Single-stranded, positive‐sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic maturation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions.

单链正义RNA病毒编码必需的复制多蛋白，该蛋白由多个结构域组成。通常对它们进行精细调节的蛋白水解成熟过程，以产生裂解中间体和终产物。多蛋白和成熟产物都起着多种关键作用，最终可以合成病毒基因组后代。尽管这些蛋白在病毒复制过程中很重要，但它们的结构特性，包括调节其众多功能的构象变化，在结构水平上却很少被描述。信息的缺乏主要是由于用适合于结构生物学方法的标准来表达大的，膜结合的，多结构域的蛋白极其困难。为了应对这一挑战，我们使用了小麦胚无细胞表达系统。我们首先建立这种方法可以合成由两种不相关的正义RNA病毒，人类诺如病毒和植物鼓膜病毒编码的病毒多蛋白。然后，我们证明了这些多蛋白具有完全的功能，并被其活性蛋白酶结构域自发自动切割，从而产生了天然的成熟产物。此外，我们表明在多蛋白中引入点突变可以抑制每种病毒的蛋白水解成熟过程。这使我们能够表达并部分纯化未切割的全长诺如病毒多蛋白和依赖于胸膜的RNA聚合酶。从而，