The virus particles of human immunodeficiency virus type 1 (HIV-1) are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane-binding domain, MA, forms a hexameric protein lattice that undergoes structural transition, following cleavage, into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated about 300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilize the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid monolayers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation and assign function to the SP2 peptide.

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

Influenza A virus causes millions of severe cases of disease during annual epidemics. The most abundant protein in influenza virions is matrix protein 1 (M1), which mediates virus assembly by forming an endoskeleton beneath the virus membrane. The structure of full-length M1, and how it oligomerizes to mediate the assembly of virions, is unknown. Here we determine the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We find that the C-terminal domain of M1 is disordered in solution but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerizing M1 into linear strands that coat the interior surface of the membrane of the assembling virion. In the M1 polymer, five histidine residues-contributed by three different monomers of M1-form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore reveal mechanisms of influenza virus assembly and disassembly.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.