Atomic structure of the translation regulatory protein NS1 of bluetongue virus

Abstract

Bluetongue virus (BTV) non-structural protein 1 (NS1) regulates viral protein synthesis and exists as tubular and non-tubular forms in infected cells but how tubules assemble and how protein synthesis is regulated are unknown. Here, we report near-atomic resolution structures of two NS1 tubular forms determined by cryo-electron microscopy. The two tubular forms are different helical assemblies of the same NS1 monomer, consisting of an N-terminal foot-, a head-and body domains connected to an extended C-terminal arm, which wraps atop the head domain of another NS1 subunit through hydrophobic interactions. Deletion of the C-terminus prevents tubule formation but not viral replication, suggesting an active non-tubular form. Two zinc finger-like motifs are present in each NS1 monomer and tubules are disrupted by divalent cation chelation and restored by cation addition, including Zn 2+ , suggesting a regulatory role of divalent cations in tubule formation. In vitro luciferase assays show that the NS1 non-tubular form upregulates BTV mRNA translation, while zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role. Thus, the non-tubular form of NS1 is sufficient for viral protein synthesis and infectious virus replication and the regulatory mechanism involved operates through divalent cation-dependent conversion between the non-tubular and tubular forms. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV

“…Cell-culture-passage-specific mutations were also identified. Eab viruses harbored unique amino-acid mutations in VP2 (G827E, body domain of VP2 [24]) and NS1 (E94G, body domain of NS1 [26]). As for Kb viruses, unique mutations were found in VP5 (T186I, unfurling domain [23]), VP6 (T33M), and NS4 (R3K) (Table 1).…”

“…During virus replication, five non-structural (NS) proteins are produced. Synthesis of NS1 generates virus-specific tubules during active infection to support the virus translation machinery [26]. NS2 proteins are important for protecting viral RNA transcripts, regulating virus genome trafficking, and the formation of viral inclusion bodies [27,28].…”

Multiple Routes of Bluetongue Virus Egress

“…NS1 is necessary for BTV replication and selectively enhances viral protein synthesis [ 6 ]. It also undergoes polymerisation producing large tubules in infected cells, whose functional consequence is unknown [ 7 ]. NS2 is responsible, and sufficient, for the generation of viral inclusion bodies (VIBS), large globular structures in cytoplasm of infected cells.…”

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication

“…Two of these are major nonstructural (NS) proteins, NS1 and NS2, which are synthesized during early infection, and each plays an essential role in virus replication. The third NS protein, NS3/NS3A, is a transmembrane protein and facilitates release of the newly assembled BTV ( 1 – 3 ). NS4 is newly identified, and its function is still not fully characterized and a fifth putative nonstructural protein ( 4 – 6 ).…”