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Several viral members of the large viral order of the Bunyavirales carry a NSm protein that acts as a virulence factor. Here we used AlphaFold to predict the structures of these NSm proteins and surprisingly found that the cytosolic domain of theNairoviridaefamily NSm (NSmcyto) is predicted to have a very similar fold to the cytosolic domain of glycoprotein N (Gncyto). This observation is particularly striking for CCHFV (a member of the Nairoviridae family), for which the NMR structure of the Gncytodomain has already been described in the literature and shows a double zinc finger. We show that while the sequence identity between the NSmcytoand Gncytodomains is generally weak, the strict conservation of the two zinc-finger forming CCCH motifs in CCHFV NSmcytoexplains the full structural conservation predicted by AlphaFold. Interestingly, a similar observation is made for the predictions of NSm in another group of the order Bunyavirales, the familyPeribunyaviridae, where the Gncytostructures have not yet been described experimentally. Our findings suggest that NSm is the result of a gene duplication event in these viruses, and indicate that such events may indeed be common in the recent evolutionary history of RNA viruses. Importantly, our predictions provide a first insight into the long-unknown structure of NSm and its link to virulence.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. Here we show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T = 4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation., Nature Communications, 14 (1), ISSN:2041-1723

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Experimentally determined protein structures often feature missing domains. One example is the C terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulated nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS is a game changer that allows to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain’s behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions. We have recently shown that Triton X-100, a detergent used during Cp purification, binds to the hydrophobic pocket with micromolar affinity. We here performed pharmacomodulation of pocket binders through systematic modifications of the three distinct chemical moieties composing the Triton X-100 molecule. Using NMR and ITC, we found that the flat aromatic moiety is essential for binding, while the number of atoms of the aliphatic chain modulates binding affinity. The hydrophilic tail, in contrast, is highly tolerant to changes in both length and type. Our data provide essential information for designing a new class of HBV antivirals targeting capsid-envelope interactions., Chemical Science, 13 (30), ISSN:2041-6520, ISSN:2041-6539