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Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB–ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB–ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB–ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB–ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB–SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ​Å. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.

Viruses with filamentous morphologies, such as tobacco mosaic virus (TMV) and M13 bacteriophage, have long been studied as multivalent nanoscaffolds for loading functional motifs. Structural assembly of the capsid proteins (CPs) of filamentous viruses often requires the presence of DNA or RNA molecules, which has limited their applications. Here, we describe a strategy for controllable assembly of flexible bio-nanotubes consisting of Escherichia coli expressed CP of baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV) in vitro. These protein-only nanotubes were studied as a new structural platform for high-density presentation of multiple active molecules on the exterior surface by direct fusion of the protein of interest to the N-terminus of HearNPV CP (HaCP). Structural characterization using cryoelectron microscopy demonstrated that the HaCP could assemble into two closely related but structurally distinct tube types, suggesting the tunable HaCP interaction network is the major contributor to the flexibility of HaCP nanotubes. Our flexible nanotubes could tolerate larger molecular modifications compared with TMV-based templates and could be used as promising candidates for versatile molecular loading applications.