Rift Valley fever virus coordinates the assembly of a programmable E3 ligase to promote viral replication.

Viruses encode strategies to degrade cellular proteins to promote infection and pathogenesis. Here, we revealed that the non-structural protein NSs of Rift Valley fever virus forms a filamentous E3 ligase to trigger efficient degradation of targeted proteins. Reconstitution in vitro and cryoelectron microscopy analysis with the 2.9-Å resolution revealed that NSs forms right-handed helical fibrils. The NSs filamentous oligomers associate with the cellular FBXO3 to form a remodeled E3 ligase. The NSs-FBXO3 E3 ligase targets the cellular TFIIH complex through the NSs-P62 interaction, leading to ubiquitination and proteasome-dependent degradation of the TFIIH complex. NSs-FBXO3-triggered TFIIH complex degradation resulted in robust inhibition of antiviral immunity and promoted viral pathogenesis in vivo. Furthermore, it is demonstrated that NSs can be programmed to target additional proteins for proteasome-dependent degradation, serving as a versatile targeted protein degrader. These results showed that a virulence factor forms a filamentous and programmable degradation machinery to induce organized degradation of cellular proteins to promote viral infection.

Neutralizing monoclonal antibodies against the Gc fusion loop region of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.

Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.

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.

Cryo-EM structures of Banna virus in multiple states reveal stepwise detachment of viral spikes.

Banna virus (BAV) is the prototype Seadornavirus, a class of reoviruses for which there has been little structural study. Here, we report atomic cryo-EM structures of three states of BAV virions-surrounded by 120 spikes (full virions), 60 spikes (partial virions), or no spikes (cores). BAV cores are double-layered particles similar to the cores of other non-turreted reoviruses, except for an additional protein component in the outer capsid shell, VP10. VP10 was identified to be a cementing protein that plays a pivotal role in the assembly of BAV virions by directly interacting with VP2 (inner capsid), VP8 (outer capsid), and VP4 (spike). Viral spikes (VP4/VP9 heterohexamers) are situated on top of VP10 molecules in full or partial virions. Asymmetrical electrostatic interactions between VP10 monomers and VP4 trimers are disrupted by high pH treatment, which is thus a simple way to produce BAV cores. Low pH treatment of BAV virions removes only the flexible receptor binding protein VP9 and triggers significant conformational changes in the membrane penetration protein VP4. BAV virions adopt distinct spatial organization of their surface proteins compared with other well-studied reoviruses, suggesting that BAV may have a unique mechanism of penetration of cellular endomembranes.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus.

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 â€‹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.

Controllable Assembly of Flexible Protein Nanotubes for Loading Multifunctional Modules.

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.

Characterization of Akabane virus from domestic bamboo rat, Southern China.

To identify the causative agents in 3 large-scale outbreaks of encephalitis and death among farmed bamboo rats (Rhizomys pruinosus). The routine bacterial culture and identification were performed. There were no significant pathogenic bacteria isolated from the brain, heart, liver, spleen, lung, or kidney of diseased bamboo rats. Using PCR-based methods, we excluded the following as causative agent: pox virus, herpesvirus, adenovirus, lymphocytic choriomeningitis virus, rabies virus, and sendai virus. Furthermore, the homogenate from the diseased bamboo rats was subjected to viral metagenomic analysis which revealed 48506 filtered viral reads annotated to Akabane virus (AKAV) with >75% nucleotide identity, suggesting the presence of AKAVs in bamboo rats. Five novel AKAV isolates were successfully isolated and characterized. Furthermore the newly isolated AKAV isolate was used to demonstrate that it can reproduce the severe encephalitic and pneumonic disease in bamboo rats and mice. The findings add to the better understanding of AKAV epidemiology and to the prevention and control of Akabane diseases in China.