Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress that is needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ₂ ] at the plasma membrane. Using x-ray crystallography, electron microscopy, and molecular dynamics, we demonstrate that PI(4,5)P ₂ binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.

Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP_1,2) are the standard of care for Ebola virus disease (EVD). Anti-GP_1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro and are protective in a mouse model of EVD. However, their neutralization mechanism is poorly understood because they target a GP_1,2 epitope that has evaded structural characterization. Using X-ray crystallography and cryo-electron tomography of mAb 3A6 complexed with its stalk–MPER epitope, we reveal a previously undescribed mechanism in which 3A6 binds to a conformation of GP_1,2 that is lifted from the virion membrane. We further show that in both domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high-viremia advanced disease stages and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. The findings reported here can guide design of next-generation highly potent anti-EBOV therapeutics and vaccines.

The matrix protein VP40 of the highly pathogenic Sudan virus (genus Orthoebolavirus) is a multifunctional protein responsible for the recruitment of viral nucleocapsids to the plasma membrane and the budding of infectious virions. In addition to its role in assembly, VP40 also downregulates viral genome replication and transcription. VP40's existence in various homo-oligomeric states is presumed to underpin its diverse functional capabilities during the viral life cycle. Given the absence of licensed therapeutics targeting the Sudan virus, our study focused on inhibiting VP40 dimers, the structural precursors to critical higher-order oligomers, as a novel antiviral strategy. We have established a crystallographic screening pipeline for the identification of small-molecule fragments capable of binding to VP40. Dimeric VP40 of the Sudan virus was recombinantly expressed in bacteria, purified, crystallized, and soaked in a solution of 96 different preselected fragments. Salicylic acid was identified as a crystallographic hit with clear electron density in the pocket between the N- and the C-termini of the VP40 dimer. The binding interaction is predominantly coordinated by amino acid residues leucine 158 (L158) and arginine 214 (R214), which are key in stabilizing salicylic acid within the binding pocket. While salicylic acid displayed minimal impact on the functional aspects of VP40, we delved deeper into characterizing the druggability of the identified binding pocket. We analyzed the influence of residues L158 and R214 on the formation of virus-like particles and viral RNA synthesis. Site-directed mutagenesis of these residues to alanine markedly affected both VP40's budding activity and its effect on viral RNA synthesis, underscoring the potential of the salicylic acid binding pocket as a drug target. In summary, our findings lay the foundation for structure-guided drug design to provide lead compounds against Sudan virus VP40.