Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core.

Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/ß1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.

AMBERff at Scale: Multimillion-Atom Simulations with AMBER Force Fields in NAMD.

All-atom molecular dynamics (MD) simulations are an essential structural biology technique with increasing application to multimillion-atom systems, including viruses and cellular machinery. Classical MD simulations rely on parameter sets, such as the AMBER family of force fields (AMBERff), to accurately describe molecular motion. Here, we present an implementation of AMBERff for use in NAMD that overcomes previous limitations to enable high-performance, massively parallel simulations encompassing up to two billion atoms. Single-point potential energy comparisons and case studies on model systems demonstrate that the implementation produces results that are as accurate as running AMBERff in its native engine.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit.

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.

Dynamic regulation of Zn(II) sequestration by calgranulin C.

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13 /N15 CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.

Multiscale Modeling of Hepatitis B Virus Capsid Assembly and Its Dimorphism.

Hepatitis B virus (HBV) is an endemic, chronic virus that leads to 800000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins: T = 3 or T = 4, respectively, in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for recently developed antivirals; nonetheless, the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of the HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T = 3, T = 4, and other experimentally observed capsid morphologies. The analysis shows that capsid polymorphism is promoted by the low HBV capsid bending modulus, where the key factors controlling polymorphism are the conformational energy landscape and protein-protein binding affinities.

Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles.

In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, i.e., to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown. With the help of mass spectrometry and molecular dynamics simulations, we found which conjugation sites in the brome mosaic virus capsid are accessible to fluorophores. Reactive external surface lysines but also those at the lumenal interface where the coat protein N-termini are located showed virtually unrestricted access to dyes. The third type of labeled lysines was situated at the intercapsomeric interfaces. Through limited proteolysis of flexible N-termini, it was determined that dyes bound to them are unlikely to be involved in the radiation brightening effect. At the same time, specific labeling of genetically inserted cysteines on the exterior capsid surface alone did not lead to radiation brightening. The results suggest that lysines situated within the more rigid structural part of the coat protein provide the chemical environments conducive to radiation brightening, and we discuss some of the characteristics of these environments.

Molecular dynamics of the viral life cycle: progress and prospects.

Molecular dynamics (MD) simulations across spatiotemporal resolutions are widely applied to study viruses and represent the central technique uniting the field of computational virology. We discuss the progress of MD in elucidating the dynamics of the viral life cycle, including the status of modeling intact extracellular virions and leveraging advanced simulations to mimic active life cycle processes. We further remark on the prospects of MD for continued contributions to the basic science characterization of viruses, especially given the increasing availability of high-quality experimental data and supercomputing power. Overall, integrative computational methods that are closely guided by experiments are unmatched in the level of detail they provide, enabling-now and in the future-new discoveries relevant to thwarting viral infection.

Integrative structural biology of HIV-1 capsid protein assemblies: combining experiment and computation.

HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS), a global pandemic that has claimed 32.7 million lives since 1981. Despite decades of research, there is no cure for the disease, with 38 million people currently infected with HIV. Attractive therapeutic targets for drug development are mature HIV-1 capsids, immature Gag polyprotein assemblies, and Gag maturation intermediates, although their complex architectures, pleomorphism, and dynamics render these assemblies challenging for structural biology. The recent development of integrative approaches, combining experimental and computational methods has enabled atomic-level characterization of structures and dynamics of capsid and Gag assemblies, and revealed their interactions with small-molecule inhibitors and host factors. These structures provide important insights that will guide the development of capsid and maturation inhibitors.

All-Atom MD Simulations of the HBV Capsid Complexed with AT130 Reveal Secondary and Tertiary Structural Changes and Mechanisms of Allostery.

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer.

Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR.

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T 1 and T 1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds.

The Integrity of the Intradimer Interface of the Hepatitis B Virus Capsid Protein Dimer Regulates Capsid Self-Assembly.

During the hepatitis B virus lifecycle, 120 copies of homodimeric capsid protein assemble around a copy of reverse transcriptase and viral RNA and go on to produce an infectious virion. Assembly needs to be tightly regulated by protein conformational change to ensure symmetry, fidelity, and reproducibility. Here, we show that structures at the intradimer interface regulate conformational changes at the distal interdimer interface and so regulate assembly. A pair of interacting charged residues, D78 from each monomer, conspicuously located at the top of a four-helix bundle that forms the intradimer interface, were mutated to serine to disrupt communication between the two monomers. The mutation slowed assembly and destabilized the dimer to thermal and chemical denaturation. Mutant dimers showed evidence of transient partial unfolding based on the appearance of new proteolytically sensitive sites. Though the mutant dimer was less stable, the resulting capsids were as stable as the wildtype, based on assembly and thermal denaturation studies. Cryo-EM image reconstructions of capsid indicated that the subunits adopted an "open" state more usually associated with a free dimer and that the spike tips were either disordered or highly flexible. Molecular dynamics simulations provide mechanistic explanations for these results, suggesting that D78 stabilizes helix 4a, which forms part of the intradimer interface, by capping its N-terminus and hydrogen-bonding to nearby residues, whereas the D78S mutation disrupts these interactions, leading to partial unwinding of helix 4a. This in turn weakens the connection from helix 4 and the intradimer interface to helix 5, which forms the interdimer interface.

Coronavirus through Delaware's Computational Microscope.

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.

Scalable Analysis of Authentic Viral Envelopes on FRONTERA.

Enveloped viruses, such as SARS-CoV-2, infect cells via fusion of their envelope with the host membrane. By employing molecular simulations to characterize viral envelopes, researchers can gain insights into key determinants of infection. Here, the Frontera supercomputer is leveraged for large-scale modeling and analysis of authentic viral envelopes, whose lipid compositions are complex and realistic. Visual Molecular Dynamics (VMD) with support for MPI is employed, overcoming previous computational limitations and enabling investigation into virus biology at an unprecedented scale. The techniques applied here to an authentic HIV-1 envelope at two levels of spatial resolution (29 million particles and 280 million atoms) are broadly applicable to the study of other viruses. The authors are actively employing these techniques to develop and characterize an authentic SARS-CoV-2 envelope. A general framework for carrying out scalable analysis of simulation trajectories on Frontera is presented, expanding the utility of the machine in humanity's ongoing fight against infectious diseases.

High-Performance Analysis of Biomolecular Containers to Measure Small-Molecule Transport, Transbilayer Lipid Diffusion, and Protein Cavities.

Compartmentalization is a central theme in biology. Cells are composed of numerous membrane-enclosed structures, evolved to facilitate specific biochemical processes; viruses act as containers of genetic material, optimized to drive infection. Molecular dynamics simulations provide a mechanism to study biomolecular containers and the influence they exert on their environments; however, trajectory analysis software generally lacks knowledge of container interior versus exterior. Further, many relevant container analyses involve large-scale particle tracking endeavors, which may become computationally prohibitive with increasing system size. Here, a novel method based on 3-D ray casting is presented, which rapidly classifies the space surrounding biomolecular containers of arbitrary shape, enabling fast determination of the identities and counts of particles (e.g., solvent molecules) found inside and outside. The method is broadly applicable to the study of containers and enables high-performance characterization of properties such as solvent density, small-molecule transport, transbilayer lipid diffusion, and topology of protein cavities. The method is implemented in VMD, a widely used simulation analysis tool that supports personal computers, clouds, and parallel supercomputers, including ORNL's Summit and Titan and NCSA's Blue Waters, where the method can be employed to efficiently analyze trajectories encompassing millions of particles. The ability to rapidly characterize the spatial relationships of particles relative to a biomolecular container over many trajectory frames, irrespective of large particle counts, enables analysis of containers on a scale that was previously unfeasible, at a level of accuracy that was previously unattainable.