Arch Virol DOI 10.1007/s00705-014-2111-6
BRIEF REPORT
A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein Lili Wei • Zhongliang Shen • Xue Zhao Yanxin Wu • Wei Liu • Junqi Zhang • Youhua Xie • Jing Liu
•
Received: 23 January 2014 / Accepted: 30 April 2014 Ó Springer-Verlag Wien 2014
Abstract A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions. Keywords
HBV Genotype HBx Protein expression
Hepatitis B virus (HBV) is the type member of the family Hepadnaviridae [6, 13]. The *3.2 kb partially doublestrand DNA genome contains four overlapping open reading frames that encode the viral structural and nonstructural proteins, including capsid core protein (HBcAg)
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2111-6) contains supplementary material, which is available to authorized users. L. Wei Z. Shen X. Zhao Y. Wu W. Liu J. Zhang Y. Xie (&) J. Liu (&) Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, People’s Republic of China e-mail: [email protected] J. Liu e-mail: [email protected]
and secreted e antigen (HBeAg), three forms of envelope proteins (large, middle and small HBsAg), polymerase and X protein (HBx) [13]. Based on sequence variations, eight genotypes of HBV (A-H) have been identified so far, some of which include multiple subgenotypes [6, 12]. Infection with hepatitis B virus can cause acute or chronic hepatitis in humans, and chronic hepatitis B may progress onto liver cirrhosis and hepatocellular carcinoma (HCC) [6]. Possible associations between certain HBV genotypes and clinical outcomes, including drug resistance, responses to treatment and development of HCC, have been suggested [10, 11, 18]. HBx has been subjected to extensive study over the years and appears to be involved in both establishment and progression of the viral life cycle upon infection [7, 16] as well as modulation of a myriad of host cellular functions [8]. Notably, a large amount of published work has linked HBx expression to the development of HCC associated with HBV infection [2]. However, most in vitro studies of HBx functions have used transiently or stably overexpressed HBx without correlating the data with the actual expression level of HBx in virus infection and replication. Consequently, apparently contradicting results are sometimes obtained by different laboratories [2]. Possible differences between HBx encoded by different genotypes of HBV might also play a part, but this has not been addressed in detail either. The lack of availability of sensitive antibodies with demonstrable reactivity against the various variants of the HBx has hindered research efforts along these lines. In an attempt to establish a sensitive method for detecting multiple genotypes of HBx protein, we prepared anti-HBx monoclonal antibodies (mAbs) by immunizing mice with purified bacterially expressed HBx encoded by a clinically isolated genotype B strain designated as 56
123
L. Wei et al. Fig. 1 Analysis of the HBx proteins of eight currently known genotypes of HBV. (A) GenBank accession numbers of the nucleotide and protein sequences of genotypes A-H HBx used for genotype reactivity tests and the genotype B HBx used for generating the monoclonal antibodies are listed. The HBx protein length in amino acids, predicted molecular weight and protein sequence identity to immunogen genotype B HBx are also listed. (B) Phylogenetic analysis of HBx protein sequences listed in A. A neighbor-joining tree was calculated using MEGA 5.1.2 (http://www.megasoftware.net) with default parameters. *, protein sequence of immunogen HBx used for bacterial expression and immunization of mice
A
B
AF100309.1
HBx Protein Accession Number AAD16255.1*
A B C D E F G H
EU859939.1 GU815641.1 D23682.1 FJ349233.1 GQ161777.1 DQ629966.1 GU565217.1 AB375162.1
ACJ66131.1 ADF06233.1 BAA04925.1 ACO89809.1 ACU24949.1 ABG23432.1 ADD62621.1 BAG50265.1
Genotype
Nucleotide Accession Number
B
HBx MW Identity with HBx (kD) Immunogen Length HBx (a.a.) 154
16.61
100%
154 154 154 154 154 154 154 154
16.69 16.63 16.71 16.8 16.64 16.7 16.86 16.85
86.83% 98.03% 85.34% 86.83% 86.09% 82.29% 74.22% 83.06%
HBx genotype C (BAA04925.1) HBx genotype E (ACU24949.1) HBx genotype D (ACO89809.1) HBx genotype A (ACJ66131.1) HBx genotype B (ADF06233.1) HBx genotype B* (AAD16255.1) HBx genotype F (ABG23432.1) HBx genotype H (BAG50265.1) HBx genotype G (ADD62621.1) 0.02
(GenBank accession no. AF100309.1). Four clones of hybridoma cells were obtained that secreted immunogenspecific antibodies as detected by ELISA and western blot (data not shown). To identify the mAbs with the broadest reactivity against different genotypes of HBx, HBxencoding sequences of HBV genotypes A-H were retrieved from GenBank (Fig. 1A), and the corresponding DNA was chemically synthesized and cloned downstream of a FLAG tag in the pcDNA3 vector (Invitrogen). Immunoblotting (IB) of lysates of transiently transfected HEK293T cells using anti-FLAG mAb showed that FLAG-tagged HBx of all eight genotypes was successfully expressed (Fig. 2A, top). The four anti-HBx mAbs that were obtained were then used to detect FLAG-tagged HBx overexpressed in HEK293T cells, and one mAb, designated 2A7, demonstrated the broadest reactivity and reacted with 7 of the 8 genotypes of HBx, with the exception being genotype G (Fig. 2A, bottom, Supplementary Figure S1A). None of the other three mAbs reacted with genotype G HBx either (data not shown). In comparison, a commercial monoclonal antiHBx mAb reacted with HBx of only five of the eight genotypes, and with two of the five very weakly (Supplementary Figure 1B). Similar broad reactivity of 2A7 was observed when it was used for immunofluorescence (IF) detection of FLAG-tagged HBx overexpressed in HEK293T cells: with the exception of genotype G, HBx encoded by the remaining seven genotypes was
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successfully detected by 2A7 (Fig. 2B). HBx-specific signals were located in both the nucleus and the cytoplasm, but predominantly in the cytoplasm, regardless of the genotype (Fig. 2B). These results demonstrated that antiHBx mAb 2A7 recognizes a linear epitope that is also structurally accessible in the native HBx protein and conserved in a majority of genotypes. Genotype G is relatively rare compared to other genotypes of HBV and apparently often co-exists in patients with other genotypes such as genotype A [5, 9]. Genotype G carries characteristic mutations, including an insertion in the HBeAg/HBcAg-encoding ORF [1, 5, 15]. Analysis of HBx protein sequences of genotypes A-H used in this work showed that HBx of all eight genotypes are of the same length and have an almost identical molecular weight of *17 kDa. Genotype B HBx shares the highest sequence identity (98.03 %) with the genotype B immunogen HBx, whereas HBx of genotypes A, C-F, and H share 82.2986.83 % identity with immunogen HBx (Fig. 1A). Genotype G HBx, however, shares a noticeably lower similarity (74.22 %) with immunogen HBx (Fig. 1A). Phylogenetic analysis of these HBx protein sequences also showed that genotype G HBx is grouped separately from all other genotypes of HBx, indicating a marked divergence between HBx of genotype G and HBx of other genotypes (Fig. 1B). The fact that anti-HBx mAb 2A7 reacts with all common genotypes of HBx except genotype G suggests that certain
Broadly reactive mAb against HBx
FLAG-tagged HBx
A pc
A
B
C
D
E
F
G
H
anti-FLAG anti-HBx 2A7
DAPI
B
anti-HBx 2A7
Merge
DAPI
pc
E
A
F
B
G
C
H
anti-HBx 2A7
Merge
D
Fig. 2 Monoclonal antibody 2A7 is reactive against seven of eight currently known genotypes of HBx. (A) FLAG-tagged HBx encoded by eight genotypes of HBV were expressed in HEK293T cells by transient transfection and analyzed by immunoblotting using monoclonal anti-FLAG (top) and anti-HBx antibody 2A7 (bottom). The genotypes used are indicated at the top. pc, pcDNA3 vector control transfection. Full images of these blots are included in
Supplenmentary Figure S1A. (B) FLAG-tagged HBx encoded by eight genotypes of HBV were expressed in HEK293T cells by transient transfection and analyzed by immunofluorescence using monoclonal anti-HBx antibody 2A7. Nuclei were stained using DAPI. The genotypes used are indicated at the left. pc, pcDNA3 vector control transfection. Scale bar, 20 lm
amino acid variations in genotype G HBx eliminate the 2A7-reactive epitope that is conserved in all other genotypes of HBx. Whether such variations might confer any functional differences upon genotype G HBx compared to other genotypes is an interesting question that warrants further study. A genotype-G-specific monoclonal antibody in combination with mAb 2A7 would also make it possible to detect HBx encoded by all known genotypes of HBV. Since detection of overexpressed FLAG-HBx demonstrated the specificity and broad reactivity of anti-HBx mAb 2A7 (Fig. 2), we went on to test whether this mAb was capable of detecting untagged HBx expressed from HBV genomes in transiently or stably transfected cultured hepatocytes. Huh-7 cells were transfected with a plasmid carrying 1.2 copies of the terminally redundant genotype C HBV genome (pWT) [17], and in IB, mAb 2A7 detected
low-level HBx expression in transfected cells (Fig. 3A). Treatment with the proteasome inhibitor MG-132 for 6 hours prior to harvesting cells resulted in a slight increase in HBx signal strength, suggesting that degradation of HBx partially involves proteasome-mediated mechanisms, which is consistent with a previous report [3]. Densitometry scanning of the HBx signals against co-transfected EGFP controls showed that the amount of expression HBx from the 1.2-copy HBV genome plasmid was only about 10 % of that from a similar amount of CMV-promoterdriven overexpression plasmid carrying identical HBx coding sequences (Fig. 3A). In IF, HBx signals were observed in cells transfected with HBx overexpression plasmid, but not in cells transfected with the 1.2-copy HBV genome plasmid, even with MG-132 treatment (Fig. 3B), confirming the low level of HBx expression in the latter.
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A
D
B
E
C
Fig. 3 Detection of HBx expression from transiently or stably transfected HBV genomes in vitro and in vivo. (A) Huh-7 cells were transiently transfected with the indicated plasmids, and HBx expression was analyzed by immunoblotting using monoclonal antibody 2A7. pWT contained 1.2 copies of the terminally redundant HBV genome, and pWT-backbone is pWT with HBV sequences deleted. HBx encoded by pWT was amplified by PCR to create overexpression plasmid pcDNA3/HBx(pWT) as a reactivity positive control. MG-132 was added to culture medium of the indicated transfections 6 hours prior to cell harvesting. Harvested cells were lysed in SDS-PAGE loading buffer, and equal volumes were loaded, except the pcDNA3/ HBx(pWT) sample, which was diluted 1:10 before loading. Densitometry scanning was performed using EGFP expressed by cotransfection as a control. (B) Huh-7 cells were transiently transfected with the indicated plasmids and HBx expression was analyzed by
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immunofluorescence using monoclonal antibody 2A7 in. (C) Immunohistochemistry detection of HBx expression in mouse liver from hydrodynamically injected HBV genome plasmids using monoclonal antibody 2A7. BALB/c mice were injected through the tail vein with pWT or pWT(1.3) in 2 ml PBS and sacrificed at 3 days post-injection. Liver sections were prepared and analyzed following a standard protocol and stained using, anti-HBcAg (DAKO) and anti-HBx 2A7 antibodies or mock treated. (D and E) HBx expression in HepG2.2.15 (2.2.15) and HepAD38 (AD38) cells with or without MG-132 treatment as indicated was analyzed by immunoblotting (D) and immunofluorescence (E) using monoclonal antibody 2A7. HepAD38 was cultured in the absence of tetracycline repression. HBx coding sequences were amplified from HepG2.2.15 cells by PCR to create pcDNA3/HBx(2.2.15) as a positive control for reactivity. Scale bar, 20 lm
Broadly reactive mAb against HBx
Transient transfection in vivo with HBV genome plasmid was then performed using a tail vein hydrodynamic injection protocol. In immunohistochemistry (IHC), mAb 2A7 detected rather low HBx expression in the liver of a pWTinjected mouse, but marked expression of HBx was detected using 2A7 in liver sections from a mouse injected with pWT(1.3), a modified pWT carrying 1.3 copies of the terminally redundant HBV genome (Fig. 3C). In addition, HBx was also detected by 2A7 in three out of four HBVpositive HCC tumor samples in IHC (Supplementary Figure S1C). These results demonstrated the usability of 2A7 mAb for detecting HBx expression in vivo. HepG2.2.15 [14] and HepAD38 [4] are two HepG2derived cell lines that were stably transfected with HBV genomes and have been widely used for the study of the HBV life cycle as well as the screening of anti-HBV drugs. Both of these cell lines have terminally redundant HBV genomes, derived from the same genotype D isolate, integrated into cellular chromosomes, and in HepAD38, HBV pregenomic RNA transcription from integrated HBV sequences is also placed under the control of an inducible Tet-off promoter. We attempted to detect, using mAb 2A7, HBx expression in these cell lines, where every single cell is supposed to express HBV proteins, including HBx. As shown in Fig. 3D, E, although 2A7 was able to detect, in both IB and IF, the overexpressed genotype D HBx amplified from HepG2.2.15 cells, endogenous HBx expression was not detected in either cell line in IB or IF, even with MG-132 treatment. These results suggested that HBx expression in these stably transfected cell lines is considerably lower than in transiently transfected cells. Research over the past decades has associated HBx with numerous functions, some of which apparently contradict each other. Consequently, although the importance of HBx for HBV both as a virus and as a tumor-inducing agent is generally accepted, controversies and uncertainties exist as to what roles HBx actually plays in the viral life cycle and how much it really contributes towards virus-host interactions such as HBV-related HCC development. In this work, we demonstrated, using a sensitive and broadly reacting anti-HBx mAb 2A7, that there are marked differences in HBx expression levels between the commonly used transient HBx overexpression transfection, transient HBV transfection and stable HBV transfection systems (Fig. 3). These results demonstrate the need for extreme caution when functional data obtained using HBx from different systems are considered and compared. We hope that HBx antibodies like 2A7 will become powerful tools that enable more-stringent and quantitative studies of HBx functions both in vitro and in vivo. Acknowledgments This work was supported by NSFC (31071143, 31170148), the National Science and Technology Major Project for
Infectious Diseases (2012ZX10002-006, 2012ZX10004-503 and 2013ZX10002-001), ‘‘973’’ program (2012CB519002), Shanghai Science and Technology Committee (11DZ2291900).
References 1. Cotelesage JJ, Osiowy C, Lawrence C, DeVarennes SL, Teow S, Beniac DR, Booth TF (2011) Hepatitis B virus genotype G forms core-like particles with unique structural properties. J Viral Hepat 18:443–448 2. Guerrieri F, Belloni L, Pediconi N, Levrero M (2013) Molecular mechanisms of HBV-associated hepatocarcinogenesis. Semin Liver Dis 33:147–156 3. Hu Z, Zhang Z, Doo E, Coux O, Goldberg AL, Liang TJ (1999) Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome complex. J Virol 73:7231–7240 4. Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT, Seeger C, King RW (1997) Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41:1715–1720 5. Li K, Zoulim F, Pichoud C, Kwei K, Villet S, Wands J, Li J, Tong S (2007) Critical role of the 36-nucleotide insertion in hepatitis B virus genotype G in core protein expression, genome replication, and virion secretion. J Virol 81:9202–9215 6. Liang TJ (2009) Hepatitis B: the virus and disease. Hepatology 49:S13–S21 7. Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, Zoulim F, Hantz O, Protzer U (2011) Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol 55:996–1003 8. Murakami S (2001) Hepatitis B virus X protein: a multifunctional viral regulator. J Gastroenterol 36:651–660 9. Osiowy C, Gordon D, Borlang J, Giles E, Villeneuve JP (2008) Hepatitis B virus genotype G epidemiology and co-infection with genotype A in Canada. J Gen Virol 89:3009–3015 10. Pujol FH, Navas MC, Hainaut P, Chemin I (2009) Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett 286:80–88 11. Schaefer S (2005) Hepatitis B virus: significance of genotypes. J Viral Hepat 12:111–124 12. Schaefer S (2007) Hepatitis B virus taxonomy and hepatitis B virus genotypes. World J Gastroenterol 13:14–21 13. Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64:51–68 14. Sells MA, Chen ML, Acs G (1987) Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci USA 84:1005–1009 15. Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R (2000) A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol 81:67–74 16. Tsuge M, Hiraga N, Akiyama R, Tanaka S, Matsushita M, Mitsui F, Abe H, Kitamura S, Hatakeyama T, Kimura T, Miki D, Mori N, Imamura M, Takahashi S, Hayes CN, Chayama K (2010) HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice. J Gen Virol 91:1854–1864 17. Yu X, Mertz JE (2003) Distinct modes of regulation of transcription of hepatitis B virus by the nuclear receptors HNF4alpha and COUP-TF1. J Virol 77:2489–2499 18. Zoulim F, Locarnini S (2009) Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137(1593–1608): e1591–e1592
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BRIEF REPORT
A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein Lili Wei • Zhongliang Shen • Xue Zhao Yanxin Wu • Wei Liu • Junqi Zhang • Youhua Xie • Jing Liu
•
Received: 23 January 2014 / Accepted: 30 April 2014 Ó Springer-Verlag Wien 2014
Abstract A highly specific and broadly reactive monoclonal antibody against hepatitis B virus X (HBx) protein was developed that detected, in both immunoblotting and immunofluorescence, HBx proteins of seven of the eight currently known genotypes of HBV, which were overexpressed in cultured cells. Evaluation of HBx expression levels in cultured hepatocytes using this monoclonal antibody showed that cells transiently and stably transfected with HBV genomes expressed far less HBx protein than cells transiently transfected with an HBx overexpression plasmid routinely used for studying HBx functions. The availability of such sensitive and broadly reactive monoclonal antibodies against HBx will enable more-quantitative studies of HBx functions. Keywords
HBV Genotype HBx Protein expression
Hepatitis B virus (HBV) is the type member of the family Hepadnaviridae [6, 13]. The *3.2 kb partially doublestrand DNA genome contains four overlapping open reading frames that encode the viral structural and nonstructural proteins, including capsid core protein (HBcAg)
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2111-6) contains supplementary material, which is available to authorized users. L. Wei Z. Shen X. Zhao Y. Wu W. Liu J. Zhang Y. Xie (&) J. Liu (&) Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, People’s Republic of China e-mail: [email protected] J. Liu e-mail: [email protected]
and secreted e antigen (HBeAg), three forms of envelope proteins (large, middle and small HBsAg), polymerase and X protein (HBx) [13]. Based on sequence variations, eight genotypes of HBV (A-H) have been identified so far, some of which include multiple subgenotypes [6, 12]. Infection with hepatitis B virus can cause acute or chronic hepatitis in humans, and chronic hepatitis B may progress onto liver cirrhosis and hepatocellular carcinoma (HCC) [6]. Possible associations between certain HBV genotypes and clinical outcomes, including drug resistance, responses to treatment and development of HCC, have been suggested [10, 11, 18]. HBx has been subjected to extensive study over the years and appears to be involved in both establishment and progression of the viral life cycle upon infection [7, 16] as well as modulation of a myriad of host cellular functions [8]. Notably, a large amount of published work has linked HBx expression to the development of HCC associated with HBV infection [2]. However, most in vitro studies of HBx functions have used transiently or stably overexpressed HBx without correlating the data with the actual expression level of HBx in virus infection and replication. Consequently, apparently contradicting results are sometimes obtained by different laboratories [2]. Possible differences between HBx encoded by different genotypes of HBV might also play a part, but this has not been addressed in detail either. The lack of availability of sensitive antibodies with demonstrable reactivity against the various variants of the HBx has hindered research efforts along these lines. In an attempt to establish a sensitive method for detecting multiple genotypes of HBx protein, we prepared anti-HBx monoclonal antibodies (mAbs) by immunizing mice with purified bacterially expressed HBx encoded by a clinically isolated genotype B strain designated as 56
123
L. Wei et al. Fig. 1 Analysis of the HBx proteins of eight currently known genotypes of HBV. (A) GenBank accession numbers of the nucleotide and protein sequences of genotypes A-H HBx used for genotype reactivity tests and the genotype B HBx used for generating the monoclonal antibodies are listed. The HBx protein length in amino acids, predicted molecular weight and protein sequence identity to immunogen genotype B HBx are also listed. (B) Phylogenetic analysis of HBx protein sequences listed in A. A neighbor-joining tree was calculated using MEGA 5.1.2 (http://www.megasoftware.net) with default parameters. *, protein sequence of immunogen HBx used for bacterial expression and immunization of mice
A
B
AF100309.1
HBx Protein Accession Number AAD16255.1*
A B C D E F G H
EU859939.1 GU815641.1 D23682.1 FJ349233.1 GQ161777.1 DQ629966.1 GU565217.1 AB375162.1
ACJ66131.1 ADF06233.1 BAA04925.1 ACO89809.1 ACU24949.1 ABG23432.1 ADD62621.1 BAG50265.1
Genotype
Nucleotide Accession Number
B
HBx MW Identity with HBx (kD) Immunogen Length HBx (a.a.) 154
16.61
100%
154 154 154 154 154 154 154 154
16.69 16.63 16.71 16.8 16.64 16.7 16.86 16.85
86.83% 98.03% 85.34% 86.83% 86.09% 82.29% 74.22% 83.06%
HBx genotype C (BAA04925.1) HBx genotype E (ACU24949.1) HBx genotype D (ACO89809.1) HBx genotype A (ACJ66131.1) HBx genotype B (ADF06233.1) HBx genotype B* (AAD16255.1) HBx genotype F (ABG23432.1) HBx genotype H (BAG50265.1) HBx genotype G (ADD62621.1) 0.02
(GenBank accession no. AF100309.1). Four clones of hybridoma cells were obtained that secreted immunogenspecific antibodies as detected by ELISA and western blot (data not shown). To identify the mAbs with the broadest reactivity against different genotypes of HBx, HBxencoding sequences of HBV genotypes A-H were retrieved from GenBank (Fig. 1A), and the corresponding DNA was chemically synthesized and cloned downstream of a FLAG tag in the pcDNA3 vector (Invitrogen). Immunoblotting (IB) of lysates of transiently transfected HEK293T cells using anti-FLAG mAb showed that FLAG-tagged HBx of all eight genotypes was successfully expressed (Fig. 2A, top). The four anti-HBx mAbs that were obtained were then used to detect FLAG-tagged HBx overexpressed in HEK293T cells, and one mAb, designated 2A7, demonstrated the broadest reactivity and reacted with 7 of the 8 genotypes of HBx, with the exception being genotype G (Fig. 2A, bottom, Supplementary Figure S1A). None of the other three mAbs reacted with genotype G HBx either (data not shown). In comparison, a commercial monoclonal antiHBx mAb reacted with HBx of only five of the eight genotypes, and with two of the five very weakly (Supplementary Figure 1B). Similar broad reactivity of 2A7 was observed when it was used for immunofluorescence (IF) detection of FLAG-tagged HBx overexpressed in HEK293T cells: with the exception of genotype G, HBx encoded by the remaining seven genotypes was
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successfully detected by 2A7 (Fig. 2B). HBx-specific signals were located in both the nucleus and the cytoplasm, but predominantly in the cytoplasm, regardless of the genotype (Fig. 2B). These results demonstrated that antiHBx mAb 2A7 recognizes a linear epitope that is also structurally accessible in the native HBx protein and conserved in a majority of genotypes. Genotype G is relatively rare compared to other genotypes of HBV and apparently often co-exists in patients with other genotypes such as genotype A [5, 9]. Genotype G carries characteristic mutations, including an insertion in the HBeAg/HBcAg-encoding ORF [1, 5, 15]. Analysis of HBx protein sequences of genotypes A-H used in this work showed that HBx of all eight genotypes are of the same length and have an almost identical molecular weight of *17 kDa. Genotype B HBx shares the highest sequence identity (98.03 %) with the genotype B immunogen HBx, whereas HBx of genotypes A, C-F, and H share 82.2986.83 % identity with immunogen HBx (Fig. 1A). Genotype G HBx, however, shares a noticeably lower similarity (74.22 %) with immunogen HBx (Fig. 1A). Phylogenetic analysis of these HBx protein sequences also showed that genotype G HBx is grouped separately from all other genotypes of HBx, indicating a marked divergence between HBx of genotype G and HBx of other genotypes (Fig. 1B). The fact that anti-HBx mAb 2A7 reacts with all common genotypes of HBx except genotype G suggests that certain
Broadly reactive mAb against HBx
FLAG-tagged HBx
A pc
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Fig. 2 Monoclonal antibody 2A7 is reactive against seven of eight currently known genotypes of HBx. (A) FLAG-tagged HBx encoded by eight genotypes of HBV were expressed in HEK293T cells by transient transfection and analyzed by immunoblotting using monoclonal anti-FLAG (top) and anti-HBx antibody 2A7 (bottom). The genotypes used are indicated at the top. pc, pcDNA3 vector control transfection. Full images of these blots are included in
Supplenmentary Figure S1A. (B) FLAG-tagged HBx encoded by eight genotypes of HBV were expressed in HEK293T cells by transient transfection and analyzed by immunofluorescence using monoclonal anti-HBx antibody 2A7. Nuclei were stained using DAPI. The genotypes used are indicated at the left. pc, pcDNA3 vector control transfection. Scale bar, 20 lm
amino acid variations in genotype G HBx eliminate the 2A7-reactive epitope that is conserved in all other genotypes of HBx. Whether such variations might confer any functional differences upon genotype G HBx compared to other genotypes is an interesting question that warrants further study. A genotype-G-specific monoclonal antibody in combination with mAb 2A7 would also make it possible to detect HBx encoded by all known genotypes of HBV. Since detection of overexpressed FLAG-HBx demonstrated the specificity and broad reactivity of anti-HBx mAb 2A7 (Fig. 2), we went on to test whether this mAb was capable of detecting untagged HBx expressed from HBV genomes in transiently or stably transfected cultured hepatocytes. Huh-7 cells were transfected with a plasmid carrying 1.2 copies of the terminally redundant genotype C HBV genome (pWT) [17], and in IB, mAb 2A7 detected
low-level HBx expression in transfected cells (Fig. 3A). Treatment with the proteasome inhibitor MG-132 for 6 hours prior to harvesting cells resulted in a slight increase in HBx signal strength, suggesting that degradation of HBx partially involves proteasome-mediated mechanisms, which is consistent with a previous report [3]. Densitometry scanning of the HBx signals against co-transfected EGFP controls showed that the amount of expression HBx from the 1.2-copy HBV genome plasmid was only about 10 % of that from a similar amount of CMV-promoterdriven overexpression plasmid carrying identical HBx coding sequences (Fig. 3A). In IF, HBx signals were observed in cells transfected with HBx overexpression plasmid, but not in cells transfected with the 1.2-copy HBV genome plasmid, even with MG-132 treatment (Fig. 3B), confirming the low level of HBx expression in the latter.
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L. Wei et al.
A
D
B
E
C
Fig. 3 Detection of HBx expression from transiently or stably transfected HBV genomes in vitro and in vivo. (A) Huh-7 cells were transiently transfected with the indicated plasmids, and HBx expression was analyzed by immunoblotting using monoclonal antibody 2A7. pWT contained 1.2 copies of the terminally redundant HBV genome, and pWT-backbone is pWT with HBV sequences deleted. HBx encoded by pWT was amplified by PCR to create overexpression plasmid pcDNA3/HBx(pWT) as a reactivity positive control. MG-132 was added to culture medium of the indicated transfections 6 hours prior to cell harvesting. Harvested cells were lysed in SDS-PAGE loading buffer, and equal volumes were loaded, except the pcDNA3/ HBx(pWT) sample, which was diluted 1:10 before loading. Densitometry scanning was performed using EGFP expressed by cotransfection as a control. (B) Huh-7 cells were transiently transfected with the indicated plasmids and HBx expression was analyzed by
123
immunofluorescence using monoclonal antibody 2A7 in. (C) Immunohistochemistry detection of HBx expression in mouse liver from hydrodynamically injected HBV genome plasmids using monoclonal antibody 2A7. BALB/c mice were injected through the tail vein with pWT or pWT(1.3) in 2 ml PBS and sacrificed at 3 days post-injection. Liver sections were prepared and analyzed following a standard protocol and stained using, anti-HBcAg (DAKO) and anti-HBx 2A7 antibodies or mock treated. (D and E) HBx expression in HepG2.2.15 (2.2.15) and HepAD38 (AD38) cells with or without MG-132 treatment as indicated was analyzed by immunoblotting (D) and immunofluorescence (E) using monoclonal antibody 2A7. HepAD38 was cultured in the absence of tetracycline repression. HBx coding sequences were amplified from HepG2.2.15 cells by PCR to create pcDNA3/HBx(2.2.15) as a positive control for reactivity. Scale bar, 20 lm
Broadly reactive mAb against HBx
Transient transfection in vivo with HBV genome plasmid was then performed using a tail vein hydrodynamic injection protocol. In immunohistochemistry (IHC), mAb 2A7 detected rather low HBx expression in the liver of a pWTinjected mouse, but marked expression of HBx was detected using 2A7 in liver sections from a mouse injected with pWT(1.3), a modified pWT carrying 1.3 copies of the terminally redundant HBV genome (Fig. 3C). In addition, HBx was also detected by 2A7 in three out of four HBVpositive HCC tumor samples in IHC (Supplementary Figure S1C). These results demonstrated the usability of 2A7 mAb for detecting HBx expression in vivo. HepG2.2.15 [14] and HepAD38 [4] are two HepG2derived cell lines that were stably transfected with HBV genomes and have been widely used for the study of the HBV life cycle as well as the screening of anti-HBV drugs. Both of these cell lines have terminally redundant HBV genomes, derived from the same genotype D isolate, integrated into cellular chromosomes, and in HepAD38, HBV pregenomic RNA transcription from integrated HBV sequences is also placed under the control of an inducible Tet-off promoter. We attempted to detect, using mAb 2A7, HBx expression in these cell lines, where every single cell is supposed to express HBV proteins, including HBx. As shown in Fig. 3D, E, although 2A7 was able to detect, in both IB and IF, the overexpressed genotype D HBx amplified from HepG2.2.15 cells, endogenous HBx expression was not detected in either cell line in IB or IF, even with MG-132 treatment. These results suggested that HBx expression in these stably transfected cell lines is considerably lower than in transiently transfected cells. Research over the past decades has associated HBx with numerous functions, some of which apparently contradict each other. Consequently, although the importance of HBx for HBV both as a virus and as a tumor-inducing agent is generally accepted, controversies and uncertainties exist as to what roles HBx actually plays in the viral life cycle and how much it really contributes towards virus-host interactions such as HBV-related HCC development. In this work, we demonstrated, using a sensitive and broadly reacting anti-HBx mAb 2A7, that there are marked differences in HBx expression levels between the commonly used transient HBx overexpression transfection, transient HBV transfection and stable HBV transfection systems (Fig. 3). These results demonstrate the need for extreme caution when functional data obtained using HBx from different systems are considered and compared. We hope that HBx antibodies like 2A7 will become powerful tools that enable more-stringent and quantitative studies of HBx functions both in vitro and in vivo. Acknowledgments This work was supported by NSFC (31071143, 31170148), the National Science and Technology Major Project for
Infectious Diseases (2012ZX10002-006, 2012ZX10004-503 and 2013ZX10002-001), ‘‘973’’ program (2012CB519002), Shanghai Science and Technology Committee (11DZ2291900).
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