| 1. |
- Brandariz-Nuñez, Alberto, et al.
(författare)
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Pressurized DNA state inside herpes capsids-A novel antiviral target
- 2020
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 16:7
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Dou, Tianyi, et al.
(författare)
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Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscope Infrared (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS)
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 92:16, s. 11297-11304
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Tidskriftsartikel (refereegranskat)abstract
- 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. 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. 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 spectroscopy (AFM-IR), 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 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 complimentary 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.
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| 3. |
- Evilevitch, Alex
(författare)
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Energetics of the DNA-Filled Head
- 2021
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Ingår i: Reference Module in Life Sciences. ; 4, s. 1-8
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Bokkapitel (refereegranskat)abstract
- Mechanical changes within cells and viruses have been shown to be of great importance for various mechano-signaling processes. In phage and herpesviruses, strong confinement of double-stranded DNA inside the capsid generates tens of atmospheres of pressure. This confinement leads to electrostatic sliding friction between neighboring DNA strands. This affects the mobility of the encapsidated genome which directly impacts the dynamics of viral DNA ejection during infection. When a cell is infected by multiple virions, the dynamics of release of viral genomes into a cell leads to competitive interactions between viral genomes, which in turn influences viral gene expression. This affects cell’s “decision” between lysogeny (dormant state where virus does not replicate) and cell lysis caused by lytic virus replication. This article reviews the energetics, structure and mobility of the intra-capsid genome, influenced by changes in physiological parameters (temperature and ionic conditions) that are important for viral replication. The mechano-regulation of DNA ejection dynamics is also analyzed and placed in the context of viral replication dynamics.
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| 4. |
- Evilevitch, Alex, et al.
(författare)
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Intranuclear HSV-1 DNA ejection induces major mechanical transformations suggesting mechanoprotection of nucleus integrity
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 119:9, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Evilevitch, Alex, et al.
(författare)
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Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly
- 2022
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 96:4, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Evilevitch, Alex, et al.
(författare)
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Reconstituted virus-nucleus system reveals mechanics of herpesvirus genome uncoating
- 2022
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Ingår i: QRB Discovery. - : Cambridge University Press (CUP). - 2633-2892. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Freeman, Krista G, et al.
(författare)
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UL25 capsid binding facilitates mechanical maturation of the Herpesvirus capsid and allows retention of pressurized DNA
- 2021
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Ingår i: Journal of Virology. - 1098-5514. ; 95:20
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Tidskriftsartikel (refereegranskat)abstract
- 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 with resulting tens of atmospheres of intra-capsid 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 multi-functional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus type 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 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 HSV-1 capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggests that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.
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| 8. |
- Laurent, Timothée, 1991-
(författare)
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Macromolecular organization of the chikungunya virus replication organelle
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The chikungunya virus is a positive-sense RNA virus responsible for the crippling chikungunya fever. It is transmitted through the bites of two species of mosquitoes: Aedes aegypti and Aedes albopictus. A key feature of this virus is that it is able to remodel the plasma membrane to form replication organelles called “spherules” in which the viral genomic RNA is replicated. There are four non-structural proteins in charge of the replication of the genome: nsP1, the capping enzyme, nsP2 the helicase, NTPase and protease, nsP3, a protein modulating the host-cell response to the infection and the RNA-dependent RNA polymerase nsP4. When I started my PhD, spherules had only been imaged using resin-embedding electron microscopy, which does not preserve macromolecular structure. It was unknown how the different non-structural proteins interacted with each other. The process leading to formation and maintenance of spherules at the plasma membrane was also not known. Using cryo-electron tomography, we could image spherules and unveil their macromolecular organization. We could identify a previously unreported two megadalton protein complex sitting at the neck of spherules, serving as an interface between the lumen of spherules and the cytoplasm. We found that nsP1 binds to negatively charged lipids at the plasma membrane. We also report that the host factor CD81, known to bind cholesterol at the plasma membrane, is a key element for the virus replication.We could establish a mathematical model highlighting the way those spherules form and are maintained at the plasma membrane. We quantified the amount of genomic RNA present in each spherule and found that a single copy was present as a double-stranded replication intermediate. We further studied the spatial organization of the viral genome in spherules and found that it occupies homogenously the lumen of these replication organelles and has a moderate preferential folding inside spherules.We aimed to characterize further the ATPase and helicase activities of nsP2 and nsP2 associated to nsP1 or nsP3 as polyproteins. These polyproteins are present in the early stages of the viral RNA replication. We estimated the kinetic parameters of the ATPase function of these proteins and showed that nsp2 had a helicase activity however; the helicase functions of P12 and P23 were severely reduced. We could show that P12 and P23 exhibited instead an ATP-independent chaperoning activity, able to partially unwind double-stranded RNA.
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| 9. |
- Villanueva-Valencia, José Ramon, et al.
(författare)
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Role of hsv-1 capsid vertex-specific component (Cvsc) and viral terminal dna in capsid docking at the nuclear pore
- 2021
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Villanueva-Valencia, Jose Ramon, et al.
(författare)
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‘SAXS-osmometer’ method provides measurement of DNA pressure in viral capsids and delivers an empirical equation of state
- 2023
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Ingår i: Nucleic Acids Research. - 1362-4962. ; 51:21, s. 11415-11427
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 11. |
- Villanueva Valencia, José Ramón, et al.
(författare)
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Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490. ; 120:45
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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