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.

scholarly journals Fusion of Large Polypeptides to Human Adenovirus Type 5 Capsid Protein IX Can Compromise Virion Stability and DNA Packaging Capacity

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Kathy L. Poulin ◽  
Emily R. McFall ◽  
Grace Chan ◽  
Natacha B. Provost ◽  
Carin Christou ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Fluorescent Proteins ◽  
Minor Component ◽  
Human Adenovirus ◽  
Heat Stability ◽  
Adenovirus Type ◽  
Dna Packaging ◽  
Wild Type ◽  
Packaging Capacity ◽  
Protein Ix

ABSTRACT The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function. IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.

scholarly journals Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases

2000 ◽  
Vol 4 (2) ◽  
pp. 147-153 ◽  
Author(s):  
LILIANA E. SCARAFIA ◽  
ANDREAS WINTER ◽  
DAVID C. SWINNEY
Keyword(s):  
Cell Types ◽  
Jurkat Cells ◽  
Lung Fibroblasts ◽  
Absolute Quantitation ◽  
Hect Domain ◽  
E3 Ligases ◽  
Quantitative Expression ◽  
C Terminus ◽  
Cellular Specificity ◽  
Carboxy Terminal

We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is “homologous to E6AP C-terminus”). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20–100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.

scholarly journals Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

Virology ◽  
2004 ◽  
Vol 320 (2) ◽  
pp. 291-300 ◽  
Author(s):  
Alexander Zakhartchouk ◽  
Wayne Connors ◽  
Andrew van Kessel ◽  
Suresh Kumar Tikoo
Keyword(s):  
Capsid Protein ◽  
Heterologous Protein ◽  
Adenovirus Type ◽  
Minor Capsid Protein ◽  
Bovine Adenovirus ◽  
C Terminus ◽  
Protein Ix ◽  
Bovine Adenovirus Type 3 ◽  
Type 3

scholarly journals Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

2010 ◽  
Vol 84 (19) ◽  
pp. 10074-10086 ◽  
Author(s):  
Kathy L. Poulin ◽  
Robert M. Lanthier ◽  
Adam C. Smith ◽  
Carin Christou ◽  
Milagros Risco Quiroz ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Virus Infection ◽  
Capsid Protein ◽  
Cell Types ◽  
Single Domain ◽  
Single Chain ◽  
Single Domain Antibody ◽  
Single Domain Antibodies ◽  
Genetic Fusion ◽  
Protein Ix

ABSTRACT Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

scholarly journals Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIA3

1973 ◽  
Vol 133 (4) ◽  
pp. 641-654 ◽  
Author(s):  
Louis S. Swart ◽  
Thomas Haylett
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Minor Component ◽  
Amino Acid Sequences ◽  
Edman Degradation ◽  
Merino Wool ◽  
C Terminus ◽  
A Minor

The complete amino acid sequences of wool protein SCMKB-IIIA3 (131 residues) and a minor component SCMKB-IIIA3A (130 residues) have been determined. The proteins are mutually homologous and have free threonine as the N-terminal residue and carboxymethylcysteine as the C-terminus. The peptides used for the sequence work were obtained by trypsin, thermolysin, pepsin and chymotrypsin digestions and were fractionated by chromatography on DEAE-cellulose, gel filtration on Sephadex G-25 and G-50, paper chromatography and electrophoresis. The Edman degradation method (employing both the Beckman Sequencer and a non-automatic procedure) was used to obtain the sequences of the peptides.

scholarly journals Activation of Adenoviral Gene Expression by Protein IX Is Not Required for Efficient Virus Replication

2004 ◽  
Vol 78 (10) ◽  
pp. 5032-5037 ◽  
Author(s):  
Kathy L. Sargent ◽  
Robert A. Meulenbroek ◽  
Robin J. Parks
Keyword(s):  
Minor Component ◽  
Transient Transfection ◽  
Minimal Effect ◽  
Protein Coding ◽  
Coding Sequences ◽  
Late Genes ◽  
Viral Promoters ◽  
Protein Ix ◽  
A Minor

ABSTRACT The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and is in part responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the DNA content is greater than ∼35 kb. More recently, pIX has been identified as a transcriptional activator and, in transient-transfection assays, was shown to enhance expression from the E1A, E4, and major late Ad promoters by as much as 70-fold. In this study, we examined the role of pIX's ability to activate transcription during Ad replication. In transient-transfection assays, pIX had a minimal effect on expression from the E1A promoter, increasing expression by only 1.4-fold. We used helper-dependent Ad vectors, which had all Ad protein coding sequences deleted with the exception of E1A and which had capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad genome did not alter the expression levels of E1A. Viruses that had pIX deleted showed a threefold reduction in virus yield and expression of late genes compared to those of a similar virus which encoded pIX. This phenotype could not be rescued by growing the virus in cells which constitutively express pIX. Our results indicate that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.

scholarly journals The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawad Hodeify ◽  
Maya Dib ◽  
Ethel Alcantara-Adap ◽  
Raphael Courjaret ◽  
Nancy Nader ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Coiled Coil ◽  
Structural Motif ◽  
Deletion Mutants ◽  
Binding Motif ◽  
Terminal Sequence ◽  
Molecular Determinant ◽  
C Terminus ◽  
The Stability ◽  
Carboxy Terminal

AbstractRegulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

scholarly journals In vitroassembly of the Rous Sarcoma Virus capsid protein into hexamer tubes under physiological conditions

2017 ◽  
Author(s):  
Soumeya A. Jaballah ◽  
Graham D. Bailey ◽  
Ambroise Desfosses ◽  
Jaekyung Hyun ◽  
Alok K. Mitra ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Rous Sarcoma Virus ◽  
Tube Formation ◽  
Maturation Process ◽  
Rous Sarcoma ◽  
C Terminus ◽  
Sarcoma Virus ◽  
Tube Assembly ◽  
A Minor

ABSTRACTDuring a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assemblesin vitroat physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.

scholarly journals VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

2016 ◽  
Vol 27 (18) ◽  
pp. 2811-2821 ◽  
Author(s):  
Benjamin F. Brinkmann ◽  
Tim Steinbacher ◽  
Christian Hartmann ◽  
Daniel Kummer ◽  
Denise Pajonczyk ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Vascular Endothelial Cells ◽  
Protein Complexes ◽  
Cell Types ◽  
Cell Junctions ◽  
Binding Motif ◽  
Lumen Formation ◽  
Vascular Lumen ◽  
Apicobasal Polarity ◽  
C Terminus

Blood vessel tubulogenesis requires the formation of stable cell-to-cell contacts and the establishment of apicobasal polarity of vascular endothelial cells. Cell polarity is regulated by highly conserved cell polarity protein complexes such as the Par3-aPKC-Par6 complex and the CRB3-Pals1-PATJ complex, which are expressed by many different cell types and regulate various aspects of cell polarity. Here we describe a functional interaction of VE-cadherin with the cell polarity protein Pals1. Pals1 directly interacts with VE-cadherin through a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. VE-cadherin clusters Pals1 at cell–cell junctions. Mutating the Pals1-binding motif in VE-cadherin abrogates the ability of VE-cadherin to regulate apicobasal polarity and vascular lumen formation. In a similar way, deletion of the Par3-binding motif at the C-terminus of VE-cadherin impairs apicobasal polarity and vascular lumen formation. Our findings indicate that the biological activity of VE-cadherin in regulating endothelial polarity and vascular lumen formation is mediated through its interaction with the two cell polarity proteins Pals1 and Par3.

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