Hepatitis C virus (HCV) was identified in 1989 by immunoscreening of an expression library with serum from a patient with post-transfusion non-A, non-B hepatitis. HCV is an enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. The HCV genome consists of a positive single-stranded RNA of approximately 9.6 kb, and encodes a large polyprotein of about 3030 amino acids. The viral protein is processed by cellular and viral proteases, resulting in structural proteins (core, E1, E2), and nonstructural proteins (P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).

About 170 million people worldwide are infected with hepatitis C virus (HCV), and HCV causes chronic liver disease at a high rate, including cirrhosis and hepatocellular carcinoma. HCV NS3 protease is essential for polyprotein processing and viral replication. NS3 protease has been considered an attractive target for HCV antiviral drugs. HCV has 6 genotypes, and only genotype 2 replicates efficiently and produces infectious viral particles in a human hepatoma cell line. However, because there is no infectious virus production system for genotype 1b, it is difficult to study new antiviral treatments for patients infected with genotype 1.

In this study, we constructed a chimeric subgenomic replicon with the backbone of the genotype 2a JFH-1 strain and NS3 protease region derived from the genotype 1b Con1 strain to examine the compatibility of NS3 protease. We found several adaptive mutations important for higher replication efficiency by colony formation assay. We introduced adaptive mutations into the full-length virus cDNA construct to examine whether the full-length clones with the adaptive mutations could replicate efficiently and produce infectious virus. In transient transfection assays, although the full-length chimera did not have the viral replication efficiency or produce infectious viral particles, adaptive mutations obtained from this study restored the viral replication efficiency. However, these adaptive mutations could not recover infectious viral particle formation and secretion.

Only a small amount of HCV core protein was detected in the culture media of cells transfected using JFH-1/N3P_Con1 chimeras with adaptive mutations. To examine whether secreted core proteins were parts of infectious viral particles in the transient transfection assay, we determined the infectivity of the culture media and analyzed them by sucrose density gradient. JFH-1 wild type had clear major peaks of HCV core protein and RNA at 1.15 mg/ml, but chimeric JFH-1/N3P_Con1 with T64N + T73P + M631V had broad peaks of HCV core protein and RNA at 1.15 mg/ml. HCV particles produced in the cell culture usually show a major pea at 1.15 mg/ml. These results suggest that chimeric virus might have weak virus particle formation or release efficiency.

To further clarify the mechanism of virus particle formation defects in adaptive mutation constructs, we determined the intracellular sublocalization of lipid droplets (LDs) and core proteins by immunofluorescence staining assay. It has been reported that sublocalization of core protein on the surface of LDs is essential for HCV infectious virus particles. In the majority of cells expressing the JFH-1 wild type, LDs accumulated in the perinuclear region, and core proteins co-localized to the surface of LDs. On the other hand, LDs and HCV core of chimeric JFH-1/N3P_Con1 virus showed identical localization in the perinuclear region. Core proteins were scattered, not localized surrounding the LDs, and LDs were small as in immature forms. This result suggests that the proper localization of core protein to produce viral particles is disrupted in the adaptive mutation RNA-transfected cells.

We then determined the cleavage efficiency of protease by a Huh7OK1/TG-Luc cell line system. This cell line also contains the C-terminal region of IPS-1, which is efficiently cleaved by HCV NS3/4A protease upstream of the transmembrane region. We are thus able to determine the cleavage efficiency of protease by luciferase assay. Chimeric JFH-1/N3P_Con1 without adaptive mutations was not active same as JFH-1 GND, but chimeric JFH-1/N3P_Con1 with T64N + T73P + M631V mutations was approximately 3 times lower than JFH-1 wt at 96 h after transfection. This result indicates that the different replication efficiency by adaptive mutations is caused by a different cleavage efficiency of NS3 protease.

To analysis the effect of NS3 protease inhibitor in this JFH-1/N3P_Con1 with adaptive mutations and JFH-1 wild type, we used the specific NS3 protease inhibitor, VX-950. VX-950 has been shown to be effective in increasing SVR rates when used with peginterferon alfa and ribavirin in patients with chronic HCV genotype 1 infection. VX-950 was more effective against NS3 protease derived from genotype 1b than that of genotype 2a. From this result, we established the assay system of NS3 protease inhibitors for genotype 1b.

In this study, we produced a JFH-1/Con1 full-length chimera that can replicate at the same levels as JFH-1 wild type in a transient assay. And protease inhibitor, VX-950, was more effective against NS3 protease derived from genotype 1b than that of genotype 2a. From this result, we established the assay system of NS3 protease inhibitors for genotype 1b. We expect this will be a useful assay system for protease inhibitors. Moreover, we can use this chimera for characterization of NS3 protease function compared to JFH-1 wild type.

C型肝炎ウイルス研究において遺伝子型1bのウイルス培養系が存在しないことが問題となっている。本研究では、培養細胞において効率よくHCV複製増殖が可能な遺伝子型2aのJFH-1株の全長ウイルス遺伝子cDNAを用いて、そのNS3プロテアーゼ領域を遺伝子型1bのCon1株に置き換えたキメラウイルスcDNAを作製した。まず、キメラウイルスのサブジェノミックレプリコン実験により複製効率を向上させる適応変異を同定した。この適合変異を全長のキメラウイルスcDNAに導入し、異なる遺伝子型由来のNS3プロテアーゼによるHCV増殖変化の機構を解析した。

その結果、以下の5点が明らかとなった。

1.ウイルス遺伝子の一過性複製実験系で、細胞内外のHCVコア蛋白質濃度を経時的に測定した。適応変異を持つキメラウイルス遺伝子を導入した細胞の細胞内コア蛋白質量はトランスフェクション後、72-96時間でJFH-1の場合とほぼ同じレベルまで増加した。その一方、上清中のコア蛋白質は96時間後に少量検出されたが、JFH-1と比較してかなり低かった。この結果は、適応変異はキメラウイルスの複製効率は回復できるが、ウイルス粒子産生能は回復できない可能性を示した。

2.キメラウイルス遺伝子導入細胞の培養上清の感染力価は検出できなかった。さらに上清中に分泌されたHCV蛋白質がウイルス粒子の一部であるかを確認するためショ糖密度勾配で解析すると、JFH-1の場合はウイルス蛋白質とRNAは1.15 mg/mlの密度にシャープなピークを示した。しかし、キメラウイルスはより軽い密度までブロードなピークであった。これらの結果は、キメラウイルスはウイルス粒子産生能が低いことを示した。

3.キメラウイルスのウイルス粒子産生能が低い理由を調べるため、HCVの感染性ウイルス粒子産生に重要な脂肪滴とHCVコア蛋白質の細胞内の局在を免疫染色で観察した。JFH-1の場合、コア蛋白質は脂肪滴周囲に局在したが、キメラウイルスの場合、脂肪滴周囲には局在せず、脂肪滴もサイズが小さいことが観察された。異なる遺伝子型のNS3プロテアーゼの置換によりウイルスゲノム複製は維持されるが、HCVコアの局在や脂肪滴の性質が変わることが明らかとなった。

4.NS3プロテアーゼは宿主のRIG-I pathwayのIPS-1を切断することが知られている。そこでIPS-1の領域を含むHuh7OK1/TG-Luc細胞を用いてNS3プロテアーゼの切断効率を解析した結果、キメラウイルスのプロテアーゼ切断効率はJFH-1に比べて3倍程度低下していた。上記1-3の結果から、NS3プロテアーゼは感染性ウイルス粒子産生に重要なことが示されたが、この結果はプロテアーゼ活性の低下がその原因である可能性を示唆した。

5.NS3プロテアーゼ阻害剤を処理すると、遺伝子型2aのJFH-1よりNS3プロテアーゼ領域を遺伝子型1bのCon1に置き換えたキメラウイルスに対する阻害効果が大きかった。従って、本研究で作製したキメラウイルスを用いた実験系は、遺伝子型1b特異的なNS3プロテアーゼ阻害剤の効果を検証可能であることが示された。

以上、本研究は遺伝子型1b特異的なNS3プロテアーゼ阻害剤実験系に有用なキメラウイルスを樹立した。さらに、HCVのNS3プロテアーゼは感染性ウイルス粒子産生に重要な因子であることを初めて示し、このキメラウイルス実験系はNS3プロテアーゼ機能のメカニズム解析に有用であると考えられ、学位の授与に相当すると認められる。