scholarly journals An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

1999 ◽  
Vol 73 (6) ◽  
pp. 4600-4610 ◽  
Author(s):  
Joseph S. Orlando ◽  
David A. Ornelles
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Protein Function ◽  
Alpha Helix ◽  
Adenovirus Type ◽  
Wild Type ◽  
Reading Frame ◽  
Α Helix ◽  
Amphipathic Alpha Helix ◽  
Adenovirus Type 5

ABSTRACT A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein

1992 ◽  
Vol 12 (12) ◽  
pp. 5673-5682 ◽  
Author(s):  
A D Bergemann ◽  
Z W Ma ◽  
E M Johnson
Keyword(s):  
Amino Acids ◽  
Alpha Helix ◽  
Element Analysis ◽  
Sequence Element ◽  
Single Stranded Dna ◽  
Reading Frame ◽  
Consensus Motif ◽  
Rich Domain ◽  
Amphipathic Alpha Helix ◽  
Dna Binding Properties

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

scholarly journals Adenovirus Type 5 E4 Open Reading Frame 4 Protein Induces Apoptosis in Transformed Cells

1998 ◽  
Vol 72 (4) ◽  
pp. 2975-2982 ◽  
Author(s):  
Ronit Shtrichman ◽  
Tamar Kleinberger
Keyword(s):  
Adenovirus Type ◽  
Open Reading Frame ◽  
Wild Type ◽  
3T3 Cells ◽  
Reading Frame ◽  
293 Cells ◽  
Lung Carcinoma Cell Line ◽  
Phosphatase 2A ◽  
Nih 3T3 ◽  
Adenovirus Type 5

ABSTRACT Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junBand c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.

scholarly journals Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

1992 ◽  
Vol 12 (12) ◽  
pp. 5673-5682 ◽  
Author(s):  
A D Bergemann ◽  
Z W Ma ◽  
E M Johnson
Keyword(s):  
Amino Acids ◽  
Alpha Helix ◽  
Element Analysis ◽  
Sequence Element ◽  
Single Stranded Dna ◽  
Reading Frame ◽  
Consensus Motif ◽  
Rich Domain ◽  
Amphipathic Alpha Helix ◽  
Dna Binding Properties

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

The Drosophila gene Bearded encodes a novel small protein and shares 3′ UTR sequence motifs with multiple Enhancer of split complex genes

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4039-4051 ◽  
Author(s):  
M.W. Leviten ◽  
E.C. Lai ◽  
J.W. Posakony
Keyword(s):  
Sequence Similarity ◽  
Notch Pathway ◽  
Alpha Helix ◽  
Sequence Motifs ◽  
Wild Type ◽  
Gain Of Function ◽  
Small Protein ◽  
Drosophila Gene ◽  
Amphipathic Alpha Helix ◽  
Enhancer Of Split

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264–283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3′ untranslated region (UTR). The wild-type Brd 3′ UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the ‘Brd box’. Moreover, the 3′ UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the ‘GY box’ (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3′ UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome

1984 ◽  
Vol 4 (2) ◽  
pp. 302-309
Author(s):  
D Hanahan ◽  
Y Gluzman
Keyword(s):  
Viral Genome ◽  
Infectious Virus ◽  
Adenovirus Type ◽  
Small Fragment ◽  
Origin Of Replication ◽  
Wild Type ◽  
Base Pairs ◽  
Bacterial Plasmid ◽  
Dna Fragment ◽  
Adenovirus Type 5

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

scholarly journals Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes

2002 ◽  
Vol 83 (6) ◽  
pp. 1311-1314 ◽  
Author(s):  
Hendrik J. Rademaker ◽  
Mohamed A. Abou El Hassan ◽  
Gijs A. Versteeg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Hepg2 Cells ◽  
Adenovirus Type ◽  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Wild Type ◽  
Genome Packaging ◽  
Firefly Luciferase Gene ◽  
Adenovirus Vectors ◽  
Initiation Of Dna Replication ◽  
Adenovirus Type 5

Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a ΔE1ΔE3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing ΔE1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.

scholarly journals Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

2000 ◽  
Vol 74 (11) ◽  
pp. 5168-5181 ◽  
Author(s):  
Michael Nevels ◽  
Susanne Rubenwolf ◽  
Thilo Spruss ◽  
Hans Wolf ◽  
Thomas Dobner
Keyword(s):  
Tumor Suppressor ◽  
Tumor Growth ◽  
Viral Protein ◽  
Alpha Helix ◽  
Adenovirus Type ◽  
Viral Oncoprotein ◽  
Oncogenic Potential ◽  
P53 Stability ◽  
Carboxy Terminal ◽  
Adenovirus Type 5

ABSTRACT Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal “oncodomain” of the viral protein.

scholarly journals Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production

2009 ◽  
Vol 83 (10) ◽  
pp. 4778-4790 ◽  
Author(s):  
Orkide Ö. Koyuncu ◽  
Thomas Dobner
Keyword(s):  
Nuclear Localization ◽  
Cell Function ◽  
Nonstructural Protein ◽  
Adenovirus Type ◽  
Arginine Methylation ◽  
Structural Protein ◽  
Modification System ◽  
Virus Growth ◽  
Arginine Residues ◽  
Adenovirus Type 5

ABSTRACT The adenovirus type 5 (Ad5) late region 4 (L4) 100-kDa nonstructural protein (L4-100K) mediates inhibition of cellular protein synthesis and selective translation of tripartite leader (TL)-containing viral late mRNAs via ribosome shunting. In addition, L4-100K has been implicated in the trimerization and nuclear localization of hexon protein. We previously proved that L4-100K is a substrate of the protein arginine methylation machinery, an emergent posttranslational modification system involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing, and DNA repair. As understood at present, L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. To identify the methylated arginine residues and assess the role of L4-100K arginine methylation, we generated amino acid substitution mutations in the RGG and GAR motifs to examine their effects in Ad-infected and plasmid-transfected cells. Arginine-to-glycine exchanges in the RGG boxes significantly diminished L4-100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation in RGG motifs is an important host cell function required for efficient Ad replication. Our data further indicate that PRMT1-catalyzed arginine methylation in the RGG boxes regulates the binding of L4-100K to hexon and promotes the capsid assembly of the structural protein as well as modulating TL-mRNA interaction. Furthermore, substitutions in GAR, but not RGG, regions affected L4-100K nuclear import, implying that the nuclear localization signal of L4-100K is located within the GAR sequence.

scholarly journals Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

2001 ◽  
Vol 75 (23) ◽  
pp. 11284-11291 ◽  
Author(s):  
David A. Einfeld ◽  
Rosanna Schroeder ◽  
Peter W. Roelvink ◽  
Alena Lizonova ◽  
C. Richter King ◽  
...  
Keyword(s):  
Adenovirus Type ◽  
Wild Type ◽  
Base Modification ◽  
Model Following ◽  
Vector Distribution ◽  
High Level ◽  
Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

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