Inhibition of HBV replication by VPS4B and its dominant negative mutant VPS4B-K180Q in vivo

Abstract

This study examined the anti-hepatitis B virus (HBV) effect of wild-type (WT) vacuolar protein sorting 4B (VPS4B) and its dominant negative (DN) mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence (ECL). HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.

Keywords

hepatitis B virus / vacuolar protein sorting / AAA ATPase

References

[1] TeH.S., JensenD.M.. Epidemiology of hepatitis B and C viruses: a global overview. Clin Liver Dis, 2010, 14(1): 1-21

[2] CavenaughJ.S., AwiD., MendyM., et al.. Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection. PLoS One, 2011, 15(2): e14626

[3] MichelM.L., DengQ.. Mancini-Bourgine M. Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges. J Hepatol, 2011, 54(6): 1286-1296

[4] BhattacharyaD., ThioC.L.. Review of hepatitis B therapeutics. Clin Infect Dis, 2010, 51(10): 1201-1208

[5] NegroF.. Adverse effects of drugs in the treatment of viral hepatitis. Best Pract Res Clin Gastroenterol, 2010, 24(2): 183-192

[6] LandsbergM.J., VajjhalaP.R., RothnagelR., et al.. Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding. Structure, 2009, 17(3): 427-437

[7] Kian ChuaP., LinM.H., ShihC.. Potent inhibition of human hepatitis B virus replication by a host factor Vps4. Virology, 2006, 354(1): 1-6

[8] LataS., SchoehnG., JainA., et al.. Helical structures of ESCRT-are disassembled by III VPS4. Science, 2008, 321(5894): 1354-1357

[9] LiL., TianY.J., ShenH., et al.. The immunization effects of HBV core antigen and surface antigen fusion protein in mice. Zhonghua Gan Zang Bing Za Zhi (Chinese), 2009, 17(4): 275-279

[10] LeiY.C., HaoY.H., ZhangZ.M., et al.. Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo. World J Gastroenterol, 2006, 12(28): 4492-4497

[11] LeiY.C., TianY.J., DingH.H., et al.. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication. World J Gastroenterol, 2006, 12(46): 7488-7496

[12] ScottA., ChungH.Y., Gonciarz-SwiatekM., et al.. Structural and mechanistic studies of VPS4 proteins. EMBO J, 2005, 24(20): 3658-3669

[13] BeyerA., ScheuringS., MüllerS., et al.. Comparative sequence and expression analyses of four mammalian VPS4 genes. Gene, 2003, 305(1): 47-59

[14] DaviesB.A., AzmiI.F., PayneJ., et al.. Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly. Mol Biol Cell, 2010, 21(19): 3396-3408

[15] ShestakovaA., HanonoA., DrosnerS., et al.. Assembly of the AAA ATPase Vps4 on ESCRT-III. Mol Biol Cell, 2010, 21(6): 1059-1071

[16] BruceE.A., MedcalfL., CrumpC.M., et al.. Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway. Virology, 2009, 390(2): 268-278

[17] CrumpC.M., YatesC., MinsonT.. Herpes simplex virus type 1 cytoplasmic envelopment requires functional Vps4. J Virol, 2007, 81(14): 7380-7387

[18] PawliczekT., CrumpC.M.. Herpes simplex virus type 1 production requires a functional ESCRT-III complex but is independent of TSG101 and ALIX expression. J Virol, 2009, 83(21): 11254-1164

[19] GarrusJ.E., von SchwedlerU.K., PornillosO.W., et al.. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell, 2001, 107(1): 55-65

[20] WatanabeT., SorensenE.M., NaitoA., et al.. Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc Natl Acad Sci USA, 2007, 104(24): 10205-10210

[21] ObitaT., SaksenaS., Ghazi-TabatabaiS., et al.. Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4. Nature, 2007, 449(7163): 735-739

[22] KiefferC., SkalickyJ.J., MoritaE., et al.. Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal protein targeting and HIV-1 budding. Dev Cell, 2008, 15(1): 62-73

[23] LambertC., DöringT., PrangeR.. Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin. J Virol, 2007, 81(17): 9050-9060

[24] LiuF., SongY., LiuD.. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther, 1999, 6(7): 1258-1266

[25] ZhangG., BudkerV., WolffJ.A.. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther, 1999, 10(10): 1735-1737

[26] YangP.L., AlthageA., ChungJ., et al.. Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci USA, 2002, 99(21): 13825-13830

[27] BonamassaB., HaiL., LiuD.. Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res, 2011, 28(4): 694-701

[28] BabstM., WendlandB., EstepaE.J., et al.. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J, 1998, 17(11): 2982-2993