 1.5 � 103 1.4 � 104 Pos Neg CE3476 Control Egypt NA NA 6 3.3 � 105 Pos Neg DE3789 Control Egypt NA NA 31 1.1 � 105 Pos Neg DHK01556 OBI Hong Kong M 47 �5 Neg Pos Neg BHK3110 OBI Hong Kong F 49 Not detectable Neg Pos Neg BHK3475 OBI Hong Kong M 51 37 Neg Pos 26 BHK6794 OBI Hong Kong M 59 91 Neg Pos 16 BHK6921 OBI Hong Kong M 50 Not detectable Neg Pos 19 BHK8442 OBI Hong Kong M 53 46 Neg Pos 98 CHK8663 OBI Hong Kong M 53 56 Neg Pos Neg BR84 OBI Italy M 44 62 Neg Pos Neg DTW0498 OBI Taiwan M 46 48 Neg Pos Neg BTW2256 OBI Taiwan M 30 56 Neg Pos 18 BTW3437 OBI Taiwan F 49 30 Neg Pos Neg BTW4576 OBI Taiwan M 55 �5 Neg Pos Neg BTW5004 OBI Taiwan F 63 7 Neg Pos Neg BTW6083 OBI Taiwan M 62 �5 Neg Pos Neg CTW6639 OBI Taiwan M 55 Not detectable Neg Pos Neg BTW8964 OBI Taiwan M 30 15 Neg Pos Neg BTW9015 OBI Taiwan M 47 Not detectable Neg Pos Neg BTW9331 OBI Taiwan M 54 6 Neg Pos Neg Ba NA, not available.

Impaired S Protein Excretion and Occult HBV Infection

July 2013 Volume 87 Number 14 jvi.asm.org 7883

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

RESULTSIn vitro HBsAg production patterns. Eighteen HBV strains car-rying multiple amino acid substitutions in the S coding regionwere selected from a repository of viral strains from blood donorswith previously characterized OBI (Table 1 and Fig. 1). EightHBsAg� strains from blood donors (HBsAg plasma concentra-tion, �104 IU/ml) were used as non-OBI controls. One clone perOBI and control sample was randomly selected for transfection inHuH-7 cells and HBsAg production in vitro characterized by eval-uating the total amount of detectable IC and EC HBsAg, the IC/ECHBsAg ratio, and the IC distribution pattern of HBsAg detected byimmunofluorescence assay (IFA).

In HBsAg� controls, the average normalized total HBsAg pro-duction was �120 ng/3 � 105 cells (range, 123 to 613 ng) at 72 hposttransfection (Fig. 2). Six control clones showed an IC/ECHBsAg ratio ranging between 0.2 and 3.0. HBsAg intracellularimmunofluorescence staining was diffuse and finely granularacross the hepatocyte cytoplasm (Fig. 3). Two genotype C controlclones (M92-cl2 and M95-cl8) showed IC/EC ratios of �10.0 (Fig.2A), and IC HBsAg concentrated in the perinuclear area as denselypacked fluorescence (data not shown).

Three distinct patterns of HBsAg production were identified incells transfected with OBI clones (Fig. 2A to D). Pattern 1 (n �5/18) was similar to the features observed in the majority of con-trols, with total HBsAg production of �120 ng/3 � 105 cells

(range, 146 to 621 ng) (Fig. 2B), IC/EC HBsAg ratio of �3 (range,0.1 and 3) (Fig. 2C), and diffuse granular fluorescent staining of ICHBsAg. Pattern 3 (n � 7/18) was characterized by total HBsAg of�50 ng (0.6 to 46 ng) and an IC/EC HBsAg ratio ranging betweennoncalculable and 7, with low or no fluorescence staining of ICHBsAg (Fig. 3). For TW6639-cl1 (HBsAg, 46 ng/3 � 105 cells;IC/EC, 7) and TW2256-cl3 (HBsAg, 31 ng/3 � 105 cells; IC/EC,2), the IC HBsAg appeared as sparsely distributed but bright greenfluorescent dots in the cytoplasm (not shown). Pattern 2 (n �6/18) was characterized by total HBsAg ranging between 60 and189 ng nearly exclusively found intracellularly. The IC/EC HBsAgratio ranged between 7 and 800 (median, 10). The intracellularHBsAg fluorescence appeared dense, compact, and essentiallylimited to the central part of the cytoplasm, adjacent to the nu-cleus, in 5 of 6 pattern 2 OBIs, as observed with M92-cl2 andM95-cl8 (Table 2). The IC HBsAg for TW9015-cl1 (HBsAg, 91ng/3 � 105 cells; IC/EC, 12) presented as diffuse but very compactfluorescence filling the entire cytosol (not shown). No significantassociation of HBsAg production patterns with HBV genotype,plasma HBV DNA load, or anti-HBs status was observed (Table 1and Fig. 2A). Figure 2D summarizes the relation between HBsAgproduction and IC/EC ratio in controls and patterns 1 to 3.

Characterization of amino acid substitutions associatedwith HBsAg impaired excretion in HuH-7 cells. In pattern 2 OBIclones, HBsAg production was perfectly detectable and reached

FIG 1 Alignment of amino acid sequences deduced from cloned S genes of genotype B to D non-OBI (HBsAg�) and OBI HBV strains. Sequences of HBsAg�

and OBI clones were aligned with consensus sequences derived from 124 wild-type/HBsAg� genotype B sequences, 95 genotype C sequences, and 150 genotypeD sequences. Residues identical to the reference consensus are indicated by dots. Site-directed mutagenesis of residues in gray boxes resulted in a change in theHBsAg excretion pattern; no change was observed when residues in open boxes were corrected.

Biswas et al.

7884 jvi.asm.org Journal of Virology

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

FIG 2 Detection of intracellular and extracellular levels of HBsAg/S protein by EIA and IFA after transfection of cloned S sequences in HuH-7 cells. (A) Averageindividual HBsAg production in HuH-7 cells transfected with 8 non-OBI controls and 18 OBI clones. Standard deviations are for three or more culture wells; thethree different production patterns observed in OBI samples are indicated. (B) Comparison of total HBsAg production observed for the non-OBI and the threeOBI patterns. Statistically significant (P � 0.05) differences determined by 1-way ANOVA/Tukey test are indicated. (C) Comparison of HBsAg IC/EC ratiosobserved for each control and OBI patterns. No statistical significance was observed due to three outliers with remarkably high ratios (OBI pattern 2 isolateHK01556-cl2 and non-OBI isolates M92-cl2 and M95-cl8, showing IC/EC ratios of 800, 75, and 23, respectively). (D) Relationship between IC/EC ratio and totalHBsAg production. Geometric means are indicated with dashed vertical and horizontal lines; gray squares and circles identify non-OBI controls and pattern 1OBIs with diffuse granular IF, black squares and triangles identify non-OBI controls and pattern 2 OBIs with dense aggregated IF, and open diamonds identifypattern 3 OBIs with low/undetectable HBsAg in IF.

July 2013 Volume 87 Number 14 jvi.asm.org 7885

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

significantly higher levels than that in pattern 3 OBI clones (P �0.003) but was lower than that in both pattern 1 OBI clones (P �0.011) and controls (P � 0.007). Nevertheless, the high IC/ECratio associated with pattern 2 suggested an impaired HBsAg ex-cretion in transfected HuH-7 cells that was investigated furtherhere.

The deduced S amino acid sequences of the six OBI genotype Bpattern 2 clones (HK01556-cl2, TW0498-cl3, HK3110-cl4,HK3475-cl6, TW9015-cl1, and HK6794-cl2) showed 12 to 25amino acid substitutions (mean, 17) compared to the HBV con-sensus genotype B sequence obtained from 124 HBsAg� se-quences (Fig. 1). Substitutions at positions 3 to 5 were not consid-ered, as they resulted from the use of the degenerated primer SPL3in the initial genomic amplification procedure. The potential as-sociation of 17 OBI-specific substitutions with pattern 2 was in-vestigated by restoring the wild-type consensual residues usingSDM (Table 2). Eight of the selected substitutions were unique tothe 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. Inaddition, Q129R and A159V were found in sequences associatedwith all three patterns.

The HBsAg production pattern was not modified when 14 se-lected amino acids were restored to the wild-type residues by SDMindividually or in combinations (Table 2). In contrast, restorationof methionine 75 in sample HK01556-cl2, tyrosine 100 in sampleHK6794-cl2, and proline 178 in samples HK3110-cl4 andHK3475-cl6 resulted in a significant increase of HBsAg produc-tion (P � 0.02 to 0.05, except HK01556-cl2) that was associatedwith increased HBsAg excretion in culture supernatants, as re-

flected in the decrease of HBsAg IC/EC ratio observed (7 to 801versus 0.125 to 4 after SDM). This finding was confirmed in allfour samples by immunofluorescence analysis, showing a changefollowing SDM from the dense intracellular accumulation ofHBsAg associated with pattern 2 to a diffuse fluorescence acrossthe cytoplasm typical of pattern 1 (Fig. 4A). These data were re-produced in the hepatocyte cell line HepG2 (data not shown). TheP178R substitution 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 lowHBsAg production that characterizes pattern 3 OBIs (Table 2).The P178R substitution was not found in the sequences of 369strains (124 genotype B, 95 genotype C, and 150 genotype D) fromHBsAg� donors, whereas M75T and Y100S were present in oneHBsAg� sequence each.

To confirm the effect of M75T, Y100S, and P178R on HBsAgexcretion, these three substitutions were introduced into the ge-

FIG 3 Immunofluorescence microscopy of HuH-7 cells expressing non-OBIand OBI HBsAg. Cell nucleus were stained with DAPI (blue) and HBsAg wasdetected with Alexa Fluor 488-labeled mouse anti-HBsAg monoclonal IgG(green). Cells transfected with a reporter plasmid expressing LacZ were used asnegative controls.

TABLE 2 Impact of OBI-specific amino acid substitutions on HBsAgproduction pattern in vitro

OBI cloneSubstitutionrepaireda

Average totalHBsAg(range) (ng)b

IC/ECratio

IFApatternc

DeducedHBsAgProductionpattern

HK01556-cl2 None 189 (93–254) 800 R 2T75 M 226 (127–310) 0.125 WT 1R105P 72 (47–103) 41 R 2P154S 214 (213–229) 464 R 2A184V 97 (83–109) 73 R 2

HK6794-cl2 None 60 (51–92) 8 R 2S100Y 325 (307–362) 3 WT 1R165W�L167S 102 (96–107) 7 R 2C182W 39 (24–70) 9 R 2

TW9015-cl1 None 91 (56–143) 12 R 2S111P�E112G 91 (82–99) 13 R 2E119G 93 (89–99) 11 R 2

TW0498-cl2 None 18 (13–23) 13 LF 3R129Q 21 (19–23) 22 LF 3

TW0498-cl3 None 135 (62–217) 15 R 2R129Q 63 14 R 2

HK3110-cl4 None 116 (88–131) 20 R 2V159A�N160K 188 (184–192) 20 R 2P176S�R178P 284 (267–302) 2 WT 1R178P 757 (692–809) 4 WT 1N226I 23 (18–25) 22 R 2

HK3475-cl6 None 75 (49–103) 7 R 2R178P 464 (409–541) 2 WT 1

TW8964-cl1 None 0.6 (0.3–0.8) NAd NF 3R178P 2 (1.8–2.1) NA NF 3

a Mutations in OBI sequences (in bold) were repaired by SDM. None, the native OBIsequence 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, retaineddense packed fluorescence close to the nucleus; LF, low level fluorescence; NF, nofluorescence detected.d NA, not applicable.

Biswas et al.

7886 jvi.asm.org Journal of Virology

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

notype B HBsAg� control M88-cl4. OBI pattern 2 was reproducedin M88-cl4 following the M75T and P178R changes but not theY100S change, as evidenced by IC/EC ratio evaluation and intra-cellular immunofluorescence analysis (Table 3 and Fig. 4B). Inaddition, the introduction of the mutation P178R by SDM in anentire HBV genotype C genome changed the HBsAg productionpattern 1 initially observed in HuH-7 transfected cells to pattern 2(data not shown). In contrast, the substitutions P111S�G112E,G119E, Q129R, I150T, S154P, and W165R introduced into the

control M88-cl4 did not substantially affect the HBsAg produc-tion pattern (Table 3).

The effect of amino acid changes at position 178 on M88-cl4HBsAg phenotype was investigated further (Table 3). Substitu-tions P178A, P178E, P178K, P178L, and P178Q resulted in a sub-stantially decreased overall average production of HBsAg (31 to 80ng) compared to M88-cl4 wt (270 ng), as observed with P178R (78ng). However, the corresponding IC/EC HBsAg ratios wereslightly higher (IC/EC � 2 to 5) than observed for M88-cl4 wt

FIG 4 Immunofluorescence staining of native and mutated HBsAg in transfected HuH-7 cells. Amino acid substitutions introduced by site-directed mutagen-esis in OBI (A) and non-OBI (B) sequences are indicated in bold.

TABLE 3 Impact of OBI-specific substitutions introduced in the HBsAg� control M88-cl4 on HBsAg production in vitro

Substitutions introducedby SDM

Avg total HBsAg (range) (ng)HBsAg IC/ECratio IFA patternc

Deduced HBsAgproductionpatternTotalb EC

Nonea 270 (203–338) 197 (167–2270) 0.33 WT 1M75T 48 (39–56) NDd NAe R 2Y100S 475 (456–487) 316 (308–321) 0.5 WT 1P111S�G112E 482 (456–500) 347 (334–354) 0.33 WT 1G119E 402 (359–433) 241 (212–256) 0.5 WT 1Q129R 439 (410–471) 278 (256–302) 0.5 WT 1I150T 102 (74–112) 71 (52–80) 0.5 WT 1S154P 337 (296–392) 214 (191–243) 0.5 WT 1W165R 138 (112–161) 70 (49–84) 1 WT 1P178R 78 (76–81) 5 (5–6) 15 R 2P178Q 63 (45–76) 11 (11–12) 5 WT IndeterminateP178L 80 (70–82) 19 (18–21) 3 WT IndeterminateP178A 31 (27–37) 11 (10–13) 2 WT IndeterminateP178K 41 (31–48) 12 (10–15) 3 WT IndeterminateP178E 136 (126–143) 99 (92–104) 0.33 WT 1a Wild type M88-cl4 control.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 fluorescence close to the nucleus; NF, no fluorescence detected.d ND, not detected.e NA, not applicable.

Impaired S Protein Excretion and Occult HBV Infection

July 2013 Volume 87 Number 14 jvi.asm.org 7887

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

(IC/EC � 0.33) but still compatible with pattern 1 definition andlower than the pattern 2-related IC/EC ratio of the P178R mutant(IC/EC � 15). The immunofluorescence (IF) results obtainedwith P178A/K/L/Q did not clearly match either pattern 1 or pat-tern 2 profiles and were considered indeterminate (Fig. 5). MutantP178E showed a pattern 1-related IC/EC HBsAg ratio and IFAprofile similar to that of M88-cl4 wt. As shown in Fig. 1, P178Qwas present in a pattern 1 and a pattern 3 OBI clone (TW5004-cl3and TW3437-cl5, respectively).

Several modeling programs predicted very similar arrange-ments of the S protein in the bilayer lipid membrane of the endo-plasmic reticulum (ER). Persson and Argos’ model as well asSOSUI’s and DAS’s identified the same four transmembrane(TM) domains for wild-type S. However, SOSUI predicted a fifthTM domain (amino acids 149 to 171) that we considered doubt-ful. Nevertheless, the three amino acid substitutions identified ascritical to HBsAg excretion had identical locations predicted bythe three models. M75T was located in the cytosolic loop separat-ing TM1 and TM2, Y100S was at the end of TM2, and P178R wasin TM3.

HBsAg excretion deficit as potential contributor to OBI phe-notype. Amino acid substitutions impairing HBsAg excretion re-flected by HBsAg quantification in vitro have been identified inindividual OBI clones. However, HBVs in HBsAg-positive as well

as OBI strains circulate as quasispecies of related variants not nec-essarily represented by a single clone tested in vitro. The geneticdiversity and the relevance of specific mutations as potential ex-planation for the OBI phenotype were therefore examined by se-quencing multiple clones from several donor samples containingpattern 2 clones (Fig. 6).

The diversity of patterns was shown in one control (M92; twoclones with pattern 2 and one clone with pattern 3) and threeOBIs: TW0498 (one pattern 2 and one pattern 3), TW8964 (onepattern 1 and one pattern 3) and HK6794 (3 pattern 1, one pattern2, and one pattern 3) (Fig. 6). In OBI HK6794, the average aminoacid diversity between clones was 20 residues (range, 11 to 32residues) without a significant relationship with the HBsAg phe-notype (data not shown). The Y100S mutations responsible forpattern 2 of clone 2 were present in a subcluster of five clonesassociated with clone 2, but Y100F was present in all 12 otherclones. In contrast, in OBIs HK01556, HK3110, and HK3475, ge-netic diversity was very limited (Fig. 6), and each of the 8 to 12clones contained Y100S (HK01556) or P178R (HK3110 andHK3475). For these three OBI strains, it is likely that these twomutations shown to prevent HBsAg excretion contribute to theOBI phenotype.

DISCUSSION

High mutation rates in the HBsAg in OBI strains from variousgeographical origins have been reported (4, 5, 8, 10). Some ofthese mutations have been functionally associated with structuralchanges in the HBsAg, leading to impaired detection by currentimmunoassays (5, 6). However, the extremely low viral load gen-erally observed in OBI carriers suggests that S mutations may alsonegatively affect either viral replication or HBsAg production incirculation. To test the second hypothesis, the effect of amino acidsubstitutions specific to genotype B to D OBI strains on HBsAgexpression was investigated in vitro.

The functional analysis of clones derived from 18 previouslycharacterized OBI strains with S mutations not present in 369HBV genotype B to D strains infecting HBsAg� blood donorsidentified three patterns of HBsAg expression (Fig. 1). These pat-terns were defined by the total HBsAg production estimated byenzyme immunoassay (EIA), the IC/EC HBsAg ratio, and the in-tracellular distribution of HBsAg detected by immunofluores-cence (Fig. 2 and 3). However, two genotype C controls (M92-cl2and M95-cl8) presented an IC/EC ratio and IFA pattern similar toOBI pattern 2, but total HBsAg was similar to pattern 1. In addi-tion, pattern 2 TW9015-cl1 and pattern 3 TW6639-cl1 andTW2256-cl3 IFA appeared to be intermediate between pattern 2and 3. Nevertheless, there were clear differences in HBsAg expres-sion properties between HBsAg�/wild-type controls and OBIstrains and between OBIs (Fig. 2A to D).

The differences in total HBsAg production between HBVstrains, used as a criterion to define the three patterns, might bedue to variations in the effectiveness of the HBsAg expressionvector transfection into HuH-7 cells despite normalization us-ing cotransfection with a reporter plasmid and the mean (standard deviation [SD]) of total IC and EC HBsAg in triplicate(Fig. 2). In addition, using a CMV promoter-driven expressionvector, protein overexpression may be responsible for the phe-notypes observed in vitro. Similarly, discrete variations inHBsAg production may be masked by high protein expressionlevels. However, similar IC and EC HBsAg levels were obtained

FIG 5 Immunofluorescence staining of HBsAg in cells transfected with wild-type and mutated M88-cl4 sequences. The position and nature of the OBI-specific amino acid substitutions introduced into the genotype B control M88-cl4 are indicated.

Biswas et al.

7888 jvi.asm.org Journal of Virology

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

when HuH-7 cells with an entire HBV genotype C genome weretransfected (data not shown). Nevertheless, total HBsAg, to-gether with the intracellular HBsAg immunofluorescence pat-tern, clearly differentiated patterns 1 and 2 from pattern 3 (Fig.2B and Fig. 3). For the latter, a low total HBsAg level (�50 ng)was supported by no or low IC HBsAg immunofluorescence(Fig. 3). Further studies are needed to establish whether thelack of HBsAg detection in pattern 3 OBIs is related to HBsAgproduction being terminated by an uncharacterized posttran-scriptional mechanism, as previously suggested (9, 10), or tomutant HBsAg unrecognized by the EIA and IFA (5, 6). The

average amino acid substitution rates in the MHR and over theS protein were similar in OBI sequences irrespective of HBsAgpattern (MHR, 6.4% in pattern 1 versus 8.6% in pattern 2versus 9.1% in pattern 3; S protein, 5.7% in pattern 1 versus7.2% in pattern 2 versus 6.9% in pattern 3) but significantlyhigher in OBI than in non-OBI sequences (0.9% and 1.9% in non-OBI MHR and S protein, respectively) (Fig. 1). However, mutationsD144E and G145R, previously associated with reduced HBsAg detec-tion (15), were present in three pattern 3 clones (TW6639-cl1 andTW3437-cl5) or in association (HK8663-cl2) and supported the im-mune detection escape hypothesis.

FIG 6 Phylogenetic analysis of the S amino acid sequences of multiple clones from OBI and non-OBI strains. Phylogenetic analysis was performed as previouslydescribed (10). HBV reference sequences of genotypes/subgenotypes A1-3 and B-H are identified by their GenBank accession numbers. For clones used in HuH-7transfection experiments, the resulting HBsAg excretion pattern is indicated by open (pattern 1), black (pattern 2) or gray (pattern 3) arrows.

Impaired S Protein Excretion and Occult HBV Infection

July 2013 Volume 87 Number 14 jvi.asm.org 7889

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

In individuals carrying pattern 1 and 2 clones, OBI did notseem related to antigenic variation in HBsAg, since when pro-duced in vitro, it was detected with commercial assays. Mutationsin other HBV regions might be responsible for OBI in individualscarrying pattern 1 clones. Mutations in the S gene may also resultin a modified reverse transcriptase region of the HBV polymerase,decreasing HBV replication (15). The reduced level of HBsAg ex-cretion observed in pattern 2 OBIs may also contribute to thegenesis of OBI by maintaining HBsAg level below the detectionlimit of currently used serological assays and by concomitantlyaffecting virion release as suggested by recent studies (5, 12, 13, 16,17). SDM experiments that involved repairing an OBI-specificmutation(s) (Table 2 and Fig. 4A) and introducing this muta-tion(s) into a control/wild-type sequence (Table 3 and Fig. 4B)identified three mutations—M75T, Y100S, and P178R—thatcontributed to decreased HBsAg excretion in vitro. The M75Tmutation was unique to HK01556-cl2 but was also present in theconsensus sequence of one genotype C HBsAg� control (n �1/369; 0.27%). The Y100S mutation was observed in one genotypeA2, six genotype B, and four genotype D OBI strains (n � 11/176;6.25%), and in one genotype D HBsAg� control (0.27%). TheP178R mutation was detected in the consensus sequence of threegenotype B and two genotype D OBI strains representing 2.8% of176 OBI strains previously studied (data not shown). The P178Rmutation was absent in 369 HBsAg� sequences of genotypes A toD and was considered OBI specific.

The rare M75T substitution was associated with HBsAg excre-tion defect. This mutation is located in the C-terminus part of theprotein 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 byuncharged residues, and the naturally occurring mutation L77Rreduced 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 theER-Golgi reported for mutant L77R (16). Mutant M75T providesadditional indirect support to a role of the cytosolic loop C termi-nus in HBsAg secretion independently of its putative role in virionmorphogenesis.

Three genotype B OBI clones—HK01556-cl2, HK6794-cl2,and TW6639-cl1—with HBsAg excretion defect contained theY100S mutation (Fig. 1 and Fig. 2). When repaired in HK6794-cl2,excretion of HBsAg was recovered and changed from pattern 2 topattern 1. However, Y100S alone did not cause HBsAg retentionwhen introduced in M88-cl4 control (Table 3) or when naturallypresent in the T75M-repaired HK01556-cl2 clone (Table 2 andFig. 4), suggesting that the negative effect of mutant Y100S onHBsAg 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) inagreement with previous reports associating this substitution withHBsAg-negative phenotype in OBI cases (12, 19–21). However, itwas also found in all pattern 2/3 clones of the M92 control, indi-cating that Y100C does not play a direct role in reducing totalHBsAg amounts or HBsAg reactivity with commercial assays, assuggested 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 TM4of the S protein (18, 22, 23). Mutations in TM2 and TM4 mayaffect (i) possible intramolecular interactions between TM do-mains within the S protein, resulting in altered protein folding anddefective insertion into the ER membrane, or (ii) intermolecularinteractions with the peptide chains of other S proteins essentialfor HBsAg morphogenesis (11, 23, 24). In the present study, theP178R substitution in the N-terminal part of the putative TM3prevented 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 placeof a proline (a residue commonly found as the first residue of analpha helix) may modify this transmembrane domain and affectthe excretion of the modified protein. The presence of a poten-tially charged residue within the TM domain of a typical integralmembrane protein was shown to result in retention and rapiddegradation in the ER (25). This is in agreement with the reducedtotal amount of HBsAg and its intracellular location characteriz-ing OBI pattern 2. These effects of charged residues appeared tocorrelate with the level of free energy required to partition chargedchains into a lipid bilayer (25), and it may explain the similar,albeit lessened, effect of substitutions with other strongly polarresidues, including lysine and glutamine (Table 3). However, lim-ited changes induced by nonpolar residues alanine and leucineand the lack of significant change with glutamate (Table 3) suggestthat these phenotypic changes may be also dependent on theirposition within the transmembrane sequence and on the nature ofthe amino acid side chains. Nevertheless, these data show that, likethe three other transmembrane domains, TM3 plays a role in themorphogenesis of HBsAg. However, the topology of the HBsAgcarboxy-terminal transmembrane domains is not preciselyknown, and structural predictions rely on models that are contin-uously refined.

Defective HBsAg secretion was also reported to be associatedwith substitutions in the MHR of OBIs (5, 13, 17, 26, 27). Some ofthese substitutions were observed in the OBI sequences studiedhere, but their negative effect if any on HBsAg phenotype re-mained unclear. For example, serine at position 126, previouslyassociated with a moderate decrease of HBsAg secretion (5), waspresent 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 cloneTW4576-cl3, pattern 2 clone TW0498-cl3, and pattern 3 clonesTW6639-cl1 and TW8964-cl1). Moreover, the correction R129Qdid not restore efficient HBsAg excretion in TW0498-cl3 (Table2), and the M88-cl4 excretion pattern was not modified by Q129R(Table 3). The substitution G145A was reported to impair HBsAgsecretion in a genotype A OBI strain, but it showed no obviouseffect in an Asian strain in another study (5, 13). This substitutionwas 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 1HK6921-cl3. Together, these data suggest that alteration of HBsAgsecretion can be due not only to a single substitution but also morefrequently to a combination of amino acid substitutions in differ-ent regions of the protein. This is supported by reports showingboth positive and negative transcomplementation effect of S mu-tations on HBsAg secretion inhibition (13, 16, 17).

It might be difficult to evaluate the exact importance of muta-tions altering HBsAg secretion in the genesis of OBI, since such

Biswas et al.

7890 jvi.asm.org Journal of Virology

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

mutations appear to be rare and strain specific in most cases. Inaddition, the HBsAg phenotype associated with these mutationswas generally examined from individual clones that may not berepresentative of the variants constituting the quasispecies in in-fected individuals, as observed for M75T carriage in HK01556sample (data not shown). In contrast, the presence of P178R inevery closely related variant within HK3110 and HK3475 quasi-species strongly supports an association between HBsAg excretiondefect and OBI phenotype in these donors. A more complex situ-ation was observed in HK6794 diverse quasispecies reflected bydiversity in the patterns of HBsAg phenotype, suggesting that theHBsAg phenotype of only few clones may not truly represent theoverall strain phenotype.

At present, the possible pathogenic role of intracellularly re-tained HBsAg in the development of liver disease in individualswith OBI can be only speculated on. However, impaired secretionof mutated misfolded/unfolded proteins is known to induce ERstress that can affect host cell physiology by activating intracellulartransduction pathways (28). In human hepatocytes, accumulationof HBV surface proteins in the ER has been reported to cause theformation of ground glass hepatocytes and to induce oxidativestress, cellular DNA damage, and mutations (29). In a transgenicmouse model, the accumulation of nonsecretable HBsAg particleswithin the hepatocyte ER appeared to cause severe and prolongedliver dysplasia and damage accompanied by continual liver in-flammation, 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 havean oncogenic potential by inducing hepatocyte stress responsepathways that may stimulate transformation processes. In thiscontext, long-term OBI carriers infected with secretion-defectiveHBV variants might be at high risk of developing HCC if sufficienttime is allowed to elapse.

In conclusion, the high genetic variability observed in the S gene ofOBI strains is associated with heterogeneous patterns of intracellularand extracellular HBsAg production in vitro. The identification ofthree new OBI-specific mutations—M75T, Y100S, and P178R—associated with HBsAg intracellular retention supports further thehypothesis that the lack of HBsAg secretion may significantly con-tribute to the multifactorial occurrence of OBI. M75T and P178Rmutants 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 potentialpathogenic role of intracellularly retained HBsAg in the develop-ment of liver disease in OBI carriers.

ACKNOWLEDGMENTS

S. Lin from the Taiwan Blood Services Foundation, Taipei, Taiwan, and C.Kit Lin from Hong Kong Red Cross Blood Transfusion Centre, HongKong, People’s Republic of China, are thanked for providing OBI samplesstudied.

S. Biswas was supported in part by a grant from NHSBT England andby a grant from Novartis Diagnostics & Vaccines.

REFERENCES1. Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DS, Co-

lombo M, Craxi A, Donato F, Ferrari C, Gaeta GB, Gerlich WH,Levrero M, Locarnini S, Michalak T, Mondelli MU, Pawlotsky JM,Pollicino T, Prati D, Puoti M, Samuel D, Shouval D, Smedile A,Squadrito G, Trépo C, Villa E, Will H, Zanetti AR, Zoulim F. 2008.

Statements from the Taormina expert meeting on occult hepatitis B virusinfection. J. Hepatol. 49:652– 657.

2. Raimondo G, Pollicino T, Romano L, Zanetti AR. 2010. A 2010 updateon occult hepatitis B infection. Pathol. Biol. 58:254 –257.

3. Pollicino T, Raffa G, Costantino L, Lisa A, Campello C, Squadrito G,Levrero M, Raimondo G. 2007. Molecular and functional analysis ofoccult hepatitis B virus isolates from patients with hepatocellular carci-noma. Hepatology 45:277–285.

4. Candotti D, Grabarczyk P, Ghiazza P, Roig R, Casamitjana N, IudiconeP, Schmidt M, Bird A, Crookes R, Brojer E, Miceli M, Amiri A, Li C,Allain JP. 2008. Characterization of occult hepatitis B virus from blooddonors carrying genotype A2 or genotype D strains. J. Hepatol. 49:537–547.

5. Huang CH, Yuan Q, Chen PJ, Zhang YL, Chen CR, Zheng QB, Yeh SH,Yu H, Xue Y, Chen YX, Liu PG, Ge SX, Zhang J, Xia NS. 2012. Influenceof mutations in hepatitis B virus surface protein on viral antigenicity andphenotype in occult HBV strains from blood donors. J. Hepatol. 57:720 –729.

6. El Chaar M, Candotti D, Crowther RA, Allain JP. 2010. Impact ofhepatitis B virus surface protein mutations on the diagnosis of occulthepatitis B virus infection. Hepatology 52:1600 –1610.

7. Chaudhuri V, Tayal R, Nayak B, Acharya SK, Panda SK. 2004. Occulthepatitis B virus infection in chronic liver disease: full-length genome andanalysis of mutant surface promoter. Gastroenterology 127:1356 –1371.

8. Fang Y, Teng X, Xu WZ, Li D, Zhao HW, Fu LJ, Zhang FM, Gu HX.2009. Molecular characterization and functional analysis of occult hepa-titis B virus infection in Chinese patients infected with genotype C. J. Med.Virol. 81:826 – 835.

9. Hass M, Hannoun C, Kalinina T, Sommer G, Manegold C, Günther S.2005. Functional analysis of hepatitis B virus reactivating in hepatitis Bsurface antigen-negative individuals. Hepatology 42:93–103.

10. Candotti D, Lin CK, Belkhiri D, Sakuldamrongpanich T, Biswas S, LinS, Teo D, Ayob Y, Allain JP. 2012. Occult hepatitis B virus infection inblood donors from South East Asia: molecular characterization and po-tential mechanisms of occurrence. Gut 61:1744 –1753.

11. Salisse J, Sureau C. 2009. A function essential to viral entry underlies thehepatitis B virus “a” determinant. J. Virol. 83:9321–9328.

12. Mello FC, Martel N, Gomes SA, Araujo NM. 2011. Expression of hep-atitis B virus surface antigen containing Y100C variant frequently detectedin occult HBV infection. Hepat. Res. Treat. 2011:695859. doi:10.1155/2011/695859.

13. Martin CM, Welge JA, Rouster SD, Shata MT, Sherman KE, BlackardJT. 2012. Mutations associated with occult hepatitis B virus infectionresult in decreased surface antigen expression in vitro. J. Vir. Hepat. 19:716 –723.

14. Persson B, Argos P. 1994. Prediction of transmembrane segments inproteins utilising multiple sequence alignments. J. Mol. Biol. 237:182–192.

15. Torresi J. 2002. The virological and clinical significance of mutations inthe overlapping envelope and polymerase genes of hepatitis B virus. J.Clin. Virol. 25:97–106.

16. Chua PK, Wang RY, Lin MH, Masuda T, Suk FM, Shih C. 2005.Reduced secretion of virions and hepatitis B virus (HBV) surface antigenof a naturally occurring HBV variant correlates with the accumulation ofthe small S envelope protein in the endoplasmic reticulum and Golgiapparatus. J. Virol. 79:13483–13496.

17. Khan N, Guarnieri M, Ahn SH, Jisu L, Zhou Y, Bang G, Kim KH,Wands JR, Tong S. 2004. Modulation of hepatitis B virus secretion bynaturally occurring mutations in the S gene. J. Virol. 78:3262–3270.

18. Loffler-Mary H, Dumortier J, Klentsch-Zimmer C, Prange R. 2000.Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop ofthe envelope proteins. Virology 270:358 –367.

19. Gutiérrez C, Devesa M, Loureiro CL, León G, Liprandi F, Pujol FH.2004. Molecular and serological evaluation of surface antigen negativehepatitis B virus infection in blood donors from Venezuela. J. Med. Virol.73:200 –207.

20. Araujo NM, Vianna COA, Soares CC, Gomes SA. 2008. A unique aminoacid substitution, L215Q, in the hepatitis B vírus small envelope protein ofa genotype F isolate that inhibits secretion of hepatitis B vírus subviralparticles. Intervirology 51:81– 86.

21. Motta JS, Mello FC, Lago BV, Perez RM, Gomes SA, Figueiredo FF.2010. Occult hepatitis B virus infection and lamivudine-resistant muta-

Impaired S Protein Excretion and Occult HBV Infection

July 2013 Volume 87 Number 14 jvi.asm.org 7891

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from

tions in isolates from renal patients undergoing hemodialysis. J. Gastro-enterol. Hepatol. 25:101–106.

22. Jenna S, Sureau C. 1999. Mutations in the carboxy-terminal domain ofthe small hepatitis B virus envelope protein impair the assembly of hepa-titis delta virus particles. J. Virol. 73:3351–3358.

23. Siegler VD, Bruss V. 2013. Role of transmembrane domains of the hep-atitis B virus small surface proteins in subviral particle biogenesis. J. Virol.87:1491–1496.

24. Patient R, Hourioux C, Roingeard P. 2009. Morphogenesis of hepatitis Bvirus and its subviral envelope particles. Cell Microbiol. 11:1561–1570.

25. Bonifacino JS, Cosson P, Shah N, Klausner RD. 1991. Role of po-tentially charged transmembrane residues in targeting proteins for re-tention and degradation within the endoplasmic reticulum. EMBO J.10:2783–2793.

26. Kalinina T, Riu A, Fischer L, Will H, Sterneck M. 2001. A dominanthepatitis B virus population defective in virus secretion because of several

S-gene mutations from a patient with fulminant hepatitis. Hepatology34:385–394.

27. Ito K, Qin Y, Guarnieri M, Garcia T, Kwei K, Mizokami M, Zhang J, LiJ, Wands JR, Tong S. 2010. Impairment of hepatitis B virus virion secre-tion by single-amino-acid substitutions in the small envelope protein andrescue by a novel glycosylation site. J. Virol. 84:12850 –12861.

28. Walter P, Ron D. 2011. The unfolded protein response: from stresspathway to homeostatic regulation. Science 334:1081–1086.

29. Hsieh YH, Su IJ, Wang HC, Chang WW, Lei HY, Lai MD, Chang WT,Huang W. 2004. Pre-S mutant surface antigens in chronic hepatitis Bvirus infection induce oxidative stress and DNA damage. Carcinogenesis10:2023–2032.

30. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, SellS, Pinkert CA, Brinster RL, Palmiter RD. 1989. Molecular pathogenesisof hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59:1145–1156.

Biswas et al.

7892 jvi.asm.org Journal of Virology

on Decem

ber 11, 2013 by NA

T IN

ST

OF

HE

ALT

H LIB

http://jvi.asm.org/

Dow

nloaded from