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 Analysis of Synthesis, Stability, Phosphorylation, and Interacting Polypeptides of the 34-Kilodalton Product of Open Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus Type 5

1999 ◽  
Vol 73 (2) ◽  
pp. 1245-1253 ◽  
Author(s):  
Dominique Boivin ◽  
Megan R. Morrison ◽  
Richard C. Marcellus ◽  
Emmanuelle Querido ◽  
Philip E. Branton
Keyword(s):  
Protein Synthesis ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Open Reading Frame ◽  
Cellular Proteins ◽  
Cell Protein ◽  
Reading Frame ◽  
Early Region ◽  
Early Region 4 ◽  
Adenovirus Type 5

ABSTRACT The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

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.

scholarly journals Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7

2012 ◽  
Vol 86 (15) ◽  
pp. 8296-8308 ◽  
Author(s):  
D. Muller ◽  
S. Schreiner ◽  
M. Schmid ◽  
P. Groitl ◽  
M. Winkler ◽  
...  
Keyword(s):  
Human Adenovirus ◽  
Adenovirus Type ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Early Region ◽  
Homeobox Protein ◽  
Early Region 4 ◽  
Adenovirus Type 5

scholarly journals Murine Cytomegalovirus Containing a Mutation at Open Reading Frame M37 Is Severely Attenuated in Growth and Virulence In Vivo

2000 ◽  
Vol 74 (23) ◽  
pp. 11099-11107 ◽  
Author(s):  
Manfred Lee ◽  
Jianqiao Xiao ◽  
Erik Haghjoo ◽  
Xiaoyan Zhan ◽  
Gerry Abenes ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Mutant ◽  
Intraperitoneal Infection ◽  
Nih 3T3

ABSTRACT A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 × 105, 2 × 105, 5 × 104, 5 × 103, and 1 × 104 PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 102 PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.

scholarly journals Truncating the i-leader open reading frame enhances release of human adenovirus type 5 in glioma cells

2011 ◽  
Vol 8 (1) ◽  
pp. 162 ◽  
Author(s):  
Sanne K van den Hengel ◽  
Jeroen de Vrij ◽  
Taco G Uil ◽  
Martine L Lamfers ◽  
Peter AE Sillevis Smitt ◽  
...  
Keyword(s):  
Glioma Cells ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Adenovirus Type 5

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Coding sequence and expression of the homeobox gene Hox 1.3

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 349-359 ◽  
Author(s):  
M. Fibi ◽  
B. Zink ◽  
M. Kessel ◽  
A.M. Colberg-Poley ◽  
S. Labeit ◽  
...  
Keyword(s):  
Homeobox Gene ◽  
Cell System ◽  
T Antigen ◽  
Open Reading Frame ◽  
Homeodomain Protein ◽  
Chromosome 11 ◽  
3T3 Cells ◽  
Exon 2 ◽  
Reading Frame ◽  
3T3 Fibroblasts

We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6. The protein sequence of 270 amino acids was deduced from the nucleotide sequence of an open reading frame containing the homeobox. The open reading frame is interrupted at the genomic level by a 960 bp intron and is organized in two exons. The Hox 1.3 protein was found to contain extensive sequence homology with the murine homeodomain protein Hox 2.1, which is encoded on chromosome 11. There are two homology with the regions in the first exon, i.e. a hexapeptide conserved in many homeobox-containing genes and the N-terminal domain, which was found to be homologous only to Hox 2.1. Furthermore, in exon 2 the homologies of the homeodomain regions are extended up to the carboxy terminus of Hox 1.3 and Hox 2.1. During prenatal murine development, maximal expression of Hox 1.3 is observed in 12-day embryonic tissue. The two transcripts carrying the Hox 1.3 homeobox are 1.9 kb and about 4 kb in length. An abundant Hox 1.3-specific 1.9 kb RNA is also found in F9 cells which were induced for parietal endoderm differentiation, whereas F9 teratocarcinoma stem cells do not stably express this specific RNA. Induction of the transcript occurs immediately after retinoic acid/cAMP treatment and the RNA level remains high for 5 days. Thus, the kinetics are different from the previously described homeobox transcripts Hox 1.1 and Hox 3.1. Interestingly, by analogy to the F9 cell system a negative correlation between transformation and Hox 1.3 expression is observed in 3T3 fibroblasts also. Untransformed 3T3 cells carry abundant 1.9 kb Hox 1.3 RNA, whereas the methylcholanthrene-transformed MB66 and LTK- cells or 3T3 cells transformed by the oncogenes src, fos or SV40 T antigen express only low levels.

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.

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