Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein

Klessig, Daniel F.; Grodzicker, Terri

doi:10.1016/0092-8674(79)90335-0

Cited by 167 publications

(121 citation statements)

References 36 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…1) or by infection with Ad2-SV40 hybrid viruses (e.g., Ad2+ND1) that contain a segment of the SV40 genome encoding only the carboxyl terminus of the SV40 large tumor antigen (2)(3)(4)(5). In addition, mutants of human adenovirus have been isolated (Ad2hr400-403, Ad5hr404, Ad2+ND3hr600-603) that grow efficiently in monkey cells as well as human cells (6)(7)(8). All nine of these independent isolates have achieved their altered host range phenotype as a result of identical point mutations in the amino terminus of the adenovirus DNA binding protein (refs.…”

mentioning

confidence: 99%

“…The striking reduction in the presence of the x and y ancillary leaders on fiber messages of abortively infected CV-1 cells correlates with the inability of abortively infected CV-1 cells to synthesize the fiber polypeptide and suggests that the x and/or y leaders may play an important role in translation of the fiber mRNA in vivo. To test the effect of the y leader on translation of the fiber message in monkey cells, we took advantage of the observation that the mutations in the DNA-binding protein of adenovirus host range mutants (e.g., Ad2hr400 or AdShr4O4) will act in trans to allow expression in monkey cells of the fiber gene carried by a coinfecting wild-type adenovirus (7). This observation was facilitated by the fact that the fiber polypeptides of the closely related Ad2 and adenovirus serotype 5 (AdS) migrate with slightly different electrophoretic mobilities in NaDodSO4/ polyacrylamide gels.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Anderson¹,

Klessig²

1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Anderson¹,

Klessig²

1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The DBP directly affects early gene turn-off as well as DNA replication, since inhibition of viral DNA synthesis with either drugs or ts lesions in other viral early genes does not affect early gene expression (9, 10). In addition, at the nonpermissive temperatui*, the ts mutants transform rat cells in culture with an enhanced frequency compared with wild-type (wt) virus (16,54).A second class of mutants which greatly enhance the ability of this human virus to grow in monkey cells defines yet a second set of functions of DBP (1,26,29). In monkey cells infected with wt human adenovirus, the viral early genes are properly expressed (3), and viral DNA replication occurs normally (43).…”

mentioning

confidence: 99%

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Klessig¹,

Brough²,

Cleghon³

1984

Mol. Cell. Biol.

View full text Add to dashboard Cite

The DNA-bindihg protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central iole in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by IBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DB3P is totic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50-to 200-fold, and within 8 h its synthesis fromb the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.A number of multifunctional proteins in euearyotes have been defined by,a combination of genetic and biochemical studies. One of the best characterized of these is the simian virus 40 (SV40) large T antigen, which exhibits separate functional domains as discerned by mapping of sets of mutations which alter one but not another of the various protein functions.The human adenovirus 72-kilodalton DNA-binding protein (DBP) also plays several independent roles during the infectious cycle of this virus. Several mutants with temperature sensitive (tsj alterations in the DBP have been indispensable in defining several of the functions of this protein. The ts alterations at the nonpermissive temperature diminish the single-strand, DNA-binding activity of the protein in vitro (52) and inhibit DNA replication in vivo (13, 47, 53) and in vitro (20). Normal turn-off of viral early genes during the late phase of the lytic cycle is also disrupted at the nonpermissive temperature in cells infected with the ts mutant (6,9,10,37,38). The DBP directly affects early gene turn-off as well as DNA replication, since inhibition of viral DNA synthesis with either drugs or ts lesions in other viral early genes does not affect early gene expression (9, 10). In addition, at the nonpermissive temperatui*, the ts mutants transform rat cells in culture with an enhanced frequency compared with wild-type (wt) virus (16,54).A second class of mutants which greatly enhance the ability of this human virus to grow in monkey cells defines yet a second set of functions of DBP (1,26,29). In monkey cells infected with wt human adenovirus, the viral early genes are properly expressed (3), and viral DNA replication occurs no...

show abstract

“…Open-reading frame 6 of the E4 region is important for the conversion of the single-stranded AAV genome into a double-stranded form, which is the substrate for subsequent steps in DNA replication (Ferrari et al, 1996;Fisher et al, 1996). Protein E2A plays a key role in viral DNA replication via binding to AAV viral DNA, promoting DNA elongation (Klessig and Grodzicker, 1979;Chang and Shenk, 1990), and displacement of the elongating strand from its template (Ward et al, 1998). Both YB1 and E2A are DNA binding proteins (DBP) but differ from each other in their cellular and viral origins, respectively.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

et al. 2014

View full text Add to dashboard Cite

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, preexisting immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAVassociated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45-and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

show abstract

Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein

Cited by 167 publications

References 36 publications

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

Contact Info

Product

Resources

About