Zhou Ting, Ren Xiaoming, Adams Rebecca L, Pyle Anna Marie
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.
Department of Chemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA.
J Virol. 2017 Dec 14;92(1). doi: 10.1128/JVI.01253-17. Print 2018 Jan 1.
Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1. Genotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important mechanical component possesses biochemical and structural properties that differ between common strains such as those of genotype 1b and a strain of HCV that replicates with exceptional efficiency (JFH-1, classified as genotype 2a). Indeed, unlike the inefficient genotype 1b NS3, which has been well studied, JFH-1 NS3 is a superhelicase with strong RNA affinity and high unwinding efficiency on a broad range of targets. Crystallographic analysis reveals architectural features that promote enhanced biochemical activity of JFH-1 NS3. These findings show that even within a single family of viruses, drift in sequence can result in the acquisition of radically new functional properties that enhance viral fitness.
丙型肝炎病毒（HCV）编码一种解旋酶，该酶对于病毒复制和组装至关重要（非结构蛋白3 [NS3]）。这种解旋酶已成为关于一般解旋酶机制的广泛基础研究的焦点，并且作为一种新型药物靶点也备受关注。尽管这种蛋白质很重要，但关于NS3的机制研究几乎完全是针对HCV 1型的变体进行的。我们对用于细胞培养中研究HCV遗传学和机制的高活性HCV毒株（如JFH - 1）中的NS3缺乏了解。因此，我们着手确定来自复制效率高的2a基因型毒株JFH - 1的NS3是否具有新的功能或结构特性。通过用于RNA结合和双链解旋的生化分析，我们发现JFH - 1 NS3与RNA的结合比先前研究的1b基因型NS3变体快得多。与其他基因型的NS3变体不同，JFH - 1 NS3以功能活性形式高亲和力结合RNA，这种形式能够立即解旋RNA双链，而无需经历激活前的限速构象变化。与其他超家族2（SF2）解旋酶不同，JFH - 1 NS3不需要长的3'突出端，并且它能解旋仅由几个核苷酸侧翼的双链，就像在折叠的HCV基因组中一样。为了理解其物理基础，我们解析了JFH - 1 NS3的晶体结构，揭示了一种新的构象，该构象包含一个开放的、带正电荷的RNA结合裂隙，该裂隙为与RNA靶标的有效相互作用做好了准备，这可能解释了HCV JFH - 1的强大复制能力。HCV的基因型与不同类型的黄病毒一样具有差异，但HCV变体的机制特征被认为是共同的。HCV复制复合体中研究最深入的成分之一是一种称为非结构蛋白3（NS3）的解旋酶。我们着手确定这种重要的机械成分在常见毒株（如1b基因型毒株）和复制效率极高的HCV毒株（JFH - 1，分类为2a基因型）之间是否具有不同的生化和结构特性。实际上，与已被充分研究的低效1b基因型NS3不同，JFH - 1 NS3是一种超级解旋酶，对广泛的靶标具有强大的RNA亲和力和高解旋效率。晶体学分析揭示了促进JFH - 1 NS3增强生化活性的结构特征。这些发现表明，即使在单一病毒家族中，序列漂移也可能导致获得全新的功能特性，从而增强病毒适应性。
