This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shen, Y.
Right arrow Articles by Hermiston, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shen, Y.
Right arrow Articles by Hermiston, T.

 Previous Article  |  Next Article 

Journal of Virology, May 2001, p. 4297-4307, Vol. 75, No. 9
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.9.4297-4307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

Yuqiao Shen,* Galila Kitzes, Julie A. Nye, Ali Fattaey, and Terry Hermistondagger

ONYX Pharmaceuticals, Inc., Richmond, California 94806

Received 1 September 2000/Accepted 6 February 2001

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.

* Corresponding author. Mailing address: ONYX Pharmaceuticals, Inc., 3031 Research Dr., Richmond, CA 94806. Phone: (510) 243-3679. Fax: (510) 222-9758. E-mail: jshen{at}onyx-pharm.com.

dagger Present address: Berlex Laboratories, Inc., Richmond, CA 94804-0099.

Journal of Virology, May 2001, p. 4297-4307, Vol. 75, No. 9
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.9.4297-4307.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Dallaire, F., Blanchette, P., Branton, P. E. (2009). A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases. J. Virol. 83: 12172-12184 [Abstract] [Full Text]  
  • Blackford, A. N., Grand, R. J. A. (2009). Adenovirus E1B 55-Kilodalton Protein: Multiple Roles in Viral Infection and Cell Transformation. J. Virol. 83: 4000-4012 [Full Text]  
  • Miller, D. L., Rickards, B., Mashiba, M., Huang, W., Flint, S. J. (2009). The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription. J. Virol. 83: 3591-3603 [Abstract] [Full Text]  
  • Schwartz, R. A., Lakdawala, S. S., Eshleman, H. D., Russell, M. R., Carson, C. T., Weitzman, M. D. (2008). Distinct Requirements of Adenovirus E1b55K Protein for Degradation of Cellular Substrates. J. Virol. 82: 9043-9055 [Abstract] [Full Text]  
  • Zheng, X., Rao, X.-M., Gomez-Gutierrez, J. G., Hao, H., McMasters, K. M., Zhou, H. S. (2008). Adenovirus E1B55K Region Is Required To Enhance Cyclin E Expression for Efficient Viral DNA Replication. J. Virol. 82: 3415-3427 [Abstract] [Full Text]  
  • Luo, K., Ehrlich, E., Xiao, Z., Zhang, W., Ketner, G., Yu, X.-F. (2007). Adenovirus E4orf6 assembles with Cullin5-ElonginB-ElonginC E3 ubiquitin ligase through an HIV/SIV Vif-like BC-box to regulate p53. FASEB J. 21: 1742-1750 [Abstract] [Full Text]  
  • Woo, J. L., Berk, A. J. (2007). Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA Nuclear Export. J. Virol. 81: 575-587 [Abstract] [Full Text]  
  • Li, X., Raikwar, S. P., Liu, Y.-H., Lee, S.-J., Zhang, Y.-P., Zhang, S., Cheng, L., Lee, S.-D., Juliar, B. E., Gardner, T. A., Jeng, M.-H., Kao, C. (2006). Combination therapy of androgen-independent prostate cancer using a prostate restricted replicative adenovirus and a replication-defective adenovirus encoding human endostatin-angiostatin fusion gene.. Molecular Cancer Therapeutics 5: 676-684 [Abstract] [Full Text]  
  • Gonzalez, R., Huang, W., Finnen, R., Bragg, C., Flint, S. J. (2006). Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts. J. Virol. 80: 964-974 [Abstract] [Full Text]  
  • Chu, R. L., Post, D. E., Khuri, F. R., Van Meir, E. G. (2004). Use of Replicating Oncolytic Adenoviruses in Combination Therapy for Cancer. Clin. Cancer Res. 10: 5299-5312 [Abstract] [Full Text]  
  • Hobom, U., Dobbelstein, M. (2004). E1B-55-Kilodalton Protein Is Not Required To Block p53-Induced Transcription during Adenovirus Infection. J. Virol. 78: 7685-7697 [Abstract] [Full Text]  
  • Stubdal, H., Perin, N., Lemmon, M., Holman, P., Bauzon, M., Potter, P. M., Danks, M. K., Fattaey, A., Dubensky, T., Johnson, L. (2003). A Prodrug Strategy Using ONYX-015-Based Replicating Adenoviruses to Deliver Rabbit Carboxylesterase to Tumor Cells for Conversion of CPT-11 to SN-38. Cancer Res. 63: 6900-6908 [Abstract] [Full Text]  
  • Yan, W., Kitzes, G., Dormishian, F., Hawkins, L., Sampson-Johannes, A., Watanabe, J., Holt, J., Lee, V., Dubensky, T., Fattaey, A., Hermiston, T., Balmain, A., Shen, Y. (2003). Developing Novel Oncolytic Adenoviruses through Bioselection. J. Virol. 77: 2640-2650 [Abstract] [Full Text]  
  • van Beusechem, V. W., van den Doel, P. B., Grill, J., Pinedo, H. M., Gerritsen, W. R. (2002). Conditionally Replicative Adenovirus Expressing p53 Exhibits Enhanced Oncolytic Potency. Cancer Res. 62: 6165-6171 [Abstract] [Full Text]  
  • Harada, J. N., Shevchenko, A., Shevchenko, A., Pallas, D. C., Berk, A. J. (2002). Analysis of the Adenovirus E1B-55K-Anchored Proteome Reveals Its Link to Ubiquitination Machinery. J. Virol. 76: 9194-9206 [Abstract] [Full Text]  
  • Lober, C., Lenz-Stoppler, C., Dobbelstein, M. (2002). Adenovirus E1-transformed cells grow despite the continuous presence of transcriptionally active p53. J. Gen. Virol. 83: 2047-2057 [Abstract] [Full Text]  
  • Gonzalez, R. A., Flint, S. J. (2002). Effects of Mutations in the Adenoviral E1B 55-Kilodalton Protein Coding Sequence on Viral Late mRNA Metabolism. J. Virol. 76: 4507-4519 [Abstract] [Full Text]  
  • Orlando, J. S., Ornelles, D. A. (2002). E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein. J. Virol. 76: 1475-1487 [Abstract] [Full Text]  
  • Querido, E., Blanchette, P., Yan, Q., Kamura, T., Morrison, M., Boivin, D., Kaelin, W. G., Conaway, R. C., Conaway, J. W., Branton, P. E. (2001). Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev. 15: 3104-3117 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»