Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS.

Structural details of a genome packaged in a viral capsid are essential for understanding how the structural arrangement of a viral genome in a capsid controls its release dynamics during infection, which critically affects viral replication. We previously found a temperature-induced, solid-like to fluid-like mechanical transition of packaged λ-genome that leads to rapid DNA ejection. However, an understanding of the structural origin of this transition was lacking. Here, we use small-angle neutron scattering (SANS) to reveal the scattering form factor of dsDNA packaged in phage λ capsid by contrast matching the scattering signal from the viral capsid with deuterated buffer. We used small-angle X-ray scattering and cryoelectron microscopy reconstructions to determine the initial structural input parameters for intracapsid DNA, which allows accurate modeling of our SANS data. As result, we show a temperature-dependent density transition of intracapsid DNA occurring between two coexisting phases-a hexagonally ordered high-density DNA phase in the capsid periphery and a low-density, less-ordered DNA phase in the core. As the temperature is increased from 20 °C to 40 °C, we found that the core-DNA phase undergoes a density and volume transition close to the physiological temperature of infection (~37 °C). The transition yields a lower energy state of DNA in the capsid core due to lower density and reduced packing defects. This increases DNA mobility, which is required to initiate rapid genome ejection from the virus capsid into a host cell, causing infection. These data reconcile our earlier findings of mechanical DNA transition in phage.

'SAXS-osmometer' method provides measurement of DNA pressure in viral capsids and delivers an empirical equation of state.

We present a novel method that provides a measurement of DNA pressure in viral capsids using small angle X-ray scattering (SAXS). This method, unlike our previous assay, does not require triggering genome release with a viral receptor. Thus, it can be used to determine the existence of a pressurized genome state in a wide range of virus systems, even if the receptor is not known, leading to a better understanding of the processes of viral genome uncoating and encapsidation in the course of infection. Furthermore, by measuring DNA pressure for a collection of bacteriophages with varying DNA packing densities, we derived an empirical equation of state (EOS) that accurately predicts the relation between the capsid pressure and the packaged DNA density and includes the contribution of both DNA-DNA interaction energy and DNA bending stress to the total DNA pressure. We believe that our SAXS-osmometer method and the EOS, combined, provide the necessary tools to investigate physico-chemical properties of confined DNA condensates and mechanisms of infection, and may also provide essential data for the design of viral vectors in gene therapy applications and development of antivirals that target the pressurized genome state.

Intranuclear HSV-1 DNA ejection induces major mechanical transformations suggesting mechanoprotection of nucleus integrity.

Maintaining nuclear integrity is essential to cell survival when exposed to mechanical stress. Herpesviruses, like most DNA and some RNA viruses, put strain on the nuclear envelope as hundreds of viral DNA genomes replicate and viral capsids assemble. It remained unknown, however, how nuclear mechanics is affected at the initial stage of herpesvirus infection-immediately after viral genomes are ejected into the nuclear space-and how nucleus integrity is maintained despite an increased strain on the nuclear envelope. With an atomic force microscopy force volume mapping approach on cell-free reconstituted nuclei with docked herpes simplex type 1 (HSV-1) capsids, we explored the mechanical response of the nuclear lamina and the chromatin to intranuclear HSV-1 DNA ejection into an intact nucleus. We discovered that chromatin stiffness, measured as Young's modulus, is increased by ∼14 times, while nuclear lamina underwent softening. Those transformations could be associated with a mechanism of mechanoprotection of nucleus integrity facilitating HSV-1 viral genome replication. Indeed, stiffening of chromatin, which is tethered to the lamina meshwork, helps to maintain nuclear morphology. At the same time, increased lamina elasticity, reflected by nucleus softening, acts as a "shock absorber," dissipating the internal mechanical stress on the nuclear membrane (located on top of the lamina wall) and preventing its rupture.

Reconstituted virus-nucleus system reveals mechanics of herpesvirus genome uncoating.

The viral replication cycle is controlled by information transduced through both molecular and mechanical interactions. Viral infection mechanics remains largely unexplored, however, due to the complexity of cellular mechanical responses over the course of infection as well as a limited ability to isolate and probe these responses. Here, we develop an experimental system consisting of herpes simplex virus type 1 (HSV-1) capsids bound to isolated and reconstituted cell nuclei, which allows direct probing of capsid-nucleus mechanics with atomic force microscopy (AFM). Major mechanical transformations occur in the host nucleus when pressurised viral DNA ejects from HSV-1 capsids docked at the nuclear pore complexes (NPCs) on the nuclear membrane. This leads to structural rearrangement of the host chromosome, affecting its compaction. This in turn regulates viral genome replication and transcription dynamics as well as the decision between a lytic or latent course of infection. AFM probing of our reconstituted capsid-nucleus system provides high-resolution topographical imaging of viral capsid docking at the NPCs as well as force volume mapping of the infected nucleus surface, reflecting mechanical transformations associated with chromatin compaction and stiffness of nuclear lamina (to which chromatin is tethered). This experimental system provides a novel platform for investigation of virus-host interaction mechanics during viral genome penetration into the nucleus.

Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly.

Most viruses undergo a maturation process from a weakly self-assembled, noninfectious particle to a stable, infectious virion. For herpesviruses, this maturation process resolves several conflicting requirements: (i) assembly must be driven by weak, reversible interactions between viral particle subunits to reduce errors and minimize the energy of self-assembly, and (ii) the viral particle must be stable enough to withstand tens of atmospheres of DNA pressure resulting from its strong confinement in the capsid. With herpes simplex virus 1 (HSV-1) as a prototype of human herpesviruses, we demonstrated that this mechanical capsid maturation is mainly facilitated through capsid binding auxiliary protein UL25, orthologs of which are present in all herpesviruses. Through genetic manipulation of UL25 mutants of HSV-1 combined with the interrogation of capsid mechanics with atomic force microscopy nano-indentation, we suggested the mechanism of stepwise binding of distinct UL25 domains correlated with capsid maturation and DNA packaging. These findings demonstrate another paradigm of viruses as elegantly programmed nano-machines where an intimate relationship between mechanical and genetic information is preserved in UL25 architecture. IMPORTANCE The minor capsid protein UL25 plays a critical role in the mechanical maturation of the HSV-1 capsid during virus assembly and is required for stable DNA packaging. We modulated the UL25 capsid interactions by genetically deleting different UL25 regions and quantifying the effect on mechanical capsid stability using an atomic force microscopy (AFM) nanoindentation approach. This approach revealed how UL25 regions reinforced the herpesvirus capsid to stably package and retain pressurized DNA. Our data suggest a mechanism of stepwise binding of two main UL25 domains timed with DNA packaging.

Role of HSV-1 Capsid Vertex-Specific Component (CVSC) and Viral Terminal DNA in Capsid Docking at the Nuclear Pore.

Penetration of the viral genome into a host cell nucleus is critical for initiation of viral replication for most DNA viruses and a few RNA viruses. For herpesviruses, viral DNA ejection into a nucleus occurs when the capsid docks at the nuclear pore complex (NPC) basket with the correct orientation of the unique capsid portal vertex. It has been shown that capsid vertex-specific component (CVSC) proteins, which are located at the twelve vertices of the human herpes simplex virus type 1 (HSV-1) capsid, interact with nucleoporins (Nups) of NPCs. However, it remained unclear whether CVSC proteins determine capsid-to-NPC binding. Furthermore, it has been speculated that terminal DNA adjacent to the portal complex of DNA-filled C-capsids forms a structural motif with the portal cap (which retains DNA in the capsid), which mediates capsid-NPC binding. We demonstrate that terminal viral DNA adjacent to the portal proteins does not present a structural element required for capsid-NPC binding. Our data also show that level of CVSC proteins on the HSV-1 capsid affects level of NPC binding. To elucidate the capsid-binding process, we use an isolated, reconstituted cell nucleus system that recapitulates capsid-nucleus binding in vivo without interference from trafficking kinetics of capsids moving toward the nucleus. This allows binding of non-infectious capsid maturation intermediates with varying levels of vertex-specific components. This experimental system provides a platform for investigating virus-host interaction at the nuclear membrane.

UL25 Capsid Binding Facilitates Mechanical Maturation of the Herpesvirus Capsid and Allows Retention of Pressurized DNA.

The maturation process that occurs in most viruses is evolutionarily driven, as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome, resulting in tens of atmospheres of intracapsid DNA pressure. Yet, the procapsid is unstable due to weak reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work, we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multifunctional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery, where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to the development of drug resistance. IMPORTANCE Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in herpes simplex virus 1 (HSV-1) capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggest that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscopy Infrared Spectroscopy (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS).

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.

Pressurized DNA state inside herpes capsids-A novel antiviral target.

Drug resistance in viruses represents one of the major challenges of healthcare. As part of an effort to provide a treatment that avoids the possibility of drug resistance, we discovered a novel mechanism of action (MOA) and specific compounds to treat all nine human herpesviruses and animal herpesviruses. The novel MOA targets the pressurized genome state in a viral capsid, "turns off" capsid pressure, and blocks viral genome ejection into a cell nucleus, preventing viral replication. This work serves as a proof-of-concept to demonstrate the feasibility of a new antiviral target-suppressing pressure-driven viral genome ejection-that is likely impervious to developing drug resistance. This pivotal finding presents a platform for discovery of a new class of broad-spectrum treatments for herpesviruses and other viral infections with genome-pressure-dependent replication. A biophysical approach to antiviral treatment such as this is also a vital strategy to prevent the spread of emerging viruses where vaccine development is challenged by high mutation rates or other evasion mechanisms.

Pressure-driven release of viral genome into a host nucleus is a mechanism leading to herpes infection.

Many viruses previously have been shown to have pressurized genomes inside their viral protein shell, termed the capsid. This pressure results from the tight confinement of negatively charged viral nucleic acids inside the capsid. However, the relevance of capsid pressure to viral infection has not been demonstrated. In this work, we show that the internal DNA pressure of tens of atmospheres inside a herpesvirus capsid powers ejection of the viral genome into a host cell nucleus. To our knowledge, this provides the first demonstration of a pressure-dependent mechanism of viral genome penetration into a host nucleus, leading to infection of eukaryotic cells.

The mobility of packaged phage genome controls ejection dynamics.

The cell decision between lytic and lysogenic infection is strongly influenced by dynamics of DNA injection into a cell from a phage population, as phages compete for limited resources and progeny. However, what controls the timing of viral DNA ejection events was not understood. This in vitro study reveals that DNA ejection dynamics for phages can be synchronized (occurring within seconds) or desynchronized (displaying minutes-long delays in initiation) based on mobility of encapsidated DNA, which in turn is regulated by environmental factors, such as temperature and extra-cellular ionic conditions. This mechano-regulation of ejection dynamics is suggested to influence viral replication where the cell's decision between lytic and latent infection is associated with synchronized or desynchronized delayed ejection events from phage population adsorbed to a cell. Our findings are of significant importance for understanding regulatory mechanisms of latency in phage and Herpesviruses, where encapsidated DNA undergoes a similar mechanical transition.

Portal Stability Controls Dynamics of DNA Ejection from Phage.

Through a unique combination of time-resolved single-molecule (cryo-TEM) and bulk measurements (light scattering and small-angle X-ray scattering), we provide a detailed study of the dynamics of stochastic DNA ejection events from phage λ. We reveal that both binding with the specific phage receptor, LamB, and thermo-mechanical destabilization of the portal vertex on the capsid are required for initiation of ejection of the pressurized λ-DNA from the phage. Specifically, we found that a measurable activation energy barrier for initiation of DNA ejection with LamB present, Ea = (1.2 ± 0.1) × 10(-19) J/phage (corresponding to ∼28 kTbody/phage at Tbody = 37 °C), results in 15 times increased rate of ejection event dynamics when the temperature is raised from 15 to 45 °C (7.5 min versus 30 s average lag time for initiation of ejection). This suggests that phages have a double fail-safe mechanism for ejection-in addition to receptor binding, phage must also overcome (through thermal energy and internal DNA pressure) an energy barrier for DNA ejection. This energy barrier ensures that viral genome ejection into cells occurs with high efficiency only when the temperature conditions are favorable for genome replication. At lower suboptimal temperatures, the infectious phage titer is preserved over much longer times, since DNA ejection dynamics is strongly inhibited even in the presence of solubilized receptor or susceptible cells. This work also establishes a light scattering based approach to investigate the influence of external solution conditions, mimicking those of the bacterial cytoplasm, on the stability of the viral capsid portal, which is directly linked to dynamics of virion deactivation.

Ionic switch controls the DNA state in phage λ.

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is 'switched on' at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.

Solid-to-fluid-like DNA transition in viruses facilitates infection.

Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid-like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA-DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection.

DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

Major capsid reinforcement by a minor protein in herpesviruses and phage.

Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres. Our data reveal that the loosely formed capsid structure is reinforced post-assembly by the minor capsid protein UL25 in HSV-1 and gpD in bacteriophage λ. Using atomic force microscopy nano-indentation analysis, we show that the capsid becomes stiffer upon binding of UL25 and gpD due to increased structural stability. At the same time the force required to break the capsid increases by ∼70% for both herpes and phage. This demonstrates a universal and evolutionarily conserved function of the minor capsid protein: facilitating the retention of the pressurized viral genome in the capsid. Since all eight human herpesviruses have UL25 orthologs, this discovery offers new opportunities to interfere with herpes replication by disrupting the precise force balance between the encapsidated DNA and the capsid proteins crucial for viral replication.

Herpes virus genome, the pressure is on.

Herpes simplex virus type 1 (HSV-1) packages its micrometers-long double-stranded DNA genome into a nanometer-scale protein shell, termed the capsid. Upon confinement within the capsid, neighboring DNA strands experience repulsive electrostatic and hydration forces as well as bending stress associated with the tight curvature required of packaged DNA. By osmotically suppressing DNA release from HSV-1 capsids, we provide the first experimental evidence of a high internal pressure of tens of atmospheres within a eukaryotic human virus, resulting from the confined genome. Furthermore, the ejection is progressively suppressed by increasing external osmotic pressures, which reveals that internal pressure is capable of powering ejection of the entire genome from the viral capsid. Despite billions of years of evolution separating eukaryotic viruses and bacteriophages, pressure-driven DNA ejection has been conserved. This suggests it is a key mechanism for viral infection and thus presents a new target for antiviral therapies.

Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids.

Viruses can be described as biological objects composed mainly of two parts: a stiff protein shell called a capsid, and a core inside the capsid containing the nucleic acid and liquid. In many double-stranded DNA bacterial viruses (aka phage), the volume ratio between the liquid and the encapsidated DNA is approximately 1:1. Due to the dominant DNA hydration force, water strongly mediates the interaction between the packaged DNA strands. Therefore, water that hydrates the DNA plays an important role in nanoindentation experiments of DNA-filled viral capsids. Nanoindentation measurements allow us to gain further insight into the nature of the hydration and electrostatic interactions between the DNA strands. With this motivation, a continuum-based numerical model for simulating the nanoindentation response of DNA-filled viral capsids is proposed here. The viral capsid is modeled as large- strain isotropic hyper-elastic material, whereas porous elasticity is adopted to capture the mechanical response of the filled viral capsid. The voids inside the viral capsid are assumed to be filled with liquid, which is modeled as a homogenous incompressible fluid. The motion of a fluid flowing through the porous medium upon capsid indentation is modeled using Darcy's law, describing the flow of fluid through a porous medium. The nanoindentation response is simulated using three-dimensional finite element analysis and the simulations are performed using the finite element code Abaqus. Force-indentation curves for empty, partially and completely DNA-filled capsids are directly compared to the experimental data for bacteriophage λ. Material parameters such as Young's modulus, shear modulus, and bulk modulus are determined by comparing computed force-indentation curves to the data from the atomic force microscopy (AFM) experiments. Predictions are made for pressure distribution inside the capsid, as well as the fluid volume ratio variation during the indentation test.

DNA bending-induced phase transition of encapsidated genome in phage λ.

The DNA structure in phage capsids is determined by DNA-DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging.