Entecavir combined with furin inhibitor simultaneously reduces hepatitis B virus replication and e antigen secretion

Virology Journal

Xiao M Peng¹  Lin Gu¹  Dong M Li²  Nai Q Zheng²  Hui Y Yang² 

[1] Liver Disease Key Laboratory of Guangdong Province, Guangzhou, China;Department of Infectious Diseases, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China

关键词: Antiviral therapy;    Furin;    Proprotein convertase;    Hepatitis B e antigen;    Viral replication;    Hepatitis B virus;   

Others  :  1148417
DOI  :  10.1186/1743-422X-11-165

 received in 2014-07-08, accepted in 2014-09-11,  发布年份 2014

【 摘 要 】

Background

The antiviral therapy of chronic hepatitis B virus (HBV) infection pursues the dual goals, virological response (undetectable serum HBV DNA) and hepatitis B e antigen (HBeAg) serological response (serum HBeAg loss/seroconversion). It is relatively difficult, however, to realize the serological response, especially for nucleotide/nucleoside analogs. Furin, a proprotein convertase, is involved in HBeAg maturation. The suppression of furin using inhibitors accordingly reduces HBeAg secretion, but possibly enhances HBV replication. For these reasons, the strategy based on the combination of nucleoside analog entecavir (ETV) and furin inhibitors to inhibit HBV replication and HBeAg secretion simultaneously were studied here.

Methods

The suppression of furin was performed using inhibitors decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine (D6R) or the expression of furin inhibitory prosegment. The influence of furin suppression on HBV replication and the effect of CMK combined with nucleoside analog entecavir (ETV) on HBV replication and HBeAg secretion was investigated in HepG2.2.15 cells. HBeAg level in media was detected using enzyme-linked immunosorbent assay. Intracellular viral antigens and HBV DNA were detected using Western and Southern blotting analyses, respectively.

Results

CMK, D6R and the expression of inhibitory prosegment all significantly reduced HBeAg secretion, but only CMK enhance HBV replication. Concordantly, only CMK post-transcriptionally accumulated cytosolic HBV replication-essential hepatitis B core antigen (HBcAg). The HBcAg-accumulating effect of CMK was further found to be resulted from its redundant inhibitory effect on the trypsin-like activity of cellular proteasomes that are responsible for HBcAg degradation. Moreover, the viral replication-enhancing effect of CMK was abrogated by ETV and ETV combined with CMK reduced HBV replication and HBeAg secretion simultaneously.

Conclusion

The suppression of furin itself does not enhance HBV replication. Nucleotide/nucleoside analogs combined with furin inhibitors may be a potential easy way to realize the dual goals of the antiviral therapy for chronic hepatitis B in the future.

【 授权许可】

   
2014 Yang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Ganem D, Prince AM: Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med 2004, 350:1118-1129.
• [2]Liaw YF: Natural history of chronic hepatitis B virus infection and long-term outcome under treatment. Liver Int 2009, 29(Suppl 1):100-107.
• [3]Chen MT, Billaud JN, Sällberg M, Guidotti LG, Chisari FV, Jones J, Hughes J, Milich DR: A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A 2004, 101:14913-149138.
• [4]Hsu YS, Chien RN, Yeh CT, Sheen IS, Chiou HY, Chu CM, Liaw YF: Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B. Hepatology 2002, 35:1522-1527.
• [5]Fattovich G, Bortolotti F, Donato F: Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol 2008, 48:335-352.
• [6]Chen YC, Chu CM, Liaw YF: Age-specific prognosis following spontaneous hepatitis B e antigen seroconversion in chronic hepatitis B. Hepatology 2010, 51:435-444.
• [7]Liaw YF, Lau GK, Kao JH, Gane E: Hepatitis B e antigen seroconversion: a critical event in chronic hepatitis B virus infection. Dig Dis Sci 2010, 55:2727-2734.
• [8]Hadziyannis SJ, Vassilopoulos D: Hepatitis B e antigen-negative chronic hepatitis B. Hepatology 2001, 34(4 Pt 1):617-624.
• [9]Zhang QQ, An X, Liu YH, Li SY, Zhong Q, Wang J, Hu HD, Zhang DZ, Ren H, Hu P: Long-term nucleos(t)ide analogues therapy for adults with chronic hepatitis B reduces the risk of long-term complications: a meta-analysis. Virol J 2011, 8:72. BioMed Central Full Text
• [10]Papatheodoridis GV, Manolakopoulos S, Touloumi G, Vourli G, Raptopoulou-Gigi M, Vafiadis-Zoumbouli I, Vasiliadis T, Mimidis K, Gogos C, Ketikoglou I, Manesis EK, HEPNET. Greece Cohort Study Group: Virological suppression does not prevent the development of hepatocellular carcinoma in HBeAg-negative chronic hepatitis B patients with cirrhosis receiving oral antiviral(s) starting with lamivudine monotherapy: results of the nationwide HEPNET. Greece cohort study. Gut 2011, 60:1109-1116.
• [11]Messageot F, Salhi S, Eon P, Rossignol JM: Proteolytic processing of the hepatitis B virus e antigen precursor. Cleavage at two furin consensus sequences. J Biol Chem 2003, 278:891-895.
• [12]Ito K, Kim KH, Lok AS, Tong S: Characterization of genotype-specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme. J Virol 2009, 83:3507-3517.
• [13]Thomas G: Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 2002, 3:753-766.
• [14]Lei RX, Shi H, Peng XM, Zhu YH, Cheng J, Chen GH: Influence of a single nucleotide polymorphism in the P1 promoter of the furin gene on transcription activity and hepatitis B virus infection. Hepatology 2009, 50:763-771.
• [15]Pang YJ, Tan XJ, Li DM, Zheng ZH, Lei RX, Peng XM: Therapeutic potential of furin inhibitors for the chronic infection of hepatitis B virus. Liver Int 2013, 33:1230-1238.
• [16]Sugiyama M, Tanaka Y, Kurbanov F, Nakayama N, Mochida S, Mizokami M: Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene. Virology 2007, 365:285-291.
• [17]López de Cicco R, Bassi DE, Zucker S, Seidah NG, Klein-Szanto AJ: Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin. Cancer Res 2005, 65:4162-4171.
• [18]Chou YC, Chen ML, Hu CP, Chen YL, Chong CL, Tsai YL, Liu TL, Jeng KS, Chang C: Transforming growth factor-beta1 suppresses hepatitis B virus replication primarily through transcriptional inhibition of pregenomic RNA. Hepatology 2007, 46(3):672-681.
• [19]Sohn SY, Kim SB, Kim J, Ahn BY: Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ proteins through destabilization of viral core and X proteins. J Gen Virol 2006, 87:1883-1891.
• [20]Duriez M, Rossignol JM, Sitterlin D: The hepatitis B virus precore protein is retrotransported from endoplasmic reticulum (ER) to cytosol through the ER-associated degradation pathway. J Biol Chem 2008, 283:32352-32360.
• [21]Braun S, Zajakina A, Aleksejeva J, Sharipo A, Bruvere R, Ose V, Pumpens P, Garoff H, Meisel H, Kozlovska T: Proteasomal degradation of core protein variants from chronic hepatitis B patients. J Med Virol 2007, 79:1312-1321.
• [22]Seifer M, Standring DN: A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface. J Virol 1994, 68:5548-5555.
• [23]Li Y, Yu Z, Zhao X, Shen SH: Identification and characterization of hepsin/-TM, a non-transmembrane hepsin isoform. Biochim Biophys Acta 2005, 1681:157-165.
• [24]Cameron A, Appel J, Houghten RA, Lindberg I: Polyarginines are potent furin inhibitors. J Biol Chem 2000, 275:36741-36749.
• [25]Wong GL, Chan HL, Mak CW, Lee SK, Ip ZM, Lam AT, Iu HW, Leung JM, Lai JW, Lo AO, Chan HY, Wong VW: Entecavir treatment reduces hepatic events and deaths in chronic hepatitis B patients With liver cirrhosis. Hepatology 2013, 58:1537-1547.
• [26]Chang TT, Liaw YF, Wu SS, Schiff E, Han KH, Lai CL, Safadi R, Lee SS, Halota W, Goodman Z, Chi YC, Zhang H, Hindes R, Iloeje U, Beebe S, Kreter B: Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B. Hepatology 2010, 52:886-893.
• [27]Lin B, Ha NB, Liu A, Trinh HN, Nguyen HA, Nguyen KK, Ahmed A, Keeffe EB, Garcia RT, Garcia G, Nguyen MH: Low incidence of hepatitis B e antigen seroconversion in patients treated with oral nucleos(t)ides in routine practice. J Gastroenterol Hepatol 2013, 28:855-860.
• [28]Ono A, Suzuki F, Kawamura Y, Sezaki H, Hosaka T, Akuta N, Kobayashi M, Suzuki Y, Saitou S, Arase Y, Ikeda K, Kobayashi M, Watahiki S, Mineta R, Kumada H: Long-term continuous entecavir therapy in nucleos(t)ide-naïve chronic hepatitis B patients. J Hepatol 2012, 57:508-514.
• [29]Shiryaev SA, Remacle AG, Ratnikov BI, Nelson NA, Savinov AY, Wei G, Bottini M, Rega MF, Parent A, Desjardins R, Fugere M, Day R, Sabet M, Pellecchia M, Liddington RC, Smith JW, Mustelin T, Guiney DG, Lebl M, Strongin AY: Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens. J Biol Chem 2007, 282:20847-20853.
• [30]Becker GL, Lu Y, Hardes K, Strehlow B, Levesque C, Lindberg I, Sandvig K, Bakowsky U, Day R, Garten W, Steinmetzer T: Highly potent inhibitors of the proprotein convertase furin as potential drugs for the treatment of infectious diseases. J Biol Chem 2012, 287:21992-22003.
• [31]Sarac MS, Peinado JR, Leppla SH, Lindberg I: Protection against anthrax toxemia by hexa-D-arginine in vitro and in vivo. Infect Immun 2004, 72:602-605.
• [32]Sarac MS, Cameron A, Lindberg I: The furin inhibitor hexa-D-arginine blocks the activation of Pseudomonas aeruginosa exotoxin A in vivo. Infect Immun 2002, 70:7136-7139.
• [33]Wu JF, Hsu HY, Ni YH, Chen HL, Wu TC, Chang MH: Suppression of furin by interferon-γ and the impact on hepatitis B virus antigen biosynthesis in human hepatocytes. Am J Pathol 2012, 181:19-25.
• [34]Biermer M, Puro R, Schneider RJ: Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid integrity through activation of NF-kappaB. J Virol 2003, 77:4033-4042.
• [35]Moravec RA, O’Brien MA, Daily WJ, Scurria MA, Bernad L, Riss TL: Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format. Anal Biochem 2009, 387:294-302.