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 amino-terminal foot, a head and body domains connected to an extended carboxy-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 Zn2+, 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, whereas zinc-finger disruption decreases viral mRNA translation, tubule formation and virus replication, confirming a functional role for the zinc-fingers. 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.
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We thank M. Turmaine for his advice and support for imaging at the UCL EM facility and C. Celma (LSHTM) for advising in the BTV reverse genetics method. This project is supported partly by grants from the US NIH (AI094386 to Z.H.Z.) and The Wellcome Trust, UK (100218, Investigator Award to P.R.). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and grants from the NIH (1S10OD018111 and 1U24 GM116792) and the National Science Foundation (DBI-1338135 and DMR-1548924). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation grant number ACI-1548562 (Comet cluster at the San Diego Supercomputing Center through allocation MCB140140).
The authors declare no competing interests.
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Kerviel, A., Ge, P., Lai, M. et al. Atomic structure of the translation regulatory protein NS1 of bluetongue virus. Nat Microbiol 4, 837–845 (2019). https://doi.org/10.1038/s41564-019-0369-x
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