A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus

Tollefson, Ann E.; Krajcsi, Péter; Yei, Soonpin; Carlin, Cathleen R.; Wold, William S.M.

doi:10.1128/jvi.64.2.794-801.1990

Cited by 53 publications

(37 citation statements)

References 44 publications

(33 reference statements)

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“…Adenovirus stocks were grown in spinner cultures of human KB cells maintained with Joklik's-modified MEM supplemented with 5% horse serum and 2 mM glutamine, and titres were determined by plaque assay using human embryonic kidney 293 cells (Graham et al, 1977) grown in D-MEM supplemented with 10% FBS and 2 mM glutamine. Two adenovirus mutants were used in this study: in724 is a splice-donor insertion mutant that overproduces the 13.7 kDa E3 protein; and dl753 has a n internal deletion of 207 nucleotides in the 13.7 kDa open reading frame and does not produce 13.7 kDa-related proteins (Hoffman et al, 1992a;Tollefson et al, 1990). Cells were acutely infected with 200 PFU per cell in serum-free MEM for 1 hr, then switched to medium containing 10% FBS and 20 kg/ml arabinofuranosyl-cytosine (ara-C; Sigma); ara-C enhances early viral protein synthesis and blocks late viral gene synthesis thereby preventing lytic infections (Kelly and Lewis, 1973).…”

Section: Methodsmentioning

confidence: 99%

“…The EGF-R1 monoclonal antibody (mAb) recognizes an extracellular peptide determinant of the human EGF receptor (Carlin and Knowles, 1984). The E3 protein-specific rabbit polyclonal antiserum was made against a synthetic 15mer corresponding to the C-terminus (Hoffman et al, 1990;Tollefson et al, 1990). mAbs specific for the transferrin receptor (OKT-9) and E1A (M73) were purchased from ATCC and Oncogene Sciences, respectively.…”

Section: Cell Labeling and Immunoprecipitationmentioning

confidence: 99%

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Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking

Hoffman

Takishima

Rosner

et al. 1993

Journal Cellular Physiology

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The ligand-activated tyrosine kinase receptor for epidermal growth factor (EGF) is down-regulated by an integral membrane protein coded for by the E3 early transcription unit of group C adenoviruses. The E3 protein appears to block recycling of constitutively internalized receptors, causing them instead to traffic to lysosomes where they are degraded. Expression of functional EGF receptors is also regulated by protein kinase C (PKC), which directly phosphorylates the EGF receptor at Thr-654. The goal of this study was to determine potential interactions between PKC and the E3 protein, since membrane-bound PKC activity is elevated by the adenovirus E1A protein. Our results show that although tumor promoters which activate PKC cause a coordinate induction of E3 protein synthesis and EGF receptor degradation, the E3 protein-induced pathway for receptor down-regulation functions independently of PKC and other kinases that are inhibited by staurosporine. This suggests that in contrast to other mechanisms that modulate receptor expression (i.e., ligand and PKC), the E3 protein is not regulated by phosphorylation but is constitutively active. We also report that adenovirus-mediated degradation is the preferred pathway in infected cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce receptor recycling.

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Section: Methodsmentioning

confidence: 99%

Section: Cell Labeling and Immunoprecipitationmentioning

confidence: 99%

Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking

Hoffman

Takishima

Rosner

et al. 1993

Journal Cellular Physiology

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“…2A, lane 1). d1759 was analyzed instead of AdS because the deletion in d1759 (A2488-2803), which reduces the sizes of mRNAs c and f (36), allows the four E3 mRNAs to be detected more clearly (in AdO, mRNAs a and f nearly comigrate). No mRNAs were detected in either total cytoplasmic ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor

Ranheim

Shisler

Horton

et al. 1993

J Virol

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The 14,700-Da protein (14.7K protein) encoded by the E3 region of adenovirus has previously been shown to protect mouse cells from cytolysis by tumor necrosis factor (TNF). Delineating the sequences in the 14.7K protein that are required for this activity may provide insight into the mechanism of protection from TNF by 14.7K as well as the mechanism of TNF cytolysis. In the present study, we examined the ability of 14.7K mutants to protect cells from lysis by TNF. In-frame deletions as well as Cys-to-Ser mutations in the 14.7K gene were generated by site-directed mutagenesis and then built into the genome of a modified adenovirus type 5 (d17001) that lacks all E3 genes. d17001, which replicates to the same titers as does adenovirus type 5 in cultured cells, has the largest E3 deletion analyzed to date. 51Cr release was used to assay TNF cytolysis. Our results indicate that most mutations in the 14.7K gene result in a loss of function, suggesting that nearly the entire

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“…Peptide P14-27 with the sequence (C) KTELRPSYGLPLLQ, corresponding to residues 14 to 27 in the predicted Ad3 16K protein, was purchased from Multiple Peptide Systems (San Diego, Calif.). The peptide was coupled to keyhole limpet hemocyanin, and rabbits were immunized with the peptide conjugate as described previously (66). Antiserum to protein pVIII was prepared in rabbits against a TrpE-pVIII fusion protein expressed in Escherichia coli; details will be presented elsewhere.…”

Section: Methodsmentioning

confidence: 99%

Region E3 of subgroup B human adenoviruses encodes a 16-kilodalton membrane protein that may be a distant analog of the E3-6.7K protein of subgroup C adenoviruses

Hawkins

Wilson‐Rawls

Wold

1995

J Virol

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There is an open reading frame in the E3 transcription unit of adenovirus type 3 (Ad3) and Ad7 that could encode a protein of 16 kDa (16K protein). Ad3 and Ad7 are members of subgroup B of human adenoviruses. Using a rabbit antipeptide antiserum, we show that the 16K protein is expressed in Ad3-and Ad7-infected cells at early and late stages of infection; it is not expressed in cells infected with an Ad7 mutant that deletes the 16K gene. The 16K protein was also transcribed and translated in vitro from DNA containing the open reading frame for the 16K protein. The 16K protein has two hydrophobic domains typical of integral membrane proteins; consistent with this, we detected 16K in the crude membrane but not the cytosol cellular fractions. Although 16K has two potential sites for Asn-linked glycosylation, the protein is not glycosylated. The 16K gene is located in the same position in region E3 as the gene for the 6.7K protein of subgroup C adenoviruses (Ad2 and Ad5). E3-6.7K is an Asn-linked integral membrane glycoprotein, localized in the endoplasmic reticulum, whose function is unknown. The 16K protein has a putative transmembrane domain located in the same place in 16K as is the transmembrane domain in 6.7K, and the C-terminal portion of 16K is partially homologous to the C-terminal cytoplasmic domain of 6.7K; we suggest that these domains in 16K and 6.7K may have a similar function. The N-terminal 102 residues in 16K are not found in 6.7K; these residues may have a function that is unique to the 16K protein. In common with all known E3 proteins, the 16K protein is dispensable for virus replication in cultured cells; this suggests that the 16K protein may function in virus-host interactions.

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A 10,400-molecular-weight membrane protein is coded by region E3 of adenovirus

Abstract: Previous studies with adenovirus mutants have indicated that a 10,400-molecular-weight (10.4K) protein predicted to be coded by an open reading frame in region E3 of adenovirus functions to down regulate the epidermal growth factor receptor (

Cited by 53 publications

References 44 publications

Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking

Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking

Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor

Region E3 of subgroup B human adenoviruses encodes a 16-kilodalton membrane protein that may be a distant analog of the E3-6.7K protein of subgroup C adenoviruses

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