RESUMEN
The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.
Asunto(s)
Infecciones por Adenoviridae , Proteínas E4 de Adenovirus , Adenovirus Humanos , Humanos , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Infecciones por Adenoviridae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismoRESUMEN
The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein-protein interactions, causing genomic instability.