4,598 results on '"Virus assembly"'
Search Results
2. Analysis of individual HIV-1 budding event using fast AFM reveals a multiplexed role for VPS4
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Shimon Harel, Yarin Altaras, Dikla Nachmias, Noa Rotem-Dai, Inbar Dvilansky, Natalie Elia, and Itay Rousso
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Protein Transport ,Vacuolar Proton-Translocating ATPases ,Endosomal Sorting Complexes Required for Transport ,Virus Assembly ,HIV-1 ,Biophysics ,Humans ,ATPases Associated with Diverse Cellular Activities - Abstract
The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Although the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (∼100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wild-type (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 38% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.
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- 2022
3. Role of NS2 specific RNA binding and phosphorylation in liquid–liquid phase separation and virus assembly
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Shah Kamranur Rahman, Khamal Kwesi Ampah, and Polly Roy
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Virus Assembly ,Genetics ,Animals ,RNA ,Phosphorylation ,Arginine ,Bluetongue virus - Abstract
Liquid–liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2–RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine–RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.
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- 2022
4. Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system
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Herman K H Fung, Shelley Grimes, Alexis Huet, Robert L Duda, Maria Chechik, Joseph Gault, Carol V Robinson, Roger W Hendrix, Paul J Jardine, James F Conway, Christoph G Baumann, and Alfred A Antson
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Adenosine Triphosphatases ,Viral Proteins ,Endodeoxyribonucleases ,Virus Assembly ,DNA Packaging ,DNA, Viral ,Genetics - Abstract
Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
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- 2022
5. Inositol Hexakisphosphate (IP6) Accelerates Immature HIV-1 Gag Protein Assembly toward Kinetically Trapped Morphologies
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Alexander J. Pak, Manish Gupta, Mark Yeager, and Gregory A. Voth
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Kinetics ,Colloid and Surface Chemistry ,Phytic Acid ,Virus Assembly ,HIV-1 ,Virion ,Gene Products, gag ,RNA, Viral ,Capsid Proteins ,General Chemistry ,Biochemistry ,Catalysis - Abstract
During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions
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- 2022
6. The unconventional way out—Egress of <scp>HCMV</scp> through multiviral bodies
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Linda Wedemann, Felix J. Flomm, and Jens B. Bosse
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Virus Assembly ,Virion ,Cytomegalovirus ,Humans ,Proteins ,Molecular Biology ,Microbiology - Abstract
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of congenital disabilities as well as a significant cause of disease in immunocompromised patients. The envelopment and egress of HCMV particles is an essential step of the viral life cycle as it determines viral spread and potentially tropism. Here we review the current literature on HCMV envelopment and egress with a particular focus on the role of virus-containing multivesicular body-like vesicles for virus egress and spread. We discuss the difficulties of determining the cellular provenance of these structures in light of viral redistribution of cellular marker proteins and provide potential paths to illuminate their genesis. Finally, we discuss how divergent egress pathways could result in virions of different tropisms.
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- 2022
7. Transient RNA Interactions Leave a Covalent Imprint on a Viral Capsid Protein
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Zahra Harati Taji, Pavlo Bielytskyi, Mikhail Shein, Marc-Antoine Sani, Stefan Seitz, and Anne K. Schütz
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Hepatitis B virus ,Colloid and Surface Chemistry ,Virus Assembly ,RNA, Viral ,Capsid Proteins ,Disulfides ,General Chemistry ,Arginine ,Biochemistry ,Phylogeny ,Catalysis - Abstract
The hepatitis B virus (HBV) is the leading cause of persistent liver infections. Its DNA-based genome is synthesized through reverse transcription of an RNA template inside the assembled capsid shell. In addition to the structured assembly domain, the capsid protein harbors a C-terminal extension that mediates both the enclosure of RNA during capsid assembly and the nuclear entry of the capsid during infection. The arginine-rich motifs within this extension, though common to many viruses, have largely escaped atomic-scale investigation. Here, we leverage solution and solid-state nuclear magnetic resonance spectroscopy at ambient and cryogenic temperatures, under dynamic nuclear polarization signal enhancement, to investigate the organization of the genome within the capsid. Transient interactions with phosphate groups of the RNA backbone confine the arginine-rich motifs to the interior capsid space. While no secondary structure is induced in the C-terminal extension, interactions with RNA counteract the formation of a disulfide bond, which covalently tethers this peptide arm onto the inner capsid surface. Electrostatic and covalent contributions thus compete in the spatial regulation of capsid architecture. This disulfide switch represents a coupling mechanism between the structured assembly domain of the capsid and the enclosed nucleic acids. In particular, it enables the redox-dependent regulation of the exposure of the C-terminal extension on the capsid surface, which is required for nuclear uptake of the capsid. Phylogenetic analysis of capsid proteins from hepadnaviruses points toward a function of this switch in the persistence of HBV infections.
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- 2022
8. The Mechanism of Action of Hepatitis B Virus Capsid Assembly Modulators Can Be Predicted from Binding to Early Assembly Intermediates
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Anna Pavlova, Leda Bassit, Bryan D. Cox, Maksym Korablyov, Christophe Chipot, Dharmeshkumar Patel, Diane L. Lynch, Franck Amblard, Raymond F. Schinazi, and James C. Gumbart
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Hepatitis B virus ,Capsid ,Virus Assembly ,Drug Discovery ,Humans ,Molecular Medicine ,Capsid Proteins ,Hepatitis B ,Virus Replication ,Antiviral Agents ,Article - Abstract
Interfering with the self-assembly of virus nucleocapsids is a promising approach for the development of novel antiviral agents. Applied to hepatitis B virus (HBV), this approach has led to several classes of capsid assembly modulators (CAMs) that target the virus by either accelerating nucleocapsid assembly or misdirecting it into noncapsid-like particles, thereby inhibiting the HBV replication cycle. Here, we have assessed the structures of early nucleocapsid assembly intermediates, bound with and without CAMs, using molecular dynamics simulations. We find that distinct conformations of the intermediates are induced depending on whether the bound CAM accelerates or misdirects assembly. Specifically, the assembly intermediates with bound misdirecting CAMs appear to be flattened relative to those with bound accelerators. Finally, the potency of CAMs within the same class was studied. We find that an increased number of contacts with the capsid protein and favorable binding energies inferred from free energy perturbation calculations are indicative of increased potency.
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- 2022
9. In vitro assembly and evaluation of Nora virus VLPs
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Darby J. Carlson, Ryan Sowle, Kellie D Licking-Murray, and Kimberly A. Carlson
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Chemistry ,Virus Assembly ,viruses ,Virion ,RNA ,General Medicine ,biochemical phenomena, metabolism, and nutrition ,medicine.disease_cause ,Virology ,Genome ,Virus ,Open reading frame ,Capsid ,Drosophila melanogaster ,Infectious Diseases ,medicine ,Animals ,RNA Viruses ,Capsid Proteins ,Persistent Infection ,ORFS ,Escherichia coli ,Gene - Abstract
Nora virus is a RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster. The genome is approximately 12,300 bases and is divided into four open reading frames (ORFs). Structurally, there are four important viral proteins: VP3, VP4A, VP4B, and VP4C. Three proteins (VP4A, VP4B, and VP4C) that form the virion's capsid are encoded by ORF 4, which produces a polyprotein that is post-translationally cleaved. The fourth protein (VP3) is encoded by ORF 3 and it is hypothesized to play a role in virion stability. The genes for these proteins were individually cloned into Escherichia coli, expressed, and the proteins were purified. Virus-like particles (VLPs) were assembled in vitro by mixing the proteins together in different combinations and measured via electron microscopy. Assemblies that contained VP4A and/or VP3 created VLPs with similar sizes to purified empty Nora virus capsids, potentially indicating that VP4A and/or VP3 are vital for Nora virus capsid structure, assembly, and/or stability. Not only does this study provide insight into the role of Nora virus proteins, but it may also lead to a deeper understanding of how Nora virus or other picorna-like viruses undergo assembly. Keywords: RNA viruses; Nora virus; picorna-like virus; virus-like particles; capsid assembly.
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- 2022
10. Tegument Assembly, Secondary Envelopment and Exocytosis
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Ian B. Hogue
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0301 basic medicine ,viruses ,Endocytic cycle ,Context (language use) ,Alphaherpesvirinae ,Biology ,Exocytosis ,Viral process ,03 medical and health sciences ,0302 clinical medicine ,Interaction network ,Autophagy ,Humans ,Virus Release ,Secretory pathway ,Endosomal Sorting Complexes Required for Transport ,Virus Assembly ,virus diseases ,Biological Transport ,General Medicine ,Viral tegument ,biochemical phenomena, metabolism, and nutrition ,Cell biology ,030104 developmental biology ,Membrane protein ,030220 oncology & carcinogenesis ,Tegument ,Host-Pathogen Interactions ,AssemblySecondary ,Envelopment ,Virus Physiological Phenomena - Abstract
Alphaherpesvirus tegument assembly, secondary envelopment, and exocytosis processes are understood in broad strokes, but many of the individual steps in this pathway, and their molecular and cell biological details, remain unclear. Viral tegument and membrane proteins form an extensive and robust protein interaction network, such that essentially any structural protein can be deleted, yet particles are still assembled, enveloped, and released from infected cells. We conceptually divide the tegument proteins into three groups: conserved inner and outer teguments that participate in nucleocapsid and membrane contacts, respectively, and 'middle' tegument proteins, consisting of some of the most abundant tegument proteins that serve as central hubs in the protein interaction network, yet which are unique to the alphaherpesviruses. We then discuss secondary envelopment, reviewing the tegument-membrane contacts and cellular factors that drive this process. We place this viral process in the context of cell biological processes, including the endocytic pathway, ESCRT machinery, autophagy, secretory pathway, intracellular transport, and exocytosis mechanisms. Finally, we speculate about potential relationships between cellular defenses against oligomerizing or aggregating membrane proteins and the envelopment and egress of viruses.
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- 2022
11. Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions
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Lewis Rolband, Damian Beasock, Yang Wang, Yao-Gen Shu, Jonathan D. Dinman, Tamar Schlick, Yaoqi Zhou, Jeffrey S. Kieft, Shi-Jie Chen, Giovanni Bussi, Abdelghani Oukhaled, Xingfa Gao, Petr Šulc, Daniel Binzel, Abhjeet S. Bhullar, Chenxi Liang, Peixuan Guo, Kirill A. Afonin, Beijing Institute of Technology (BIT), College of Life Sciences, Nankai University (NKU), Indiana University - Purdue University Indianapolis (IUPUI), Indiana University System, Scuola Internazionale Superiore di Studi Avanzati / International School for Advanced Studies (SISSA / ISAS), Laboratoire Analyse, Modélisation et Matériaux pour la Biologie et l'Environnement (LAMBE - UMR 8587), Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-CY Cergy Paris Université (CY), and Arizona State University [Tempe] (ASU)
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RNA nanotechnology ,ISRNN ,Biomotors ,Biophysics ,Drug delivery ,Therapies ,Virus assembly ,Biochemistry ,Settore FIS/03 - Fisica della Materia ,Computer Science Applications ,Structural Biology ,Genetics ,[CHIM]Chemical Sciences ,Biotechnology - Abstract
The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.
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- 2022
12. Identification of the tail assembly chaperone genes of T4-Like phages suggests a mechanism other than translational frameshifting for biogenesis of their encoded proteins
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Maria Vladimirov, Vasu K. Gautam, and Alan R. Davidson
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viruses ,chemical and pharmacologic phenomena ,Genome, Viral ,medicine.disease_cause ,Genome ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Virology ,Escherichia coli ,medicine ,Bacteriophage T4 ,Amino Acid Sequence ,030212 general & internal medicine ,Gene ,Conserved Sequence ,030304 developmental biology ,Genetics ,0303 health sciences ,Translational frameshift ,Bacteria ,Sequence Homology, Amino Acid ,biology ,Mechanism (biology) ,Virus Assembly ,Virion ,Computational Biology ,Frameshifting, Ribosomal ,Viral Tail Proteins ,Lambda phage ,biology.organism_classification ,stomatognathic diseases ,Chaperone (protein) ,biology.protein ,Sequence Alignment ,Biogenesis ,Molecular Chaperones - Abstract
Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4).
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- 2022
13. Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing
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Jinsheng Zhou, Adam Zlotnick, and Stephen C. Jacobson
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Hepatitis B virus ,Capsid ,Virus Assembly ,Virion ,Capsid Proteins ,Article ,Analytical Chemistry - Abstract
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 to 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 the particle-size measurements when the 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.
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- 2021
14. A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors
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Eirini Vamva, Alex Griffiths, Conrad A Vink, Andrew M L Lever, and Julia C Kenyon
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HEK293 Cells ,AcademicSubjects/SCI00010 ,Virus Assembly ,Genetic Vectors ,HIV-1 ,RNA and RNA-protein complexes ,Genetics ,Humans ,Nucleic Acid Conformation ,RNA, Viral ,Regulatory Sequences, Nucleic Acid - Abstract
Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5′ leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5–gag interaction and a more stable structure with a U5–AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5′ region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.
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- 2021
15. Bunyavirus SFTSV exploits autophagic flux for viral assembly and egress
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Xue-Jie Yu, Li-Na Yan, Chuan-Min Zhou, Jia-Min Yan, Wen-Kang Zhang, and Yong-Jun Jiao
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Phlebovirus ,Orthobunyavirus ,Severe Fever with Thrombocytopenia Syndrome ,Virus Assembly ,viruses ,Autophagy ,RNA ,Cell Biology ,Golgi apparatus ,Biology ,Exocytosis ,Cell biology ,Nucleoprotein ,symbols.namesake ,Viral life cycle ,Cell culture ,symbols ,Humans ,Molecular Biology ,Flux (metabolism) - Abstract
Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging negatively stranded enveloped RNA bunyavirus that causes SFTS with a high case fatality rate of up to 30%. Macroautophagy/autophagy is an evolutionarily conserved process involved in the maintenance of host homeostasis, which exhibits anti-viral or pro-viral responses in reaction to different viral challenges. However, the interaction between the bunyavirus SFTSV and the autophagic process is still largely unclear. By establishing various autophagy-deficient cell lines, we found that SFTSV triggered RB1CC1/FIP200-BECN1-ATG5-dependent classical autophagy flux. SFTSV nucleoprotein induced BECN1-dependent autophagy by disrupting the BECN1-BCL2 association. Importantly, SFTSV utilized autophagy for the viral life cycle, which not only assembled in autophagosomes derived from the ERGIC and Golgi complex, but also utilized autophagic vesicles for exocytosis. Taken together, our results suggest a novel virus-autophagy interaction model in which bunyavirus SFTSV induces classical autophagy flux for viral assembly and egress processes, suggesting that autophagy inhibition may be a novel therapy for treating or releasing SFTS.
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- 2021
16. CryoEM structure and assembly mechanism of a bacterial virus genome gatekeeper
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Igor Orlov, Stéphane Roche, Sandrine Brasilès, Natalya Lukoyanova, Marie-Christine Vaney, Paulo Tavares, Elena V. Orlova, Centre for Integrative Biology - CBI (Inserm U964 - CNRS UMR7104 - IGBMC), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Glasgow, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Birkbeck College [University of London], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University College of London [London] (UCL), Part of this work was supported by institutional funding from CNRS., and We thank Dr. D. Houldershaw and Y. Goudetsidis for computer support at Birkbeck throughout the duration of the project. Cryo-EM samples were prepared and optimised at the ISMB EM facility at Birkbeck College, with financial support from Wellcome Trust (079605/2/06/2). We acknowledge Diamond Light Source for access and support of the cryo-EM facilities at the UK National electron Bio-Imaging Centre (eBIC, under proposal EM14704) funded by the Wellcome Trust, the Medical Research Council UK, and the Biotechnology and Biological Sciences Research Council. We are grateful to Dr. Y. Chaban for his assistance with the data collection at the eBIC. We thank Dr. A. Isidro (VMS, CNRS, Gif-sur-Yvette, France) for a sample of HSV-1 C-capsids and Dr. R. Lurz (MPIMG, Berlin, Germany) for kindly providing the micrographs shown in Fig. 6.
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Viral Proteins ,Multidisciplinary ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Virus Assembly ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Cryoelectron Microscopy ,Electron microscopy ,Phage biology ,General Physics and Astronomy ,Bacteriophages ,Genome, Viral ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology - Abstract
Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit β-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage.
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- 2022
17. Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System
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Hyunwook Lee, Alexis J. Baxter, Carol M. Bator, Bentley A. Fane, and Susan L. Hafenstein
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Capsid ,Virus Assembly ,Structure and Assembly ,Virology ,Insect Science ,Cryoelectron Microscopy ,Microviridae ,Immunology ,Capsid Proteins ,Microvirus ,Microbiology ,Ecosystem ,Bacteriophage phi X 174 - Abstract
Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8’s C-terminus and a portion of VP1’s N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.
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- 2022
18. Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation
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Johan A. den Boon, Hong Zhan, Nuruddin Unchwaniwala, Mark Horswill, Kailey Slavik, Janice Pennington, Amanda Navine, and Paul Ahlquist
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Mammals ,Viral Proteins ,Infectious Diseases ,RNA Replication ,Virology ,Virus Assembly ,Animals ,RNA, Viral ,positive-strand RNA virus ,nodavirus ,RNA replication complex ,cryo-EM tomography ,crown complex ,Capsid Proteins ,Drosophila ,Virus Replication ,RNA, Double-Stranded - Abstract
Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are “crowned” by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.
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- 2022
19. Capsid-specific nanobody effects on HIV-1 assembly and infectivity
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Ce Ann Romanaggi, Ayna Alfadhli, Robin Lid Barklis, Timothy A. Bates, Fikadu G. Tafesse, Ilaria Merutka, and Eric Barklis
- Subjects
viruses ,Human immunodeficiency virus (HIV) ,Biology ,Virus Replication ,medicine.disease_cause ,gag Gene Products, Human Immunodeficiency Virus ,Article ,Virus ,Cell Line ,Capsid ,Protein Domains ,Virology ,medicine ,Humans ,Virus Release ,Infectivity ,Virus Assembly ,Virion ,Single-Domain Antibodies ,Antigen recognition ,Cell biology ,HIV-1 ,biology.protein ,Capsid Proteins ,Antibody - Abstract
The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.
- Published
- 2021
20. Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA
- Author
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Christian Shema Mugisha, Tung Dinh, Abhishek Kumar, Kasyap Tenneti, Jenna E. Eschbach, Keanu Davis, Robert Gifford, Mamuka Kvaratskhelia, and Sebla B. Kutluay
- Subjects
Aspartic Acid ,Virus Assembly ,Virology ,Static Electricity ,Mutation ,HIV-1 ,Virion ,RNA, Viral ,Genomics ,Lipids ,Microbiology ,RNA, Guide, Kinetoplastida - Abstract
HIV-1 integrase (IN) has a non-catalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit IN-gRNA binding and result in non-infectious viruses marked by mislocalization of the gRNA within virions. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA. However, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R and D279R substitutions was sufficient to restore IN-RNA binding and infectivity for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding. Finally, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Taken together, our findings highlight the essential role of CTD in gRNA binding and ALLINI sensitivity, and reveal the importance of pliable electrostatic interactions between the IN- CTD and the gRNA.IMPORTANCEIn addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues within its C-terminal domain (CTD) and regulates proper virion maturation. Here we show that compensatory mutations in nearby acidic residues (i.e. D256N and D270N) restore the ability to bind gRNA for IN variants bearing substitutions in these positively charged CTD residues. Similarly, charge reversals through individual D-to-R and D-to-K substitutions at these positions enabled the respective IN mutants to bind gRNA and restore virion infectivity. Further, we show that specific residues within the IN-CTD required for RNA binding also influence sensitivity to allosteric integrase inhibitors, a class of novel IN- targeting compounds that target the non-catalytic function of IN. Taken together, our findings reveal the importance of electrostatic interactions in IN-gRNA binding and provide key evidence for a crucial role of the IN-CTD in allosteric integrase inhibitor mechanism of action.
- Published
- 2022
21. Synthesis and Concomitant Assembly of Adeno-Associated Virus-like Particles in
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Dinh To, Le, Marco T, Radukic, Kathrin, Teschner, Lukas, Becker, and Kristian M, Müller
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Capsid ,Virus Assembly ,Escherichia coli ,Humans ,Capsid Proteins ,Dependovirus ,HeLa Cells - Abstract
Virus-like particles (VLPs) have been used for numerous pharmaceutical applications, particularly vaccination and drug delivery. Recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy, has been proposed as a vaccine scaffold, but high production costs limit its use. Here we establish intracellular production of AAV VLPs in
- Published
- 2022
22. Liquid–liquid phase separation mediates the formation of herpesvirus assembly compartments
- Author
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Sheng Zhou, Zhifei Fu, Ziwei Zhang, Xing Jia, Guangjun Xu, Long Sun, Fei Sun, Pu Gao, Pingyong Xu, and Hongyu Deng
- Subjects
Organelles ,Cytoplasm ,Viral Proteins ,Virus Assembly ,Virion ,RNA ,Cell Biology ,Herpesviridae - Abstract
Virus assembly, which takes place during the late stage of viral replication, is essential for virus propagation. However, the underlying mechanisms remain poorly understood, especially for viruses with complicated structures. Here, we use correlative light and electron microscopy to examine the formation of cytoplasmic virion assembly compartments (cVACs) during infection by a γ-herpesvirus. These cVACs are membraneless organelles with liquid-like properties. Formation of cVACs during virus infection is mediated by ORF52, an abundant tegument protein. ORF52 undergoes liquid–liquid phase separation (LLPS), which is promoted by both DNA and RNA. Disrupting ORF52 phase separation blocks cVACs formation and virion production. These results demonstrate that phase separation of ORF52 is critical for cVACs formation. Our work defines herpesvirus cVACs as membraneless compartments that are generated through a process of LLPS mediated by a tegument protein and adds to the cellular processes that are facilitated by phase separation.
- Published
- 2022
23. N 6 -Methyladenine Modification of Hepatitis Delta Virus Regulates Its Virion Assembly by Recruiting YTHDF1
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Geon-Woo Kim, Jae-Su Moon, Severin O. Gudima, and Aleem Siddiqui
- Subjects
Hepatitis delta Antigens ,Hepatitis B virus ,Adenine ,Virus Assembly ,Immunology ,Virion ,RNA-Binding Proteins ,Hep G2 Cells ,Methyltransferases ,Microbiology ,Virus-Cell Interactions ,Viral Envelope Proteins ,Virology ,Insect Science ,Humans ,RNA, Viral ,Hepatitis Delta Virus - Abstract
Hepatitis delta virus (HDV) is a defective satellite virus that uses hepatitis B virus (HBV) envelope proteins to form its virions and infect hepatocytes via the HBV receptors. Concomitant HDV/HBV infection continues to be a major health problem, with at least 25 million people chronically infected worldwide. N(6)-methyladenine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally, and this modification regulates various biological processes. We have previously described a wider range of functional roles of m6A methylation of HBV RNAs, including its imminent regulatory role in the encapsidation of pregenomic RNA. In this study, we present evidence that m6A methylation also plays an important role in the HDV life cycle. Using the methylated RNA immunoprecipitation (MeRIP) assay, we identified that the intracellular HDV genome and antigenome are m6A methylated in HDV- and HBV-coinfected primary human hepatocytes and HepG2 cell expressing sodium taurocholate cotransporting polypeptide (NTCP), while the extracellular HDV genome is not m6A methylated. We observed that HDV genome and delta antigen levels are significantly decreased in the absence of METTL3/14, while the extracellular HDV genome levels are increased by depletion of METTL3/14. Importantly, YTHDF1, an m6A reader protein, interacts with the m6A-methylated HDV genome and inhibits the interaction between the HDV genome and antigens. Thus, m6A of the HDV genome negatively regulates virion production by inhibiting the interaction of the HDV genome with delta antigens through the recruitment of YTHDF1. This is the first study that provides insight into the functional roles of m6A in the HDV life cycle. IMPORTANCE The functional roles of N(6)-methyladenine (m6A) modifications in the HBV life cycle have been recently highlighted. Here, we investigated the functional role of m6A modification in the HDV life cycle. HDV is a subviral agent of HBV, as it uses HBV envelope proteins to form its virions. We found that m6A methylation also occurs in the intracellular HDV genome and antigenome but not in the extracellular HDV genome. The m6A modification of the HDV genome recruits m6A reader protein (YTHDF1) onto the viral genome. The association of YTHDF1 with the HDV genome abrogates the interaction of delta antigens with the HDV genome and inhibits virion assembly. This study describes the unique effects of m6A on regulation of the HDV life cycle.
- Published
- 2022
24. A Glu-Glu-Tyr Sequence in the Cytoplasmic Tail of the M2 Protein Renders Influenza A Virus Susceptible to Restriction of the Hemagglutinin-M2 Association in Primary Human Macrophages
- Author
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Sukhmani Bedi, Rajat Mudgal, Amanda Haag, and Akira Ono
- Subjects
Host Microbial Interactions ,Macrophages ,Virus Assembly ,Structure and Assembly ,Immunology ,Glutamic Acid ,Neuraminidase ,Microbiology ,Actins ,Viroporin Proteins ,Viral Matrix Proteins ,Hemagglutinins ,Ribonucleoproteins ,Influenza A virus ,Virology ,Insect Science ,Humans ,Tyrosine ,Amino Acid Sequence - Abstract
Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components, including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes, although particle formation is observed with expression of only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDMs) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. Both defects are specific to MDMs and can be reversed upon disruption of F-actin. However, the relationship between the two defects is unclear. Here, we examined whether M2 contributes to particle assembly in MDMs and if so, which region of M2 determines the susceptibility to the MDM-specific and actin-dependent suppression. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus failed to form budding structures at the cell surface even after F-actin was disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that a single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74 to 76) in the M2 cytoplasmic tail allowed the HA-M2 association to occur efficiently even in MDMs with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identified M2 as a target of MDM-specific restriction of IAV assembly, which requires the Glu-Glu-Tyr sequence in the cytoplasmic tail. IMPORTANCE Human MDMs represent a cell type that is nonpermissive to particle formation of influenza A virus (IAV). We previously showed that close proximity association between viral HA and M2 proteins is blocked in MDMs. However, whether MDMs express a restriction factor against IAV assembly or whether they lack a dependency factor promoting assembly remained unknown. In the current study, we determined that the M2 protein is necessary for particle formation in MDMs but is also a molecular target of the MDM-specific suppression of assembly. Substitutions in the M2 cytoplasmic tail alleviated the block in both the HA-M2 association and particle production in MDMs. These findings suggest that MDMs express dependency factors necessary for assembly but also express a factor(s) that inhibits HA-M2 association and particle formation. High conservation of the M2 sequence rendering the susceptibility to the assembly block highlights the potential for M2 as a target of antiviral strategies.
- Published
- 2022
25. Structures of a large prolate virus capsid in unexpanded and expanded states generate insights into the icosahedral virus assembly
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Qianglin Fang, Wei-Chun Tang, Andrei Fokine, Marthandan Mahalingam, Qianqian Shao, Michael G. Rossmann, and Venigalla B. Rao
- Subjects
Capsid ,Multidisciplinary ,Protein Domains ,Virus Assembly ,Cryoelectron Microscopy ,Virion ,Bacteriophage T4 ,Capsid Proteins - Abstract
Many icosahedral viruses assemble proteinaceous precursors called proheads or procapsids. Proheads are metastable structures that undergo a profound structural transition known as expansion that transforms an immature unexpanded head into a mature genome-packaging head. Bacteriophage T4 is a model virus, well studied genetically and biochemically, but its structure determination has been challenging because of its large size and unusually prolate-shaped, ∼1,200-Å-long and ∼860-Å-wide capsid. Here, we report the cryogenic electron microscopy (cryo-EM) structures of T4 capsid in both of its major conformational states: unexpanded at a resolution of 5.1 Å and expanded at a resolution of 3.4 Å. These are among the largest structures deposited in Protein Data Bank to date and provide insights into virus assembly, head length determination, and shell expansion. First, the structures illustrate major domain movements and ∼70% additional gain in inner capsid volume, an essential transformation to contain the entire viral genome. Second, intricate intracapsomer interactions involving a unique insertion domain dramatically change, allowing the capsid subunits to rotate and twist while the capsomers remain fastened at quasi-threefold axes. Third, high-affinity binding sites emerge for a capsid decoration protein that clamps adjacent capsomers, imparting extraordinary structural stability. Fourth, subtle conformational changes at capsomers’ periphery modulate intercapsomer angles between capsomer planes that control capsid length. Finally, conformational changes were observed at the symmetry-mismatched portal vertex, which might be involved in triggering head expansion. These analyses illustrate how small changes in local capsid subunit interactions lead to profound shifts in viral capsid morphology, stability, and volume.
- Published
- 2022
26. Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport
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Yuki Takamatsu, Tomoki Yoshikawa, Takeshi Kurosu, Shuetsu Fukushi, Noriyo Nagata, Masayuki Shimojima, Hideki Ebihara, Masayuki Saijo, and Takeshi Noda
- Subjects
Structure and Assembly ,Virus Assembly ,Immunology ,Virion ,Biological Transport ,Hemorrhagic Fever, Ebola ,Ebolavirus ,Microbiology ,Viral Proteins ,Virology ,Insect Science ,Humans ,Phosphorylation ,Nucleocapsid ,Transcription Factors - Abstract
Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.
- Published
- 2022
27. Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding
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Monica L Husby, Souad Amiar, Laura I Prugar, Emily A David, Caroline B Plescia, Kathleen E Huie, Jennifer M Brannan, John M Dye, Elsje Pienaar, and Robert V Stahelin
- Subjects
Viral Matrix Proteins ,Mammals ,Fendiline ,Virus Assembly ,Genetics ,Animals ,Cluster Analysis ,Phosphatidylserines ,Hemorrhagic Fever, Ebola ,Ebolavirus ,Molecular Biology ,Biochemistry - Abstract
Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.
- Published
- 2022
28. Adaptation of HIV-1/HIV-2 Chimeras with Defects in Genome Packaging and Viral Replication
- Author
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Jonathan M. O. Rawson, Olga A. Nikolaitchik, Jennifer A. Yoo, Xayathed Somoulay, Matthew A. Brown, Franck S. Mbuntcha Bogni, Vinay K. Pathak, Ferri Soheilian, Ryan L. Slack, Stefan G. Sarafianos, and Wei-Shau Hu
- Subjects
Viral Proteins ,Chimera ,Virology ,Virus Assembly ,HIV-2 ,HIV-1 ,Humans ,RNA, Viral ,Amino Acid Sequence ,Genome, Viral ,Virus Replication ,Microbiology - Abstract
Frequent recombination is a hallmark of retrovirus replication. In rare cases, recombination occurs between distantly related retroviruses, generating novel viruses that may significantly impact viral evolution and public health. These recombinants may initially have substantial replication defects due to impaired interactions between proteins and/or nucleic acids from the two parental viruses. However, given the high mutation rates of retroviruses, these recombinants may be able to evolve improved compatibility of these viral elements. To test this hypothesis, we examined the adaptation of chimeras between two distantly related human pathogens: HIV-1 and HIV-2. We constructed HIV-1-based chimeras containing the HIV-2 nucleocapsid (NC) domain of Gag or the two zinc fingers of HIV-2 NC, which are critical for specific recognition of viral RNA. These chimeras exhibited significant defects in RNA genome packaging and replication kinetics in T cells. However, in some experiments, the chimeric viruses replicated with faster kinetics when repassaged, indicating that viral adaptation had occurred. Sequence analysis revealed the acquisition of a single amino acid substitution, S18L, in the first zinc finger of HIV-2 NC. This substitution, which represents a switch from a conserved HIV-2 residue to a conserved HIV-1 residue at this position, partially rescued RNA packaging and replication kinetics. Further analysis revealed that the combination of two substitutions in HIV-2 NC, W10F and S18L, almost completely restored RNA packaging and replication kinetics. Our study demonstrates that chimeras of distantly related retroviruses can adapt and significantly enhance their replication by acquiring a single substitution.
- Published
- 2022
29. The influenza A virus genome packaging network - complex, flexible and yet unsolved
- Author
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Celia Jakob, Rithu Paul-Stansilaus, Martin Schwemmle, Roland Marquet, and Hardin Bolte
- Subjects
Influenza A virus ,Virus Assembly ,Influenza, Human ,Genetics ,Humans ,RNA, Viral ,Viral Genome Packaging ,Genome, Viral - Abstract
The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA–RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA–RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA–RNA interactions. We speculate that identifying such functional intersegmental RNA–RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.
- Published
- 2022
30. Visualizing molecular interactions that determine assembly of a bullet-shaped vesicular stomatitis virus particle
- Author
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Simon Jenni, Joshua A. Horwitz, Louis-Marie Bloyet, Sean P. J. Whelan, and Stephen C. Harrison
- Subjects
Multidisciplinary ,Virus Assembly ,viruses ,Virion ,General Physics and Astronomy ,Vesiculovirus ,General Chemistry ,Vesicular stomatitis Indiana virus ,General Biochemistry, Genetics and Molecular Biology ,Ribonucleoproteins ,Animals ,RNA ,RNA, Viral ,Vesicular Stomatitis - Abstract
SUMMARYVesicular stomatitis virus (VSV) is a negative-strand RNA virus with a non-segmented genome, closely related to rabies virus. Both have characteristic bullet-like shapes. We report the structure of intact, infectious VSV particles determined by cryogenic electron microscopy. By compensating for polymorphism among viral particles with computational classification, we obtained a reconstruction of the shaft (“trunk”) at 3.5 Å resolution, with lower resolution for the rounded tip. The ribonucleoprotein (RNP), genomic RNA complexed with nucleoprotein (N), curls into a dome-like structure with about eight gradually expanding turns before transitioning into the regular helical trunk. Two layers of matrix (M) protein link the RNP with the membrane. Radial inter-layer subunit contacts are fixed within single RNA-N-M1-M2 modules, but flexible lateral and axial interactions allow assembly of polymorphic virions. Together with published structures of recombinant N in various states, our results suggest a mechanism for membrane- coupled self-assembly of VSV and its relatives.
- Published
- 2022
31. Identification of Arginine Finger as the Starter of the Biomimetic Motor in Driving Double-Stranded DNA
- Author
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Chenxi Liang and Peixuan Guo
- Subjects
Arginine ,Protein subunit ,ATPase ,General Physics and Astronomy ,phi29 DNA packaging ,Random hexamer ,Article ,viral assembly ,chemistry.chemical_compound ,Adenosine Triphosphate ,Biomimetics ,Transcription (biology) ,DNA Packaging ,Translocase ,General Materials Science ,DNA-packaging motor ,Adenosine Triphosphatases ,sequential action ,biology ,Chemistry ,Virus Assembly ,Walker motifs ,General Engineering ,Cell biology ,revolving motor ,DNA, Viral ,biology.protein ,asymmetrical hexameric ATPase ,inchworm ,DNA - Abstract
Nanomotors in nanotechnology may be as important as cars in daily life. Biomotors are nanoscale machines ubiquitous in living systems to carry out ATP-driven activities such as walking, breathing, blinking, mitosis, replication, transcription, and trafficking. The sequential action in an asymmetrical hexamer by a revolving mechanism has been confirmed in dsDNA packaging motors of phi29, herpesviruses, bacterial dsDNA translocase FtsK, and Streptomyces TraB for conjugative dsDNA transfer. These elaborate, delicate, and exquisite ring structures have inspired scientists to design biomimetics in nanotechnology. Many multisubunit ATPase rings generate force via sequential action of multiple modules, such as the Walker A, Walker B, P-loop, arginine finger, sensors, and lid. The chemical to mechanical energy conversion usually takes place in sequential order. It is commonly believed that ATP binding triggers such conversion, but how the multimodule motor starts the sequential process has not been explicitly investigated. Identification of the starter is of great significance for biomimetic motor fabrication. Here, we report that the arginine finger is the starter of the motor. Only one amino acid residue change in the arginine finger led to the impediment and elimination of all following steps. Without the arginine finger, the motor failed to assemble, bind ATP, recruit DNA, or hydrolyze ATP and was eventually unable to package DNA. However, the loss of ATPase activity due to an inactive arginine finger can be rescued by an arginine finger from the adjacent subunit of Walker A mutant through trans-complementation. Taken together, we demonstrate that the formation of dimers triggered by the arginine finger initiates the motor action rather than the general belief of initiation by ATP binding.
- Published
- 2021
32. Cellular factors involved in the hepatitis C virus life cycle
- Author
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Hui-Chun Li, Shih-Yen Lo, and Chee-Hing Yang
- Subjects
Translation ,Hepatitis C virus ,Assembly ,Replication ,Review ,Hepacivirus ,Biology ,Virus Replication ,medicine.disease_cause ,Viral Assembly ,Intracellular pathogen ,Viral entry ,medicine ,Animals ,Humans ,Cellular factor ,Protein translation ,Life Cycle Stages ,Virus Assembly ,Gastroenterology ,virus diseases ,Translation (biology) ,General Medicine ,Hepatitis C ,Virology ,digestive system diseases ,Release - Abstract
The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.
- Published
- 2021
33. Integrative structural biology of HIV-1 capsid protein assemblies: combining experiment and computation
- Author
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Tatyana Polenova, Angela M. Gronenborn, Juan R. Perilla, and Jodi A. Hadden-Perilla
- Subjects
0301 basic medicine ,Acquired Immunodeficiency Syndrome ,Virus Assembly ,Virus Integration ,viruses ,030106 microbiology ,Human immunodeficiency virus (HIV) ,Gag Polyprotein ,Host factors ,Computational biology ,Biology ,medicine.disease_cause ,gag Gene Products, Human Immunodeficiency Virus ,Article ,03 medical and health sciences ,Capsid ,030104 developmental biology ,Structural biology ,Drug development ,Virology ,HIV-1 ,medicine ,Humans ,Capsid Proteins - Abstract
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.
- Published
- 2021
34. Virus-induced activation of the rac1 protein at the site of respiratory syncytial virus assembly is a requirement for virus particle assembly on infected cells
- Author
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Boon Huan Tan, Laxmi Iyer Ravi, Richard J. Sugrue, Timothy J. C. Tan, School of Biological Sciences, Lee Kong Chian School of Medicine (LKCMedicine), Defense Medical and Environment Research Institute, and DSO National Laboratories
- Subjects
rac1 GTP-Binding Protein ,RHOA ,viruses ,Cell ,Morphogenesis ,RAC1 ,macromolecular substances ,CDC42 ,Inclusion bodies ,Virus ,Cell Line ,03 medical and health sciences ,Virology ,medicine ,Humans ,Actin ,030304 developmental biology ,0303 health sciences ,biology ,Virus Assembly ,030302 biochemistry & molecular biology ,Biological sciences [Science] ,Actins ,medicine.anatomical_structure ,Respiratory Syncytial Virus, Human ,biology.protein ,Respiratory Syncytial Virus - Abstract
The distributions of the rac1, rhoA and cdc42 proteins in respiratory syncytial virus (RSV) infected cells was examined. All three rhoGTPases were detected within inclusion bodies, and while the rhoA and rac1 proteins were associated with virus filaments, only the rac1 protein was localised throughout the virus filaments. RSV infection led to increased rac1 protein activation, and using the rac1 protein inhibitor NS23766 we provided evidence that the increased rac1 activation occurred at the site of RSV assembly and facilitated F-actin remodeling during virus morphogenesis. A non-infectious virus-like particle (VLP) assay showed that the RSV VLPs formed in lipid-raft microdomains containing the rac1 protein, together with F-actin and filamin-1 (cell proteins associated with virus filaments). This provided evidence that the virus envelope proteins are trafficked to membrane microdomains containing the rac1 protein. Collectively, these data provide evidence that the rac1 protein plays a direct role in the RSV assembly process. National Medical Research Council (NMRC) National Research Foundation (NRF) We thank National Medical Research Council of Singapore, and National Research Foundation of Singapore for providing funding.
- Published
- 2021
35. Packaging of DNA origami in viral capsids: towards synthetic viruses
- Author
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Stanislav Kler, Ran Zalk, Alexander Upcher, and Idit Kopatz
- Subjects
Capsid ,Virus Assembly ,DNA, Viral ,Viruses ,Virion ,General Materials Science ,Capsid Proteins ,DNA ,Simian virus 40 - Abstract
We report a new type of nanoparticle, consisting of a nucleic acid core (7500 nt) folded into a 35 nm DNA origami sphere, encapsulated by a capsid composed of all three SV40 virus capsid proteins. Compared to the prototype reported previously, whose capsid consists of VP1 only, the new nanoparticle closely adopts the unique intracellular pathway of the native SV40, suggesting that the proteins of the synthetic capsid retain their native viral functionality. Some of the challenges in the design of such near-future composite drugs destined for gene delivery are discussed.
- Published
- 2022
36. The Herpes Simplex Virus Tegument Protein pUL21 is Required for Viral Genome Retention Within Capsids
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Ethan C. M. Thomas, Maike Bossert, and Bruce W. Banfield
- Subjects
Capsid ,Virology ,Virus Assembly ,Immunology ,Genetics ,Humans ,Parasitology ,Herpes Simplex ,Herpesvirus 1, Human ,Genome, Viral ,Molecular Biology ,Microbiology - Abstract
During virion morphogenesis herpes simplex virus nucleocapsids transit from the nucleoplasm to the cytoplasm, through a process called nuclear egress, where the final stages of virion assembly occur. Coupled to nuclear egress is a poorly understood quality-control mechanism that preferentially selects genome-containing C-capsids, rather than A- and B-capsids that lack genomes, for transit to the cytoplasm. We and others have reported that cells infected with HSV strains deleted for the tegument protein pUL21 accumulate both empty A-capsids and C-capsids in the cytoplasm of infected cells. Quantitative microscopy experiments indicated that C-capsids were preferentially selected for envelopment at the inner nuclear membrane and that nuclear integrity remained intact in cells infected with pUL21 mutants, prompting alternative explanations for the accumulation of A-capsids in the cytoplasm. More A-capsids were also found in the nuclei of cells infected with pUL21 mutants compared to their wild type (WT) counterparts, suggesting pUL21 might be required for optimal genome packaging or genome retention within capsids. In support of this, more viral genomes were prematurely released into the cytoplasm during pUL21 mutant infection compared to WT infection and led to enhanced activation of cellular cytoplasmic DNA sensors. Mass spectrometry and western blot analysis of WT and pUL21 mutant capsids revealed an increased association of the known pUL21 binding protein, pUL16, with pUL21 mutant capsids, suggesting that premature and/or enhanced association of pUL16 with capsids might result in capsid destabilization. Further supporting this idea, deletion of pUL16 from a pUL21 mutant strain rescued genome retention within capsids. Taken together, these findings suggest that pUL21 regulates pUL16 addition to nuclear capsids and that premature, and/or, over-addition of pUL16 impairs HSV genome retention within capsids.ImportancepUL21 is a conserved, multifunctional alphaherpesvirus protein involved in cell-to-cell spread of infection, transport of capsids along microtubules, and regulation of the phosphorylation status of both viral and host proteins. pUL21 thereby controls diverse processes such as nuclear egress of capsids and cellular lipid trafficking. This study provides additional insight into HSV-1 and HSV-2 pUL21 activities and suggests that, by binding pUL16, pUL21 prevents pUL16 from interacting prematurely with nuclear capsids that would otherwise lead to capsid destabilization and premature ejection of viral genomes. Thus, prevention of pUL21 interaction with pUL16 may prove to be a useful strategy for interfering with virion assembly.
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- 2022
37. Discovery and antiviral profile of new sulfamoylbenzamide derivatives as HBV capsid assembly modulators
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Leda Ivanova Bencheva, Lorena Donnici, Luca Ferrante, Adolfo Prandi, Roberta Sinisi, Marilenia De Matteo, Pietro Randazzo, Matteo Conti, Pietro Di Lucia, Elisa Bono, Leonardo Giustini, Maria Vittoria Orsale, Alexandros Patsilinakos, Edith Monteagudo, Matteo Iannacone, Vincenzo Summa, Luca G. Guidotti, Raffaele De Francesco, Romano Di Fabio, Ivanova Bencheva, Leda, Donnici, Lorena, Ferrante, Luca, Prandi, Adolfo, Sinisi, Roberta, De Matteo, Marilenia, Randazzo, Pietro, Conti, Matteo, Di Lucia, Pietro, Bono, Elisa, Giustini, Leonardo, Vittoria Orsale, Maria, Patsilinakos, Alexandro, Monteagudo, Edith, Iannacone, Matteo, Summa, Vincenzo, Guidotti, Luca G, De Francesco, Raffaele, and Di Fabio, Romano
- Subjects
Hepatitis B virus ,Virus Assembly ,Organic Chemistry ,Clinical Biochemistry ,Pharmaceutical Science ,Settore BIO/19 - Microbiologia Generale ,Virus Replication ,Chronic hepatitis B ,Settore CHIM/08 - Chimica Farmaceutica ,Biochemistry ,Antiviral Agents ,Mice ,Capsid assembly modulator (CAM) ,Hepatic B virus (HBV) ,Animals ,Capsid Proteins ,Capsid ,Drug Discovery ,Molecular Medicine ,Molecular Biology - Abstract
Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.
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- 2022
38. Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis
- Author
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Olaf Isken, Minh Tu Pham, Hella Schwanke, Felicia Schlotthauer, Ralf Bartenschlager, and Norbert Tautz
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Virus Assembly ,Immunology ,Virion ,Hepacivirus ,Viral Nonstructural Proteins ,Virus Replication ,Microbiology ,Hepatitis C ,Cell Line ,Virology ,Genetics ,Morphogenesis ,Humans ,Parasitology ,Molecular Biology ,Peptide Hydrolases - Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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- 2022
39. Virus Hijacks Host Proteins and Machinery for Assembly and Budding, with HIV-1 as an Example
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Chih-Yen Lin, Aspiro Urbina, Wen-Hung Wang, Arunee Thitithanyanont, and Sheng-Fan Wang
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Infectious Diseases ,Endosomal Sorting Complexes Required for Transport ,Virology ,Virus Assembly ,Calcium-Binding Proteins ,HIV-1 ,Cell Cycle Proteins ,gag Gene Products, Human Immunodeficiency Virus ,Virus Release - Abstract
Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus–host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix–Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.
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- 2022
40. Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly
- Author
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Justine Kniert, Theodore dos Santos, Heather E. Eaton, Woo Jung Cho, Greg Plummer, and Maya Shmulevitz
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Mammals ,Virus Assembly ,Immunology ,Reoviridae ,Microbiology ,Cell Line ,Kinetics ,Viral Proteins ,Virology ,Genetics ,Animals ,RNA, Viral ,Parasitology ,Capsid Proteins ,Cycloheximide ,Molecular Biology - Abstract
Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins µ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a µ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC µ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating µ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.NON-TECHNICAL AUTHOR SUMMARYIt is important to understand how viruses replicate and assemble to discover antiviral therapies and to modify viruses for applications like gene therapy or cancer therapy. Reovirus is a harmless virus being tested as a cancer therapy. Reovirus has two coats of proteins, an inner coat and an outer coat. To replicate, reovirus particles need only the inner coat, but to become infectious they require the outer coat. Strangely, inner and outer coat proteins are all made by the virus at once, so it was unknown what determines whether newly made viruses will contain just the inner coat to continue to replicate, or both coats to transmit to new hosts. Our experiments reveal that the inner coat proteins are located in a different area of an infected cell versus the outer coat proteins. The location therefore determines if the newly made viruses contain just the inner coat versus both coats. Reoviruses have evolved extravagant mechanisms to be able to efficiently take on the best composition required for replication and transmission.
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- 2022
41. Atomic view of the HIV-1 matrix lattice; implications on virus assembly and envelope incorporation
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Alexandra B. Samal, Todd J. Green, and Jamil S. Saad
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Multidisciplinary ,Protein Domains ,Virus Assembly ,Cell Membrane ,HIV-1 ,Virion ,gag Gene Products, Human Immunodeficiency Virus - Abstract
During the late phase of HIV type 1 (HIV-1) infection cycle, the virally encoded Gag polyproteins are targeted to the inner leaflet of the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag binding to the PM is mediated by interactions of the N-terminally myristoylated matrix (myrMA) domain with phosphatidylinositol 4,5-bisphosphate. Formation of a myrMA lattice on the PM is an obligatory step for the assembly of immature HIV-1 particles and envelope (Env) incorporation. Atomic details of the myrMA lattice and how it mediates Env incorporation are lacking. Herein, we present the X-ray structure of myrMA at 2.15 Å. The myrMA lattice is arranged as a hexamer of trimers with a central hole, thought to accommodate the C-terminal tail of Env to promote incorporation into virions. The trimer–trimer interactions in the lattice are mediated by the N-terminal loop of one myrMA molecule and α-helices I–II, as well as the 310 helix of a myrMA molecule from an adjacent trimer. We provide evidence that substitution of MA residues Leu13 and Leu31, previously shown to have adverse effects on Env incorporation, induced a conformational change in myrMA, which may destabilize the trimer–trimer interactions within the lattice. We also show that PI(4,5)P2 is capable of binding to alternating sites on MA, consistent with an MA–membrane binding mechanism during assembly of the immature particle and upon maturation. Altogether, these findings advance our understanding of a key mechanism in HIV-1 particle assembly.
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- 2022
42. Identification and characterization of key residues in Zika virus envelope protein for virus assembly and entry
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Xiao Ma, Zhenghong Yuan, and Zhigang Yi
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Epidemiology ,Zika Virus Infection ,Flavivirus ,Virus Assembly ,Immunology ,General Medicine ,Zika Virus ,Virus Replication ,Microbiology ,Infectious Diseases ,Viral Envelope Proteins ,Virology ,Drug Discovery ,Humans ,Parasitology - Abstract
Zika virus (ZIKV), a family member in the
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- 2022
43. CP-MAS and Solution NMR Studies of Allosteric Communication in CA-assemblies of HIV-1
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Nicastro, Giuseppe, Lucci, Massimo, Oregioni, Alain, Kelly, Geoff, Frenkiel, Tom A, and Taylor, Ian A
- Subjects
Model organisms ,Capsid ,Allosteric Regulation ,Structural Biology ,Virus Assembly ,HIV-1 ,Humans ,Infectious Disease ,Capsid Proteins ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Imaging ,Structural Biology & Biophysics - Abstract
Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA. Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cβ backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.
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- 2022
44. Atomic model of Vesicular Stomatitis Virus and Mechanism of Assembly
- Author
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Kang Zhou, Zhu Si, Peng Ge, Jun Tsao, Ming Luo, and Z. Hong Zhou
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Multidisciplinary ,Virus Assembly ,General Physics and Astronomy ,Animals ,RNA ,RNA, Viral ,General Chemistry ,Vesiculovirus ,Nucleocapsid Proteins ,Vesicular Stomatitis ,General Biochemistry, Genetics and Molecular Biology ,Vesicular stomatitis Indiana virus ,Glycoproteins - Abstract
Like other negative-strand RNA viruses (NSVs) such as influenza and rabies, vesicular stomatitis virus (VSV) has a three-layered organization: a layer of matrix protein (M) resides between the membrane envelope, studded by glycoprotein (G), and the nucleocapsid, composed of the nucleocapsid protein (N) and the encapsidated genomic RNA. Lack of in situ atomic structures of these viral components has limited our understanding of the virion assembly mechanism. Here, by cryoEM and sub-particle reconstruction, we have determined the in situ structures of M and N inside VSV at 3.47 Å resolution. In the virion, N and M have a stoichiometry of 1:2. The in situ structures of both N and M differ from their crystal structures in their N-terminal segments and oligomerization loops. N-RNA, N-N, and N-M-M interactions govern the formation of the capsid. A double layer of M contributes to packaging of the helical nucleocapsid: the inner M (IM) joins neighboring turns of the N helix, while the outer M (OM) contacts G and the membrane envelope. The pseudo-crystalline organization of G is further mapped by cryoET. The mechanism of VSV assembly is delineated by the network interactions of these viral components.
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- 2022
45. Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents
- Author
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Yoon Jun Kim, Pyeong Hwa Jeong, Woochan Kim, Yong-Chul Kim, Yuri Cho, Sung-Gyoo Park, Minji Park, and Jung Ah Kang
- Subjects
Male ,Hepatitis B virus ,Drug Evaluation, Preclinical ,Mice, SCID ,Antiviral Agents ,01 natural sciences ,Virus ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,Hepatitis B, Chronic ,In vivo ,Drug Discovery ,medicine ,Animals ,Humans ,Structure–activity relationship ,Tenofovir ,IC50 ,030304 developmental biology ,Mice, Inbred ICR ,0303 health sciences ,Binding Sites ,Chemistry ,Virus Assembly ,Drug Synergism ,Hepatitis B ,medicine.disease ,Molecular biology ,Reverse transcriptase ,In vitro ,0104 chemical sciences ,Molecular Docking Simulation ,010404 medicinal & biomolecular chemistry ,Pyrimidines ,Capsid ,DNA, Viral ,Molecular Medicine ,Capsid Proteins ,Half-Life - Abstract
Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.
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- 2021
46. Reprogramming Virus Coat Protein Carboxylate Interactions for the Patterned Assembly of Hierarchical Nanorods
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Reza Ghodssi, James N. Culver, Madhu Kappagantu, Sangwook Chu, and Adam D. Brown
- Subjects
Polymers and Plastics ,Ionic bonding ,Bioengineering ,02 engineering and technology ,Coat protein ,010402 general chemistry ,01 natural sciences ,Virus ,Biomaterials ,chemistry.chemical_compound ,Materials Chemistry ,Tobacco mosaic virus ,Carboxylate ,Nanotubes ,Virus Assembly ,RNA ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Tobacco Mosaic Virus ,chemistry ,Biophysics ,RNA, Viral ,Capsid Proteins ,Nanorod ,0210 nano-technology ,Reprogramming - Abstract
The self-assembly system of the rod-shaped tobacco mosaic virus (TMV) has been studied extensively for nanoscale applications. TMV coat protein assembly is modulated by intersubunit carboxylate groups whose electrostatic repulsion limits the assembly of virus rods without incorporating genomic RNA. To engineer assembly control into this system, we reprogrammed intersubunit carboxylate interactions to produce self-assembling coat proteins in the absence of RNA and in response to unique pH and ionic environmental conditions. Specifically, engineering a charge attraction at the intersubunit E50-D77 carboxylate group through a D77K substitution stabilized the coat proteins assembly into virus-like rods. In contrast, the reciprocal E50K modification alone did not confer virus-like rod assembly. However, a combination of R46G/E50K/E97G substitutions enabled virus-like rod assembly. Interestingly, the D77K substitution displays a unique pH-dependent assembly-disassembly profile, while the R46G/E50K/E97G substitutions confer a novel salt concentration dependency for assembly control. In addition, these unique environmentally controlled coat proteins allow for the directed assembly and disassembly of chimeric virus-like rods both in solution and on substrate-attached seed rods. Combined, these findings provide a controllable means to assemble functionally discrete virus-like rods for use in nanotechnology applications.
- Published
- 2021
47. Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles
- Author
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Elaine M. Mihelc, Susan C. Baker, and Jason Lanman
- Subjects
Electron Microscope Tomography ,Biology ,Endoplasmic Reticulum ,Virus Replication ,medicine.disease_cause ,Membrane rearrangement ,Article ,Replication organelle ,Cell Line ,Mice ,Viral Proteins ,03 medical and health sciences ,Mouse hepatitis virus ,Virology ,Electron microscopy ,medicine ,Animals ,Double membrane vesicle ,030304 developmental biology ,Coronavirus ,Murine hepatitis virus ,0303 health sciences ,Budding ,Virus Assembly ,Vesicle ,Endoplasmic reticulum ,030302 biochemistry & molecular biology ,Virion ,biology.organism_classification ,Cell biology ,ERAD tuning ,Membrane ,Electron tomography ,Viral replication ,Cell culture ,Coronavirus Infections ,Lysosomes ,Viral Replication Compartments - Abstract
Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection., Highlights • Large volume electron tomography provides a broad view of coronavirus membrane rearrangements. • Double membrane vesicles form by budding from the ER. • Double membrane vesicles are trafficked to lysosomes for degradation. • The reticulovesicular network breaks down late in infection. • The main virus assembly site shifts from the ERGIC to the ER during infection.
- Published
- 2021
48. The role of host cell Rab GTPases in influenza A virus infections
- Author
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Shengjun Wang, Li Shen, Jiaqi Gu, Jing Wu, Lingxiang Mao, Yiwen Chen, Xiaonan Jia, and Yiqian Yin
- Subjects
0301 basic medicine ,Microbiology (medical) ,Viral pathogenesis ,GTPase ,Biology ,Virus Replication ,medicine.disease_cause ,Microbiology ,Viral Proteins ,03 medical and health sciences ,0302 clinical medicine ,Viral entry ,Influenza, Human ,Influenza A virus ,medicine ,Humans ,Small GTPase ,Virus Assembly ,Critical factors ,SUPERFAMILY ,Virus Internalization ,Virology ,030104 developmental biology ,Ribonucleoproteins ,rab GTP-Binding Proteins ,030220 oncology & carcinogenesis ,Host-Pathogen Interactions ,Rab - Abstract
Influenza A virus (IAV) is a crucial cause of respiratory infections in humans worldwide. Therefore, studies should clarify adaptation mechanisms of IAV and critical factors of the viral pathogenesis in human hosts. GTPases of the Rab family are the largest branch of the Ras-like small GTPase superfamily, and they regulate almost every step during vesicle-mediated trafficking. Evidence has shown that Rab proteins participate in the lifecycle of IAV. In this mini-review, we outline the regulatory mechanisms of different Rab proteins in the lifecycle of IAV. Understanding the role of Rab proteins in IAV infections is important to develop broad-spectrum host-targeted antiviral strategies.
- Published
- 2021
49. CRISPR-Guided Programmable Self-Assembly of Artificial Virus-Like Nucleocapsids
- Author
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Carlos Calcines-Cruz, Ilya J. Finkelstein, and Armando Hernandez-Garcia
- Subjects
Dna template ,viruses ,Bioengineering ,02 engineering and technology ,Computational biology ,Article ,Virus ,Viral Assembly ,chemistry.chemical_compound ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,General Materials Science ,Nucleocapsid ,Chemistry ,Virus Assembly ,Mechanical Engineering ,Virion ,DNA ,General Chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Template ,Nucleic acid ,Self-assembly ,0210 nano-technology - Abstract
Designer virus-inspired proteins drive the manufacturing of more effective and safer gene-delivery systems as well as simpler models to study viral assembly. However, the self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate was further increased when dCas12a was dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus non-cognate DNA templates by the synthetic polypeptide. Our CRISPR-guided VLPs could help develop programmable bio-inspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.
- Published
- 2021
50. Intracellular host cell membrane remodelling induced by SARS‐CoV‐2 infection in vitro
- Author
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Wanderley de Souza, Luiza M. Higa, Amilcar Tanuri, Fabio Luis Monteiro, Lucio Ayres Caldas, Fabiana A. Carneiro, Ingrid Augusto, and Kildare Miranda
- Subjects
Electron Microscope Tomography ,Coronavirus morphogenesis ,Nuclear Envelope ,viruses ,Cell ,Biology ,Endoplasmic Reticulum ,Virus Replication ,SARS‐CoV‐2 ,03 medical and health sciences ,0302 clinical medicine ,Microscopy, Electron, Transmission ,Chlorocebus aethiops ,Electron microscopy ,medicine ,Animals ,Humans ,Endomembrane system ,Vero Cells ,030304 developmental biology ,Host cell membrane ,0303 health sciences ,SARS-CoV-2 ,Virus Assembly ,Endoplasmic reticulum ,COVID-19 ,Intracellular Membranes ,Cell Biology ,General Medicine ,Membrane budding ,Cell biology ,medicine.anatomical_structure ,Cytoplasm ,Vero cell ,030217 neurology & neurosurgery ,Intracellular ,Research Article - Abstract
Background Information Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS‐CoV‐2 isolate from São Paulo state (Brazil). Results Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. Conclusions and Significance This study contributes to a better understanding of the cell biology of SARS‐CoV‐2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus‐induced membrane remodelling., Research article: Schematic view of the route of nascent severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) through the compartments of the viral factory. SARS‐CoV‐2 infection induces an intense intracellular membrane remodelling in Vero cells. This alteration protects and delimits the locus of viral replication, morphogenesis and maturation. The intermediate steps of SARS‐CoV‐2 morphogenesis were approached by transmission electron microscopy and electron tomography, revealing and describing different membrane structures and the assembly of viral particles by budding through the endoplasmic reticulum‐Golgi intermediate compartment.
- Published
- 2021
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