In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes

Abstract

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.

De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation

“…Thus, we purified the full-length pORF1 that represents the major form of the protein, the fragments observed by Western-blot being produced in negligible amounts. This observation contrasts to what we observed with other viral replication polyproteins that are spontaneously and quantitatively processed through their active protease 46 .…”

“…Genotype 1 and 3 HEV s-ORF1 were expressed in a wheat-germ cell-free expression system, with uncoupled transcription and translation steps as previously described 45 and adapted for viral replication polyproteins 46 . We used a home-made wheat-germ extract prepared from non-treated durum wheat seeds as previously described 47 .…”

“…8A, lanes P). The solubility of the protein can be improved by the addition of detergents within the translation mixture 46,99 . Therefore, we screened different detergents, and we found that the pORF1 solubility is strongly increased in presence of Digitonin, GDN or Brij58 for both genotypes 1 and 3 (Fig.…”

Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro

“…High-throughput applications will, however, require improved P protein production methods. A promising option is a more easily upscalable in vitro translation system such as wheat germ extract (WGE) [ 70 ]. Though earlier reported as much inferior to RRL for production of active DHBV P protein [ 22 ] our recent data indicate WGE as a viable alternative for expression of P protein [ 15 ].…”