scholarly journals Near Atomic Structure of an Atadenovirus Reveals a Conserved Capsid-Binding Motif and Intergenera Variations in Cementing Proteins

2020 ◽  
Author(s):  
Roberto Marabini ◽  
Gabriela N. Condezo ◽  
Josué Gómez-Blanco ◽  
Carmen San Martín
Keyword(s):  
Duplication Event ◽  
Specific Protein ◽  
Mammalian Host ◽  
Binding Motif ◽  
Coat Proteins ◽  
Human Adenoviruses ◽  
High Resolution Structure ◽  
Attachment Proteins ◽  
Protein Ix ◽  
Capsid Stability

AbstractLittle is known about the basic biology of non-human adenoviruses, which could be alternative vectors free of issues posed by preexisting immunity to human adenoviruses. We present the cryo-EM structure of a lizard atadenovirus, LAdV-2, at 3.4 Å resolution. This is the first high resolution structure of an adenovirus with non-mammalian host, and of an adenovirus not belonging to the Mastadenovirus genus. Atadenovirus capsids contain genus specific proteins LH3, p32k, and LH2, and are more thermostable than the more studied human adenoviruses. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide in the region. This polypeptide, as well as α-helical clusters located beneath the icosahedral facet, likely correspond to proteins LH2 and p32k. The external genus specific protein LH3, with a trimeric β-helix fold typical of bacteriophage host attachment proteins, contacts the hexon shell surface via a triskelion structure identical to that used by protein IX in human AdV, revealing a conserved capsid-binding motif and a possible gene duplication event. Altogether, this work shows how the network of minor coat proteins differs between AdV genera and relates to virus evolution and capsid stability properties.

scholarly journals Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins

2021 ◽  
Vol 7 (14) ◽  
pp. eabe6008
Author(s):  
Roberto Marabini ◽  
Gabriela N. Condezo ◽  
Mart Krupovic ◽  
Rosa Menéndez-Conejero ◽  
Josué Gómez-Blanco ◽  
...  
Keyword(s):  
High Resolution ◽  
Duplication Event ◽  
Specific Protein ◽  
Binding Motif ◽  
Coat Proteins ◽  
New Information ◽  
High Resolution Structure ◽  
Specific Proteins ◽  
Protein Ix ◽  
Ancient Gene Duplication

Of five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide. This polypeptide, and α-helical clusters beneath the facet, likely correspond to genus-specific proteins LH2 and p32k. Another genus-specific protein, LH3, with a fold typical of bacteriophage tailspikes, contacts the capsid surface via a triskelion structure identical to that used by mastadenovirus protein IX, revealing a conserved capsid-binding motif and an ancient gene duplication event. Our data also suggest that mastadenovirus E1B-55 K was exapted from the atadenovirus-like LH3 protein. This work provides new information on the evolution of adenoviruses, emphasizing the importance of minor coat proteins for determining specific physicochemical properties of virions and most likely their tropism.

scholarly journals Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses

2021 ◽  
Vol 7 (9) ◽  
pp. eabd9421
Author(s):  
Marta Pérez-Illana ◽  
Marta Martínez ◽  
Gabriela N. Condezo ◽  
Mercedes Hernando-Pérez ◽  
Casandra Mangroo ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Viral Gastroenteritis ◽  
Coat Proteins ◽  
Structural Variations ◽  
Starting Point ◽  
Enteric Adenovirus ◽  
Protein Ix ◽  
Capsid Stability ◽  
Harsh Conditions ◽  
Enteric Adenoviruses

Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base, and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and nonhuman mastadenoviruses. Knowledge of the structure of enteric adenoviruses provides a starting point for the design of vectors suitable for oral delivery or intestinal targeting.

scholarly journals Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural divergence among human adenoviruses

2020 ◽  
Author(s):  
Marta Pérez-Illana ◽  
Marta Martínez ◽  
Gabriela N. Condezo ◽  
Mercedes Hernando-Pérez ◽  
Casandra Mangroo ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Viral Gastroenteritis ◽  
Coat Proteins ◽  
Successful Infection ◽  
Starting Point ◽  
Enteric Adenovirus ◽  
Protein Ix ◽  
Capsid Stability ◽  
Harsh Conditions ◽  
Enteric Adenoviruses

AbstractEnteric adenoviruses are one of the main causes of viral gastroenteritis in the world. To carry out a successful infection, the virions must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We have determined the structure of a human enteric adenovirus, HAdV-F41, at 4.0 Å resolution by single particle averaging cryo-electron microscopy, and compared it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and non-human mastadenoviruses. Knowledge of the structure of enteric adenoviruses can provide a starting point for the design of vectors suitable for oral delivery or intestinal targeting.

scholarly journals Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

2002 ◽  
Vol 76 (14) ◽  
pp. 6893-6899 ◽  
Author(s):  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
David T. Curiel
Keyword(s):  
Heparan Sulfate ◽  
Capsid Protein ◽  
Minor Component ◽  
Cell Types ◽  
Binding Motif ◽  
Flag Epitope ◽  
C Terminus ◽  
Protein Ix ◽  
A Minor ◽  
Carboxy Terminal

ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Dictyostelium prespore-specific gene Dp87 encodes a sorus matrix protein

1994 ◽  
Vol 107 (3) ◽  
pp. 397-403 ◽  
Author(s):  
H. Nakao ◽  
A. Yamamoto ◽  
I. Takeuchi ◽  
M. Tasaka
Keyword(s):  
Matrix Protein ◽  
Specific Protein ◽  
Specific Gene ◽  
Coat Proteins ◽  
Fruiting Bodies ◽  
Protein Fragment ◽  
Microscopic Studies ◽  
Spore Coat Proteins ◽  
Space Matrix ◽  
Membranous Material

In this paper we report on the characteristics of the product of a prespore-specific gene (Dp87) of Dictyostelium discoideum. Polyclonal antibody was made against a bacterially synthesized Dp87-encoded protein fragment. Using this antibody, the product was characterized by immunochemical and immunocytological methods. It was shown that the Dp87-encoded protein is a prespore-specific protein with a molecular mass of 83 kDa, which first appears at the standing slug stage and persists in mature fruiting bodies. Western blot studies revealed the presence of an additional 81 kDa protein prior to the appearance of the 83 kDa protein from the tipped aggregate to the standing slug stage, thus indicating the former to be a precursor protein. Immunocytochemical and immunoelectron microscopic studies showed that the protein is bound to ER at the early stages of development when only the 81 kDa protein is present. At the later stages when the 83 kDa protein predominates, however, it becomes localized in prespore-specific vacuoles (PSVs) and is associated with the inner fibrous material of PSVs, but not with the peripheral membranous material. This is in contrast to spore coat proteins, which are localized in PSVs from the beginning of their appearance and associated with both structures of PSVs. In mature fruiting bodies, most Dp87 protein is localized to the interspore space (matrix) of the sori, with some left on the surface of the stalk tube. Disruptants of the Dp87 gene were also produced. Although they contained neither 81 kDa nor 83 kDa protein, they showed no phenotypic defects as compared to the parental strain.

scholarly journals A hydrophobic network: Intersubunit and intercapsomer interactions stabilizing the bacteriophage P22 capsid

2019 ◽  
Author(s):  
Kunica Asija ◽  
Carolyn M. Teschke
Keyword(s):  
Coat Protein ◽  
Hydrophobic Interactions ◽  
Protein Structures ◽  
Antiviral Drug ◽  
Coat Proteins ◽  
Bacteriophage P22 ◽  
Outer Periphery ◽  
Phage P22 ◽  
P Domain ◽  
Capsid Stability

AbstractdsDNA tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P-domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCESimilarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra-and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra-and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

scholarly journals Functional Analysis of Adenovirus Protein IX Identifies Domains Involved in Capsid Stability, Transcriptional Activity, and Nuclear Reorganization

2001 ◽  
Vol 75 (15) ◽  
pp. 7131-7141 ◽  
Author(s):  
Manuel Rosa-Calatrava ◽  
Linda Grave ◽  
Francine Puvion-Dutilleul ◽  
Bruno Chatton ◽  
Claude Kedinger
Keyword(s):  
Self Assembly ◽  
Transcriptional Activity ◽  
Nuclear Retention ◽  
Nuclear Reorganization ◽  
Infected Cells ◽  
Nuclear Structures ◽  
The Stability ◽  
Protein Ix ◽  
Capsid Stability ◽  
Expression Program

ABSTRACT The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology.

scholarly journals Evolution of a σ–(c-di-GMP)–anti-σ switch

2021 ◽  
Vol 118 (30) ◽  
pp. e2105447118
Author(s):  
Maria A. Schumacher ◽  
Kelley A. Gallagher ◽  
Neil A. Holmes ◽  
Govind Chandra ◽  
Max Henderson ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Duplication Event ◽  
Binding Motif ◽  
Ancestral State ◽  
Binding Motifs ◽  
Type Iv ◽  
Small Proteins ◽  
Single Motif ◽  
Internal Gene ◽  
Antiparallel Coiled Coil

Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG–(c-di-GMP)2–WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2–(c-di-GMP)2–WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2–(c-di-GMP)2–WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

scholarly journals Conservation of S20 as an Ineffective and Disposable IFNγ-Inducing Determinant of Plasmodium Sporozoites Indicates Diversion of Cellular Immunity

2021 ◽  
Vol 12 ◽  
Author(s):  
Calvin Hon ◽  
Johannes Friesen ◽  
Alyssa Ingmundson ◽  
Diana Scheppan ◽  
Julius C. R. Hafalla ◽  
...  
Keyword(s):  
Genetic Background ◽  
Cell Epitope ◽  
Specific Protein ◽  
Phase Iii ◽  
Mammalian Host ◽  
Pivotal Phase ◽  
Phase Iii Clinical Trials ◽  
Immunogenic Proteins

Despite many decades of research to develop a malaria vaccine, only one vaccine candidate has been explored in pivotal phase III clinical trials. This candidate subunit vaccine consists of a portion of a single Plasmodium antigen, circumsporozoite protein (CSP). This antigen was initially identified in the murine malaria model and shown to contain an immunodominant and protective CD8+ T cell epitope specific to the H-2Kd (BALB/c)-restricted genetic background. A high-content screen for CD8+ epitopes in the H2Kb/Db (C57BL/6)-restricted genetic background, identified two distinct dominant epitopes. In this study, we present a characterization of one corresponding antigen, the Plasmodium sporozoite-specific protein S20. Plasmodium berghei S20 knockout sporozoites and liver stages developed normally in vitro and in vivo. This potent infectivity of s20(-) sporozoites permitted comparative analysis of knockout and wild-type parasites in cell-based vaccination. Protective immunity of irradiation-arrested s20(-) sporozoites in single, double and triple immunizations was similar to irradiated unaltered sporozoites in homologous challenge experiments. These findings demonstrate the presence of an immunogenic Plasmodium pre-erythrocytic determinant, which is not essential for eliciting protection. Although S20 is not needed for colonization of the mammalian host and for initiation of a blood infection, it is conserved amongst Plasmodium species. Malarial parasites express conserved, immunogenic proteins that are not required to establish infection but might play potential roles in diverting cellular immune responses.

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