| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
We tried to identify cellular proteins that specifically bind to the 3' X trail of the hepatitis C virus genome on yeast tri-hybrid system. However, we could not obtain putative proteins which interact with the 3' X trail, although UV-crosslinking study showed that several proteins, such as 38, 95, 110 kDa proteins, bound to the tail. We next checked whether purified HCV proteins such as core, NS3, 4B, 5A and 5B interact with the 3' X tail. These proteins did not show binding to the 3' X tail and only NS5B bound to poly (U) stretch which resides just upstream of the 3' X tail. We next tried to obtain active NS5B gene product to reconstitute the activity of genomic replication. NS5B sequence was obtained from HCV-infected MT-2C cells of 8 days post-infection. Among several expression system tested with mammalian cells and E. coli, we found that only maltose binding protein (MBP)-fused NS5B was collected as a soluble protein. The MBP-NS5B was soluble in the absence of detergent. The affinity-purified MBP-NS5B showed a high level of poly (A), oligo (U) dependent UMP incorporation activity. Surprisingly, E. coli ribosomes were associated with the affinity-purified active MBP-NS5B. This binding was dependent on the sequences of NS5B either of amino acid residues 1-107 or 498-591. Preliminary experiments suggested that NS5B also bound with human ribosomes prepared from HeLa G cells.
Our results in addition to those by others suggested that the following factor, NS5B and other NS proteins, HCV RNA including the 3' X tail, cellular proteins which interact with the 3' X tail, and ribosomes, form a complex after termination of viral translation. The viral replication and translation may be regulated in this complex.
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