scholarly journals Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Tina Motwani ◽  
Carolyn M. Teschke
Keyword(s):  
Virus Assembly ◽  
Genetic Material ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Portal Protein ◽  
Double Stranded Dna ◽  
Dsdna Viruses

ABSTRACTTailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed anin vitroP22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCEThe existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assemblyin vivoindicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

scholarly journals The architect of virus assembly: the portal protein complex nucleates procapsid assembly in bacteriophage P22

2019 ◽  
Author(s):  
Tina Motwani ◽  
Carolyn M. Teschke
Keyword(s):  
Protein Complex ◽  
Virus Assembly ◽  
Genetic Material ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Portal Protein ◽  
Dsdna Viruses

AbstractThe genetic material of tailed dsDNA bacteriophages, herpesviruses and adenoviruses is packaged into a precursor capsid through a 12-mer ring-shaped protein complex called the portal protein, located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and HSV-1, the dodecameric portal protein forms a nucleation complex with scaffolding proteins to initiate procapsid assembly, thereby ensuring incorporation of only one portal complex per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We recently developed anin vitroP22 assembly assay where portal protein is co-assembled into procapsid-like particles. We also showed that scaffolding protein catalyzes oligomerization of monomeric portal protein into 12-mer rings, and possibly forming a scaffolding-protein nucleation complex that results in one portal complex per P22 procapsid. Here, we present evidence substantiating that P22 portal protein, similar to the other dsDNA viruses, can act as an assembly nucleator. We find that the presence of P22 portal protein is able to increase the rate of particle assembly. Additionally, we show that P22 portal protein proper contributes to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature likely conserved among these classes of dsDNA viruses.

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

scholarly journals Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation

2010 ◽  
Vol 84 (17) ◽  
pp. 8821-8828 ◽  
Author(s):  
Manli Wang ◽  
Era Tuladhar ◽  
Shu Shen ◽  
Hualin Wang ◽  
Monique M. van Oers ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Critical Role ◽  
Nucleocapsid Proteins ◽  
Double Stranded Dna ◽  
C Terminus ◽  
Sequence Elements ◽  
Eukaryotic Organisms ◽  
Dsdna Viruses ◽  
Virion Formation

ABSTRACT The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alpha baculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.

scholarly journals Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

2019 ◽  
Vol 116 (9) ◽  
pp. 3556-3561 ◽  
Author(s):  
Oliver W. Bayfield ◽  
Evgeny Klimuk ◽  
Dennis C. Winkler ◽  
Emma L. Hesketh ◽  
Maria Chechik ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Integrity ◽  
Thermus Thermophilus ◽  
Dna Packaging ◽  
Dna Viruses ◽  
Portal Protein ◽  
Single Vertex ◽  
Double Stranded Dna ◽  
Β Sheets

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

scholarly journals Perspectives on Viral RNA Genomes and the RNA Folding Problem

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1126
Author(s):  
Susan J. Schroeder
Keyword(s):  
Defense Mechanisms ◽  
Rna Folding ◽  
Dynamic Equilibrium ◽  
Virus Assembly ◽  
Viral Rna ◽  
Particle Assembly ◽  
Virus Particles ◽  
Chemical Probing

Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.

Structure and assembly of the capsid of bacteriophage P22

1976 ◽  
Vol 276 (943) ◽  
pp. 37-49 ◽  
Keyword(s):  
Coat Protein ◽  
Protein Gene ◽  
Scaffolding Protein ◽  
Bacteriophage P22 ◽  
Particle Assembly ◽  
Thin Sections ◽  
X Ray Scattering ◽  
Phage P22 ◽  
Infected Cells ◽  
Protein Shell

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.

scholarly journals An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

2015 ◽  
Vol 89 (24) ◽  
pp. 12457-12466 ◽  
Author(s):  
Wei Zhao ◽  
Paul J. Jardine ◽  
Shelley Grimes
Keyword(s):  
Viral Genome ◽  
Virus Assembly ◽  
Molecular Motor ◽  
Protein A ◽  
Dna Packaging ◽  
Viral Dna ◽  
High Biological Activity ◽  
Content Type ◽  
Double Stranded Dna

ABSTRACTDuring assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in theBacillus subtilisbacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activityin vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages.IMPORTANCEDuring virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs.

scholarly journals Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly

2016 ◽  
Vol 90 (12) ◽  
pp. 5700-5714 ◽  
Author(s):  
Katrina J. Heyrana ◽  
Boon Chong Goh ◽  
Juan R. Perilla ◽  
Tam-Linh N. Nguyen ◽  
Matthew R. England ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electrostatic Interactions ◽  
Virus Assembly ◽  
Particle Assembly ◽  
Rous Sarcoma ◽  
Charged Residues ◽  
Sarcoma Virus ◽  
Capsid Maturation ◽  
Immature Particle

ABSTRACTExtensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessedin vivoandin vitroand analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection.IMPORTANCEThe production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

scholarly journals Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1504
Author(s):  
Justin C. Leavitt ◽  
Eddie B. Gilcrease ◽  
Brianna M. Woodbury ◽  
Carolyn M. Teschke ◽  
Sherwood R. Casjens
Keyword(s):  
Dna Packaging ◽  
Bacteriophage P22 ◽  
Portal Protein ◽  
Dna Molecule ◽  
Double Stranded Dna ◽  
Phage P22

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.

scholarly journals A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia

2021 ◽  
Vol 22 (21) ◽  
pp. 11902
Author(s):  
Yu-Chien Chen ◽  
Szu-Yu Tung ◽  
Chia-Wei Huang ◽  
Soo-Wah Gan ◽  
Bo-Chi Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cyst Wall ◽  
Giardia Lamblia ◽  
Genetic Material ◽  
Cyst Formation ◽  
Positive Regulator ◽  
Double Stranded Dna ◽  
Recombination Processes

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.

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