[转移帖]Michael Nassal 弗来堡 ——HBV相关实验室及人物介绍系列

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原帖由bigben发表于 2009-10-8 20:57：

Michael Nassal
AG Nassal
Research Interests:
Hepatitis B virus (HBV) is one of the most successful viral pathogens of humans. An estimated 2 billion people show markers of infection, and 400 million have progressed into a chronic carrier state, with a greatly increased associated risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma. Although HBV infection can be be prevented by prophylactic vaccination, current therapies for chronic hepatitis B (type I interferons; currently 4 nucleos(t)ide analogs) are effective in only a fraction of patients. Our research is aimed at understanding the molecular basics of HBV infection, using biochemical in vitro systems, reverse genetics in cell culture, and in vivo infection in the duck HBV (DHBV) model system, and thus to also contribute to improved treatments of chronic hepatitis B.

Specific research topics:
I. Replication mechanism of HBV
HBV and its related animal viruses, collectively called hepadnaviruses, replicate by chaperone-assisted protein-primed reverse transcription. Using recently developed in vitro systems that reconstitute the initiation reaction from purified components, we analyze the interaction between the hepadnaviral reverse transcriptase (P protein) and a specific RNA stem-loop structure, epsilon, that acts as encapsidation signal and replication origin for first strand DNA synthesis, and how this interaction is affected by individual chaperones. Complementary, cell culture systems are employed to verify the biochemical data in the context of the full viral genome.

II. Structure and function of the hepadnaviral nucleocapsid
Hepadnavirions consist of an outer envelope containing two (avian HBVs) or three (mammalian HBVs) surface proteins, and an inner "core particle" harboring the viral genome. These nucleocapsids initially contain the specifically encapsidated pregenomic RNA (pgRNA) which is then, by the co-encapsidated P protein, converted into relaxed-circular (RC) DNA. RC DNA is an about 3 kb circular, only partially double-stranded and not covalently closed molecule with P protein covalently linked to the long (-) DNA strand. The capsid shell is formed by a single core protein species; the protein forms stable dimers 120 of which assemble into the icosahedral capsid structure. The core protein also provides a C terminal nucleic acid binding domain that is required for proper reverse transcription. recombinantly expressed core protein assembles spontaneously into genome-less capsid-like particles (CLPs) providing access to biochemical and biophysical studies of the capsid structure; they can also serve as a potent immune-enhancing nanocarrier for heterologous molecules (see below). Core function during replication is investigated by reverse genetics in cell culture.

III. Host tropism of hepadnaviruses
All hepadnaviruses show a strong tissue-tropism for the liver and a narrow host range; human HBV, for instance, efficiently infects only humans and the Great Apes, imposing severe restrictions on experimental infection. The large surface protein is thought to play a pivotal role in infection and tissue- and host specificity but the cellular interaction partners are not known. As infection model systems we are using primary hepatocytes from tupaias (tree shrews) that, for unknown reasons, are susceptible to human HBV, and Pekin ducks which are the natural host of DHBV to analyze the role of the surface proteins, as well as of the core protein and cis-acting elements on the viral nucleic acids in determining host tropism.

IV. Novel therapeutic approaches for hepatitis B
The in vitro, cell culture, and in vivo models also provide a platform to investigate the potential of new approaches towards improved therapies for chronic hepatitis B. Examples include ribozymes, capsid-targeted viral interference, antisense and RNA interference but also small compound inhibitors of the viral reverse transcriptase, and recently molecules that target capsid assembly; in addition, drugs targeting the RNA encapsidation signal and its productive interaction with the reverse transcriptase may soon be available.

V. Recombinant HBV capsid as a nanoparticulate presentation platform for heterologous               molecules
HBV core particles (serologically termed HBcAg) but also recombinant HBV CLPs are exceptionally strong immunogens. This property can be transferred to heterologous molecules that are presented on the surface of the CLPs, as is known for some time for small heterologous peptides. We have demonstrated that even select complete proteins, e.g. GFP, can be displayed in native form on HBV CLPs, and that these CLPs induce very strong anti-GFP antibody responses when used for vaccination. Since then we have been constantly improving the system and, for instance, demonstrated induction of protective immunity, in mouse models, against Lyme disease by CLPs that display different outer surface proteins of Borrelia burgdorferi. Apart from vaccines, such CLPs also hold potential for applications in Materials Sciences.

Publikationen ab 1999:
P. Bellecave, J. Gouttenoire, M. Gajer, V. Brass, G. Koutsoudakis, H. E. Blum, R. Bartenschlager, M. Nassal, D. Moradpour (2009) Hepatitis B and C virus coinfection: A novel model system reveals the absence of direct viral interference. Hepatology, 50, 46-55.

K. Dallmeier, U. Schultz, M. Nassal (2008) Heterologous replacement of the supposed host determining region of avihepadnaviruses: high in vivo infectivity despite low infectivity for hepatocytes. PLoS Pathog, 4, e1000230.

A. Walker, C. Skamel, J. Vorreiter, M. Nassal (2008) Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins. J Biol Chem, 238, 33508-33515.

L. Halverscheid, N.K. Mannes, R. Weth, M. Kleinschmidt, U. Schultz, K. Reifenberg, R. Schirmbeck, M. Nassal, H.E. Blum, J. Reimann, M. Geissler(2008) Transgenic mice replicating hepatitis B virus but lacking expression of the major HBsAg. J Med Virol, 80, 583-590.

A.J. Gehring, D. Sun, P.T. Kennedy, T. Nolte, E. Hoen, S.G. Lim, S. Wasser, C. Selden, M.K. Maini, D.M. Davis, M. Nassal, A. Bertoletti (2007) The level of viral antigen presented by hepatocytes influences CD8 T-cell function. J Virol, 81, 2940-2949.

V. Christen, F. Duong, C. Bernsmeier, D. Sun, M. Nassal and M.H. Heim (2007) Inhibition of alpha interferon signaling by hepatitis B virus. J Virol, 81, 159-165.

M. Nassal, I. Leifer, I. Wingert, K. Dallmeier, S. Prinz and J. Vorreiter (2007) A structural model for duck hepatitis B virus core protein derived by extensive mutagenesis. J Virol, 81, 13218-13229.

M. Stahl, M. Retzlaff, M. Nassal and J. Beck (2007) Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system. Nucleic Acids Res, 35, 6124-6136.

M. Stahl, J. Beck and M. Nassal (2007) Chaperones activate hepadnavirus reverse transcriptase by transiently exposing a C-proximal region in the terminal protein domain that contributes to epsilon RNA binding. J Virol, 81, 13354-13364.

D. Sun and M. Nassal (2006) Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus. J Hepatol, 45, 636-645.

C. Skamel, M. Ploss, B. Böttcher, T. Stehle, R. Wallich, M.M. Simon and M. Nassal (2006) Hepatitis B virus capsid-like particles can display the complete, dimeric outer surface protein C and stimulate production of protective antibody responses against Borrelia burgdorferi infection. J Biol Chem, 281, 17474-17481.

B. Böttcher, M. Vogel, M. Ploss and M. Nassal (2006) High plasticity of the hepatitis B virus capsid revealed by conformational stress. J Mol Biol, 356, 812-822.

M. Vogel, M. Diez, J. Eisfeld and M. Nassal (2005) In vitro assembly of mosaic hepatitis B virus capsid-like particles (CLPs): rescue into CLPs of assembly-deficient core protein fusions and FRET-suited CLPs. FEBS Lett, 579, 5211-5216.

M. Nassal, C. Skamel, P.A. Kratz, R. Wallich, T. Stehle and M.M Simon (2005) A fusion product of the complete Borrelia burgdorferi outer surface protein A (OspA) and the hepatitis B virus capsid protein is highly immunogenic and induces protective immunity similar to that seen with an effective lipidated OspA vaccine formula. Eur J Immunol, 35, 655-665.

T.F. Baumert, C. Yang, P. Schurmann, J. Köck, C. Ziegler, C. Grullich, M. Nassal, T.J. Liang, H.E. Blum and F. von Weizsäcker (2005) Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes. Hepatology, 41, 247-256

J. Köck, M. Nassal, K. Deres, H.E. Blum, F. von Weizsacker (2004) Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA. J Virol.78:13812-8

M. Vogel, J. Vorreiter, M. Nassal (2005) Quaternary structure is critical for protein display on capsid-like particles (CLPs): Efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins. Proteins. 58, 478-488.

K. Hu, J. Beck, M. Nassal (2004) SELEX-derived aptamers of the duck hepatitis B virus RNA encapsidation signal distinguish critical and non-critical residues for productive initiation of reverse transcription. Nucleic Acids Res. 32:4377-89.

A.C. Jacquard, M. Nassal, C. Pichoud, S. Ren, U. Schultz, S. Guerret, M. Chevallier, P. Werle, S. Peyrol, C. Jamard, L.T. Rimsky, C. Trepo, F. Zoulim (2004) Evaluation of a combination of clevudine and emtricitabine with adenovirus mediated delivery of interferon gamma in the woodchuck model of HBV infection. Antimicrobial Agents Chemother; 48:2683-92

J. Beck, M. Nassal (2003) "Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatits B virus reverse transcriptase, an assumed Hsp90 client protein." J. Biol. Chem., 278, 36128-38

S. Kreft, M. Nassal (2003) "hRUL138, a novel human RNA binding ubiquitin ligase." J. Cell Science 15, 605-16

F.v. Weizsäcker, J. Köck, S. Wieland, J. Beck, M. Nassal, H.E. Blum (2002) "Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex." Hepatology 35, 209-16

J. Beck, M. Vogel, M. Nassal (2002) "dNTP versus rNTP discrimination by phenylalanine 451 in duck hepatitis B virus P protein indicates a common structure of the dNTP binding pocket with other reverse transcriptases." Nucleic Acid Res. 30, 1679-87

P. Chouteau, J. LeSeyec, I. Cannie, M. Nassal, C. Guguen-Guillouzo, P. Gripon (2001) A short N-proximal region in the large surface protein harbors a determinant that contributes to the species specificity of human hepatitits B virus. J. Virol. 75, 11565-72

J. Beck, M. Nassal (2001) "Reconstitution of a functional hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in E. coli." J. Virol. 75, 7410-19

J. Köck, C. Möcklin, S. McNelly, H.E. Blum, M. Nassal, F. v. Weizsäcker (2001) Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus. J. Virol. 75, 5084-89

S. Ren, M. Nassal (2001) Hepatitis B virion and cccDNA formation in primary tupaia hepatocytes and human heptoma cell lines upon HBV genome transduction with replication-defective adeno vectors. J. Virol. 75, 1104-16

G. Beterams, M. Nassal (2001) Significant interference with hepatitis B virus replication by a core-nuclease fusion protein. J. Biol. Chem., 276, 8875-83

G. Beterams, B. Böttcher, M. Nassal (2000) Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids. FEBS Lett. 481,169-76

S.G. Schaaf, J. Beck, M. Nassal (1999) "A small 2´-OH- and base-dependent recognition element downstream of the initiation site in the RNA encapsidation signal is essential for hepatitis B virus replication initiation". J. Biol. Chem., 274, 37787-94

U. Protzer-Knolle, M. Nassal, P.-W. Chiang, M. Kirschfink, H. Schaller (1999) "Interferon Gene Transfer by a Novel Hepatitits B Virus Vector Efficiently Suppresses wild-type Virus Replication". Proc.Natl.Acad.Sci.USA 96, 10818-23

P. Kratz, B. Böttcher, M. Nassal (1999) "Native Display of Complete Foreign Protein Domains on the Surface of Hepatitis B Virus Capsids". Proc.Natl.Acad.Sci.USA 96, 1915-20

Reviews ab 1998:
M. Nassal (2009) New insights into HBV replication: new opportunities for improved therapies. Future Virology, 4, 47-53.

K. Dallmeier, M. Nassal (2008) Hepadnaviruses have a narrow host range - do they? In Comparative Hepatitis - Birkhäuser Advances in Infectious Diseases, 303-340

M. Nassal (2008) Hepatitis B viruses: reverse transcription a different way. Virus Res. 134(1-2):235-49

M. Nassal, C. Skamel, M. Vogel, PA. Kratz, T. Stehle, R. Wallich, MM. Simon (2008) Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: new particulate Lyme disease vaccines. Int J Med Microbiol.298(1-2):135-42

J. Beck, M. Nassal (2007) Hepatitis B virus replication. World J Gastroenterol. ;13:48-64

M. Nassal, K. Dallmeier, U. Schultz, D. Sun (2005) Phenotyping hepatitis B virus variants: from transfection towards a small animal in vivo infection model. J Clin Virol, 34 Suppl 1, S89-95

U. Schultz, E. Grgacic, M. Nassal (2004) "Duck hepatitis B virus – an invaluable model system for HBV infection." Adv. Virus Res. 63:1-70

F. v. Weizsäcker, J. Köck, S. MacNelly, S. Ren, H. E. Blum, M. Nassal (2003) "The tupaia model for the study of hepatitis B virus: direct infection and HBV genome transduction of primary tupaia hepatocytes" Meth Mol Med 96:153-61

J. Beck, M. Nassal (2004) "In vitro reconstitution of e-dependent duck hepatitits B virus replication initiation" Meth Mol Med 95:315-25

P. Pumpens, E. Grens, M. Nassal (2002) "Molecular Epidemiology and Immunology of Hepatitis B Virus Infection - An Update." Intervirology 45, 218-232

M. Nassal (2002) "Ca2+ - the clue to hepatitis B virus X protein function?". Hepatology 36, 755-757; invited Comment on Bouchard et al. (2001) Science 294, 2376-8: Calcium signaling by HBx protein in hepatitis B virus replication.

M. Nassal (2000) "Mechanisms of Hepatitis B Virus Replication". Proc. of the Falk Workshop "Chronic Hepatitis: New Concepts of Pathogenesis, Diagnosis and Treatment." Kluwer Academic Press, pp 3-22

M. Nassal (2000) "Macromolecular Interactions in Hepatitis B Virus Replication and Particle Assembly". Frontiers in Molecular Biology Vol. 26: DNA virus replication; pp 1-40. Oxford University Press

M. Nassal (1999) "Hepatitis B Virus Replication: Novel Roles for Virus-Host Interactions." Intervirology 42, 100-116 (Sonderausgabe: Viral hepatitis update)

R. Ulrich, M. Nassal, H. Meisel, D. Krüger (1998) "Core Particles of Hepatitis B Virus as Carrier for Foreign Epitopes." Adv. Virus Res. 50, 141-182

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