Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa

Instituto Di Ricerche Di Biologia Molecolare P. Angeletti

Istituto Di Richerche Di Biologia Molecolare P. Angeletti S.p.a.

Istituto Di Ricerche Di Biologia Moleculare S.p.a.

Istituto Di Ricerehe Di Biologia Molecolare P. Angeletti S.p.a.

Raffaele De Francesco

Licia Tomei

Sven-Erik Behrens

Elisabetta Bianchi

Antonello Pessi

Giacomo Paonessa

Giovanni Migliaccio

Christian Steink&uuml;hler

Andrea Urbani

Marina Taliani

Frank Narjes

Christina Failla

The present invention features nucleic acid containing one or more adaptive mutations, and HCV replicon enhanced cells. Adaptive mutations are mutations that enhance HCV replicon activity. HCV replicon enhanced cells are cells having an increased ability to maintain an HCV replicon.

Hepatitis c virus replicons and replicon enhanced cells

The present invention relates to the molecular biology and virology of the hepatitis C virus (HCV). An object of the present invention is a method to reproduce in vitro the RNA-dependent RNA polymerase activity of HCV that makes use of sequences contained in the HCV NS5B protein.

Method for identifying a HCV RNA-dependent RNA polymerase inhibitor

This is a method for reproducing in vitro the RNA-dependent RNA polymerase activity associated with hepatitis C virus. The method is characterized in that sequences contained in NS5B are used in the reaction mixture. The terminal nucleotidyl transferase activity, a further property of the NS5B protein, can also be reproduced using this method. The method takes advantage of the fact that the NS5B protein, either purified to apparent homogeneity or present in extracts of overproducing organisms, can catalyze the addition of ribonucleotides to the 3&prime;-termini of exogenous or endogenous RNA molecules. The invention also relates to a composition of matter that comprises sequences contained in NS5B, and to the use of these compositions for the set up of an enzymatic test capable of selecting, for therapeutic purposes, compounds that inhibit the enzymatic activity associated with NS5B.

Method for producing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities encoded by hepatitis C virus (HCV)

The process according to the present invention allows expression and isolation of polypeptides with the proteolytic activity of HCV NS3 protease in a pure, catalytically active form, and in amounts that are sufficient for discovery of NS3 protease inhibitors and for determination of the three-dimensional structure of the NS3 protease. A further subject of the present invention is a procedure that defines the chemical and physical conditions necessary for completion of the proteolytic activity of the above polypeptides. The invention further comprises new compositions of matter (expression vectors) containing nucleotide sequences capable of expressing the above mentioned polypeptides in culture cells. Finally, new compounds of matter are defined, suitable to measure the above proteolytic activity, and useful to develop NS3 protease inhibitors and therefore therapeutic agents for use against HCV. The figure shows the kinetic parameters of HCV NS3 protease using the S3 depsipeptide substrate (SEQ ID NO:45).

Methodology to produce, and purify and assay polypeptides with the proteolytic activity of the HCV NS3 protease

Method for reproducing in vitro the Proteolytic activity of the NS3

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Rome, Italy

Raffaele De Francesco