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J. Biol. Chem., Vol. 278, Issue 32, 29744-29751, August 8, 2003

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Homogenous Hepatitis A Virus Particles

PROTEOLYTIC RELEASE OF THE ASSEMBLY SIGNAL 2A FROM PROCAPSIDS BY FACTOR Xa^*

Andrea Rachow , Verena Gauss-Müller   and Christian Probst ¶ ||

From the Institute of Medical Molecular Biology, University of Luebeck, 23538 Luebeck, Germany and the ^¶November AG, D-91056 Erlangen, Germany

Among the picornaviridae, hepatitis A virus (HAV) is unique in that its assembly is driven by domain 2A of P1-2A, the precursor of the structural proteins (Probst, C., Jecht, M., and Gauss-Müller, V. (1999) J. Biol. Chem. 274, 4527–4531). Whereas infected individuals excrete in stool mature HAV capsids with VP1 as the major structural protein, its C-terminal extended form VP1-2A is the main component of immature procapsids produced in HAV-infected cells in culture. Obviously, a postassembly proteolytic step is required to remove the primary assembly signal 2A from VP1-2A of procapsids. Mutants of VP1-2A were expressed in COS7 cells to determine the cleavage site in VP1-2A and to test for the cleavage potential of viral and host proteinases (factor Xa and thrombin). Site-specific in vitro cleavage by factor Xa and thrombin occurred in procapsids that contained VP1-2A with engineered cognate cleavage sites for these proteinases. Interestingly, factor Xa but not thrombin liberated mature VP1 also from native procapsids in an assembly-dependent manner. The data show that domain 2A, which is required for pentamerization of its precursor polypeptides and thus for the primary step of HAV assembly, is removed from the surface of immature procapsid by a host proteinase. Moreover, our data open a novel avenue to produce homogenous HAV particles from recombinant intermediates by in vitro treatment with exogenously added proteases such as factor Xa or thrombin.

Received for publication, January 15, 2003 , and in revised form, May 27, 2003.

^* This work was supported by the November AG and the Deutsche Forschungsgemeinschaft (Transferproject 24 and Ga304/6-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Euroimmun, Gross Grönau, Germany.

To whom correspondence should be addressed. Tel.: 49-451-500-4085; Fax: 49-451-500-3637; E-mail: gaussmue{at}molbio.mu-luebeck.de.

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				Y. Kusov, T. Kanda, A. Palmenberg, J.-Y. Sgro, and V. Gauss-Muller Silencing of Hepatitis A Virus Infection by Small Interfering RNAs. J. Virol., June 1, 2006; 80(11): 5599 - 5610. [Abstract] [Full Text] [PDF]