Butkiewicz N, Yao N, Zhong W, Wright-Minogue J, Ingravallo P, Zhang R, Durkin J, Standring D N, Baroudy B M, Sangar D V, Lemon S M, Lau J Y, Hong Z
Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
J Virol. 2000 May;74(9):4291-301. doi: 10.1128/jvi.74.9.4291-4301.2000.
GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.
GB病毒B（GBV - B）与丙型肝炎病毒（HCV）密切相关，可在绢毛猴（柽柳猴属物种）中引发急性肝炎，这使其成为在小型猴模型中体内测试抗HCV抑制剂的一种有吸引力的替代病毒。据报道，GBV - B的非结构蛋白3（NS3）丝氨酸蛋白酶与其在HCV中的对应物具有相似的底物特异性。HCV多蛋白连接点（NS4A/4B、NS4B/5A和NS5A/5B）的真实蛋白水解加工可由GBV - B NS3蛋白酶以不依赖HCV NS4A辅因子的方式完成。我们使用体外翻译的GBV - B底物进一步表征了全长GBV - B NS3蛋白的蛋白酶活性及其对辅因子的需求。仅在存在源自GBV - B NS4A中央区域的辅因子肽时，才能很容易地检测到NS4A/4B和NS5A/5B连接点处的切割。有趣的是，GBV - B底物也可以被HCV NS3蛋白酶以依赖HCV NS4A辅因子的方式切割，这支持了HCV和GBV - B具有相似的NS3蛋白酶特异性，同时保留病毒特异性辅因子需求的观点。这一关于严格病毒特异性辅因子需求的发现与HCV和GBV - B的NS4A辅因子区域缺乏序列同源性一致。支持GBV - B蛋白酶活性的最小辅因子区域被定位到GBV - B NS4A的中央区域（氨基酸Phe22和Val36之间），该区域与HCV的辅因子区域重叠。丙氨酸替代分析表明，两个氨基酸Val27和Trp31对辅因子活性至关重要，这一发现让人联想到HCV NS4A辅因子中的两个关键残基Ile25和Ile29。GBV - B NS3蛋白酶结构域和NS4A辅因子复合物的模型表明，GBV - B在其NS3蛋白酶活性的激活和调节方面可能采用了类似的结构策略。最后，构建了一种嵌合的HCV/GBV - B双功能NS3，它由N端的HCV蛋白酶结构域和C端的GBV - B RNA解旋酶结构域组成。嵌合蛋白保留了两种酶活性，这可能会导致开发出一种依赖HCV蛋白酶功能的嵌合GBV - B病毒。
