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Journal of Virology, July 2003, p. 7764-7778, Vol. 77, No. 14
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.14.7764-7778.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutations within the ADP (E3-11.6K) Protein Alter Processing and Localization of ADP and the Kinetics of Cell Lysis of Adenovirus-Infected Cells

Ann E. Tollefson, Abraham Scaria,{dagger} Baoling Ying, and William S. M. Wold*

Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104

Received 10 July 2002/ Accepted 21 April 2003

ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic NendoCexo nuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2 adp gene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, 1402 S. Grand Blvd., St. Louis, MO 63104. Phone: (314) 577-8435. Fax: (314) 773-3403. E-mail: woldws{at}slu.edu.

{dagger} Present address: Genzyme Corporation, Framingham, MA 01701.

Journal of Virology, July 2003, p. 7764-7778, Vol. 77, No. 14
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.14.7764-7778.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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