Adenovirus early region 4 (E4) encodes proteins critical in a variety of processes required for a successful viral infection. While E4 is not required for DNA replication, three E4 proteins (E4 11k, product of E4 ORF3; E4 34k, product of E4 ORF6; and the product of E4 ORF4) regulate replication in infected cells. In addition, most of the viral DNA produced by E4 mutants lacking E4 11k and E4 34k is structurally abnormal, consisting of concatemers of the viral genome up to 6 or more monomers in length. Concatemeric viral DNA is not observed in cells infected by wild-type virus. Eukaryotic cells possess efficient double strand break repair (DSBR) systems for rejoining DNAs broken by radiation and other agents. To test the hypothesis that the concatenated viral DNA seen in E4 mutant infections arises by end-to-end joining of linear intracellular viral DNAs by DSBR, concatemer formation was examined in cells lacking the DNA-dependent protein kinase (DNA PK), an essential component of the cellular DSBR system. No concatemers were observed in E4 mutant infections of DNA PK- cells, consistent with the hypothesis that concatemers arise by DSBR and suggestion that in wild-type infections, E4 prevents concatenation by inhibiting DSBR. Further, E4 34k inhibited V(D)J recombination, a process that requires DSBR, in a plasmid-based assay. Finally, immunoprecipitation experiments showed that both the E4 11k and E4 34k proteins associate physically with DNA PK. Together, these data strongly suggest that both E4 proteins inhibit DSBR, possibly by a mechanism that involves binding to DNA PK. It is likely that suppression of concatemer formation increases the efficiency of viral DNA replication. Additionally, since DNA PK is a proximal element in the pathway that induces p53 activity in response to DNA, inhibition of DNA PK may be anti-apoptotic in infected cells. Thus, the interaction of E4 with DSBR may contribute in two distinct and novel ways to the success of an adenoviral infection. The goal of the work proposed here is to develop an understanding of the physical nature of the interactions between these E4 products and DNA PK, and to determine the significance to the viral life cycle of this newly-recognized aspects of E4 function.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA082127-02
Application #
6489312
Study Section
Virology Study Section (VR)
Program Officer
Wong, May
Project Start
2001-01-19
Project End
2005-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
2
Fiscal Year
2002
Total Cost
$241,776
Indirect Cost
Name
Johns Hopkins University
Department
Microbiology/Immun/Virology
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Jayaram, Sumithra; Ketner, Gary; Adachi, Noritaka et al. (2008) Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF. Nucleic Acids Res 36:5773-86
Jayaram, Sumithra; Gilson, Timra; Ehrlich, Elana S et al. (2008) E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection. Virology 382:163-70
Smeaton, Michael B; Miller, Paul S; Ketner, Gary et al. (2007) Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining. Nucleic Acids Res 35:e152
Berg, Michael; Gambhira, Ratish; Siracusa, Mark et al. (2007) HPV16 L1 capsid protein expressed from viable adenovirus recombinants elicits neutralizing antibody in mice. Vaccine 25:3501-10
Baker, Amy; Rohleder, Kent J; Hanakahi, Les A et al. (2007) Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation. J Virol 81:7034-40
Berg, Michael; Difatta, Julie; Hoiczyk, Egbert et al. (2005) Viable adenovirus vaccine prototypes: high-level production of a papillomavirus capsid antigen from the major late transcriptional unit. Proc Natl Acad Sci U S A 102:4590-5
Mohammadi, Elham S; Ketner, Elizabeth A; Johns, David C et al. (2004) Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line. Nucleic Acids Res 32:2652-9