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HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CA_NTD-CA_NTDinter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45A^aα9-helices compared to WT, E45A/R132T, WT_PF74, WT_Nup153, and WT_CPSF6decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.

 

Enveloped viruses are enclosed by a lipid membrane inside of which are all of the components necessary for the virus life cycle; viral proteins, the viral genome and metabolites. Viral envelopes are lipid bilayers that adopt morphologies ranging from spheres to tubes. The envelope is derived from the host cell during viral replication. Thus, the composition of the bilayer depends on the complex constitution of lipids from the host-cell’s organelle(s) where assembly and/or budding of the viral particle occurs. Here, molecular dynamics (MD) simulations of authentic, asymmetric HIV-1 liposomes are used to derive a unique level of resolution of its full-scale structure, mechanics and dynamics. Analysis of the structural properties reveal the distribution of thicknesses of the bilayers over the entire liposome as well as its global fluctuations. Moreover, full-scale mechanical analyses are employed to derive the global bending rigidity of HIV-1 liposomes. Finally, dynamical properties of the lipid molecules reveal important relationships between their 3D diffusion, the location of lipid-rafts and the asymmetrical composition of the envelope. Overall, our simulations reveal complex relationships between the rich lipid composition of the HIV-1 liposome and its structural, mechanical and dynamical properties with critical consequences to different stages of HIV-1’s life cycle. 

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CA_CTD), by binding to and stabilizing the CA_CTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CA_CTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

 

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

 

The Perilla/Hadden-Perilla research team at the University of Delaware presents an overview of computational structural biology, their efforts to model the SARS-CoV-2 viral particle, and their perspective on how their work and training endeavors can contribute to public health.