Compositions and Methods for Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

Abstract

The present invention provides a cell line capable producing infectious hepatitis C virus 1a (HCV 1a) particles in culture. Disclosed are compositions and methods for an HCV 1a (clone H77) transfected immortal human hepatocyte (IHH) capable of generating infectious HCV 1a virus particles in culture. Also disclosed are methods of using the cell line, or HCV 1a virus particles derived from said cell line, to screen for potential therapeutic agents which interfere with HCV 1a virus propagation to treat hepatic disease.

1 ) A method of producing infective hepatitis C 1a (HCV 1a) particles comprising culturing a cell, the cell comprising,
 a) a human hepatocyte comprising HCV 1a cDNA,   b) the HCV 1a cDNA consisting of HCV 1a cDNA encoding the HCV 1a core   protein,   c) the human hepatocyte being immortal,   d) the human hepatocyte further comprising a full length hepatitis C virus 1a   genome,   e) the full length hepatitis C virus 1a genome consisting of RNA,   f) the immortalized human hepatic cell replicating the HCV 1a RNA genome and generating infective hepatitis C virus 1a virus particles.   
     
     
         2 ) The method according to  claim 1 , wherein the full length hepatitis C virus 1a genome consisting of RNA, is transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         3 ) The method according to  claim 1 , wherein the full length hepatitis C virus 1a viral RNA genome comprises at least 99 percent homology with RNA transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         4 ) The method according to  claim 1 , wherein the full length hepatitis C virus 1a viral RNA genome comprises at least 99 percent identity with RNA transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         5 ) A method of producing infective hepatitis C 1a (HCV 1a) particles comprising, culturing a cell, the cell comprising,
 a) a human hepatocyte comprising HCV 1a cDNA,   b) the HCV 1a cDNA consisting of HCV 1a cDNA encoding the HCV 1a core protein,   c) the human hepatocyte being immortal,   d) contacting the cell with infective HCV 1a particles,   e) culturing the cell contacted with the infective HCV 1a particles,   f) the cell contacted with the infective HCV 1a particles replicating the HCV 1a RNA genome and generating infective hepatitis C virus 1a virus particles.   
     
     
         6 ) The method according to  claim 5 , wherein the full length hepatitis C virus 1a genome consisting of RNA, is transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         7 ) The method according to  claim 5 , wherein the full length hepatitis C virus 1a viral RNA genome comprises at least 99 percent homology with RNA transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         8 ) The method according to  claim 5 , wherein the full length hepatitis C virus 1a viral RNA genome comprises at least 99 percent identity with RNA transcribed from the sequence set forth in SEQ ID NO:1. 
     
     
         9 ) A method of screening therapeutic agents for the treatment of hepatitis comprising,
 a) culturing the cell of  claim 1 ,   b) contacting the cell with a with a potential therapeutic agent,   c) monitoring hepatitis C virus 1a (HCV 1a) production.   
     
     
         10 ) The method as in  claim 9 , wherein monitoring HCV 1a production comprises measuring replication of the HCV 1a genome using reverse transcriptase and polymerase chain reaction. 
     
     
         11 ) The method as in  claim 9 , wherein monitoring HCV 1a production comprises measuring HCV 1a polypeptide production using Western blots. 
     
     
         12 ) The method as in  claim 9 , wherein monitoring HCV 1a production comprises the localization of virus particles within the cell using histological methods. 
     
     
         13 ) The method as in  claim 9 , wherein monitoring HCV 1a production comprises the localization of virus particles by electron microscopy. 
     
     
         14 ) The method as in  claim 12 , wherein localization of virus particles comprises immunochemical techniques. 
     
     
         15 ) The method as in  claim 9 , wherein monitoring HCV 1a production comprises determining infectivity of HCV 1a virus particles produced by the cell.