A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation.

SARS-CoV-2 Nucleocapsid protein ( N ) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation ( LLPS ). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 µM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

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.

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.

Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

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.

In-Plane, In-Series Nanopores with Circular Cross Sections for Resistive-Pulse Sensing.

Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus capsids. Current pulses from T = 3 capsids (∼31 nm diameter) and T = 4 capsids (∼35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (<45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.

Hysteresis in Hepatitis B Virus (HBV) Requires Assembly of Near-Perfect Capsids.

The hepatitis B virus (HBV) must release its contents to initiate infection, making capsid disassembly critical to the viral life cycle. Capsid assembly proceeds through a cascade of weak interactions between copies of capsid protein (Cp) to yield uniform particles. However, there is a hysteresis to capsid dissociation that allows capsids to persist under conditions where they could not assemble. In this study, we have sought to define the basis of hysteresis by examining urea-induced dissociation of in vitro-assembled HBV capsids. In general, capsid samples show a mixture of two pools, differentiated by stability. Labile capsid dissociation corresponds to an ∼5 µM pseudocritical concentration of assembly (pcc), the same as that observed in assembly reactions. Dissociation of the stable pool corresponds to a subfemtomolar pcc, indicative of hysteresis. The fraction of stable capsids in an assembly reaction increases with the integrity of the Cp preparation and when association is performed at a higher ionic strength, which modifies the Cp conformation. Labile complexes are more prevalent when assembly conditions yield many kinetically trapped (incomplete and overgrown) products. Cp isolated from stable capsids reassembles into a mixture of stable and labile capsids. These results suggest that hysteresis arises from an ideal capsid lattice, even when some of the substituents in that lattice have defects. Consistent with structural studies that show a subtle difference between Cp dimers and Cp in capsid, we propose that hysteresis arises when HBV capsids undergo a lattice-dependent structural transition.

Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Core Protein-Directed Antivirals and Importin ß Can Synergistically Disrupt Hepatitis B Virus Capsids.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impß) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

Virus self-assembly proceeds through contact-rich energy minima.

Self-assembly of supramolecular complexes such as viral capsids occurs prominently in nature. Nonetheless, the mechanisms underlying these processes remain poorly understood. Here, we uncover the assembly pathway of hepatitis B virus (HBV), applying fluorescence optical tweezers and high-speed atomic force microscopy. This allows tracking the assembly process in real time with single-molecule resolution. Our results identify a specific, contact-rich pentameric arrangement of HBV capsid proteins as a key on-path assembly intermediate and reveal the energy balance of the self-assembly process. Real-time nucleic acid packaging experiments show that a free energy change of ~1.4 kBT per condensed nucleotide is used to drive protein oligomerization. The finding that HBV assembly occurs via contact-rich energy minima has implications for our understanding of the assembly of HBV and other viruses and also for the development of new antiviral strategies and the rational design of self-assembling nanomaterials.

HBV Core Protein Is in Flux between Cytoplasmic, Nuclear, and Nucleolar Compartments.

Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly.

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry.

Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a capsid with different surface chemistries.

The unique potency of Cowpea mosaic virus (CPMV) in situ cancer vaccine.

The immunosuppressive tumor microenvironment enables cancer to resist immunotherapies. We have established that intratumoral administration of plant-derived Cowpea mosaic virus (CPMV) nanoparticles as an in situ vaccine overcomes the local immunosuppression and stimulates a potent anti-tumor response in several mouse cancer models and canine patients. CPMV does not infect mammalian cells but acts as a danger signal that leads to the recruitment and activation of innate and subsequently, adaptive immune cells. In the present study we addressed whether other icosahedral viruses or virus-like particles (VLPs) of plant, bacteriophage and mammalian origin can be similarly employed as intratumoral immunotherapy. Our results indicate that CPMV in situ vaccine outperforms Cowpea chlorotic mottle virus (CCMV), Physalis mosaic virus (PhMV), Sesbania mosaic virus (SeMV), bacteriophage Qß VLPs, or Hepatitis B virus capsids (HBVc). Furthermore, ex vivo and in vitro assays reveal unique features of CPMV that makes it an inherently stronger immune stimulant.

Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry is a single particle technique where the masses of individual ions are determined from simultaneous measurements of each ion's m/z ratio and charge. The ions pass through a conducting cylinder, and the charge induced on the cylinder is detected. The cylinder is usually placed inside an electrostatic linear ion trap so that the ions oscillate back and forth through the cylinder. The resulting time domain signal is analyzed by fast Fourier transformation; the oscillation frequency yields the m/z, and the charge is determined from the magnitudes. The mass resolving power depends on the uncertainties in both quantities. In previous work, the mass resolving power was modest, around 30-40. In this work we report around an order of magnitude improvement. The improvement was achieved by coupling high-accuracy charge measurements (obtained with dynamic calibration) with higher resolution m/z measurements. The performance was benchmarked by monitoring the assembly of the hepatitis B virus (HBV) capsid. The HBV capsid assembly reaction can result in a heterogeneous mixture of intermediates extending from the capsid protein dimer to the icosahedral T = 4 capsid with 120 dimers. Intermediates of all possible sizes were resolved, as well as some overgrown species. Despite the improved mass resolving power, the measured peak widths are still dominated by instrumental resolution. Heterogeneity makes only a small contribution. Resonances were observed in some of the m/z spectra. They result from ions with different masses and charges having similar m/z values. Analogous resonances are expected whenever the sample is a heterogeneous mixture assembled from a common building block.

Viral structural proteins as targets for antivirals.

Viral structural proteins are emerging as effective targets for new antivirals. In a viral lifecycle, the capsid must assemble, disassemble, and respond to host proteins, all at the right time and place. These reactions work within a narrow range of conditions, making them susceptible to small molecule interference. In at least three specific viruses, this approach has had met with preliminary success. In rhinovirus and poliovirus, compounds like pleconaril bind capsid and block RNA release. Bevirimat binds to Gag protein in HIV, inhibiting maturation. In Hepatitis B virus, core protein allosteric modulators (CpAMs) promote spontaneous assembly of capsid protein leading to empty and aberrant particles. Despite the biological diversity between viruses and the chemical diversity between antiviral molecules, we observe common features in these antivirals' mechanisms of action. These approaches work by stabilizing protein-protein interactions.

Dynamics of Hepatitis B Virus Capsid Protein Dimer Regulate Assembly through an Allosteric Network.

While there is an effective vaccine for Human Hepatitis B Virus (HBV), 257 million people have chronic infections for which there is no cure. The assembly process for the viral capsid is a potential therapeutic target. In order to understand the capsid assembly process, we investigated the dimeric building blocks of the capsid. To understand what blocks assembly, we took advantage of an assembly incompetent mutant dimer, Cp149-Y132A, located in the interdimer interface. This mutation leads to changes in protein dynamics throughout the structure of the dimer as measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). To further understand how the HBV capsid assembles, the homologue woodchuck HBV (WHV) capsid protein dimer (Cp) was used. WHV is more stable than HBV in HDX-MS and native mass spectrometry experiments. Because the WHV Cp assembles more rapidly into viral capsids than HBV, it was suspected that an increase in stability of the intradimer interface and/or in the contact region leads to increased assembly rates. The differences in dynamics when comparing HBV and human Cp149-Y132A as well as the differences in dynamics when comparing the HBV and WHV Cps allowed us to map an allosteric network within the HBV dimer. Through a careful comparison of structure, stability, and dynamics using four different capsid protein dimers, we conclude that protein subunit dynamics regulate HBV capsid assembly.

Revealing in real-time a multistep assembly mechanism for SV40 virus-like particles.

Many viruses use their genome as template for self-assembly into an infectious particle. However, this reaction remains elusive because of the transient nature of intermediate structures. To elucidate this process, optical tweezers and acoustic force spectroscopy are used to follow viral assembly in real time. Using Simian virus 40 (SV40) virus-like particles as model system, we reveal a multistep assembly mechanism. Initially, binding of VP1 pentamers to DNA leads to a significantly decreased persistence length. Moreover, the pentamers seem able to stabilize DNA loops. Next, formation of interpentamer interactions results in intermediate structures with reduced contour length. These structures stabilize into objects that permanently decrease the contour length to a degree consistent with DNA compaction in wild-type SV40. These data indicate that a multistep mechanism leads to fully assembled cross-linked SV40 particles. SV40 is studied as drug delivery system. Our insights can help optimize packaging of therapeutic agents in these particles.