Characterization of the 55-Residue Protein Encoded by the 9S E1A mRNA of Species C Adenovirus (original) (raw)
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Regulation of the 26S proteasome by adenovirus E1A
The EMBO Journal, 2000
We have identi®ed the N-terminus of adenovirus early region 1A (AdE1A) as a region that can regulate the 26S proteasome. Speci®cally, in vitro and in vivo co-precipitation studies have revealed that the 19S regulatory components of the proteasome, Sug1 (S8) and S4, bind through amino acids (aa) 4±25 of Ad5 E1A. In vivo expression of wild-type (wt) AdE1A, in contrast to the N-terminal AdE1A mutant that does not bind the proteasome, reduces ATPase activity associated with anti-S4 immunoprecipitates relative to mock-infected cells. This reduction in ATPase activity correlates positively with the ability of wt AdE1A, but not the N-terminal deletion mutant, to signi®cantly reduce the ability of HPV16 E6 to target p53 for ubiquitin-mediated proteasomal degradation. AdE1A/ proteasomal complexes are present in both the cytoplasm and the nucleus, suggesting that AdE1A interferes with both nuclear and cytoplasmic proteasomal degradation. We have also demonstrated that wt AdE1A and the N-terminal AdE1A deletion mutant are substrates for proteasomal-mediated degradation. AdE1A degradation is not, however, mediated through ubiquitylation, but is regulated through phosphorylation of residues within a C-terminal PEST region (aa 224±238).
The biology of the adenovirus E1B 55K protein
FEBS Letters, 2019
The adenovirus E1B 55K (E1B) protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved.
Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome
Oncogene, 1999
Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirusmediated transformation and oncogenesis. Whilst E1A is generally considered to exert its in¯uence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein halflife. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identi®ed as mammalian SUG1, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUG1. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad E1A binds strongly to SUG1 but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that puri®ed Ad12 E1A inhibits the activity of the puri®ed 20S but not 26S proteasomes. We have also demonstrated that SUG1 can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.
PLOS ONE
The E1B 55kDa produced by human adenovirus type 5 is a multifunctional protein that participates in the regulation of several steps during the viral replication cycle. Previous studies suggest this protein plays an important role in postranscriptional regulation of viral and cellular gene expression, as it is required for the selective accumulation of maximal levels of viral late mRNA in the cytoplasm of the infected cell; however the molecular mechanisms that are altered or regulated by this protein have not been elucidated. A ribonucleoprotein motif that could implicate the direct interaction of the protein with RNA was initially predicted and tested in vitro, but the interaction with RNA could not be detected in infected cells, suggesting the interaction may be weak or transient. Here it was determined that the E1B 55kDa interacts with RNA in the context of the viral infection in non-transformed human cells, and its contribution to the adenovirus replication cycle was evaluated. Using recombinant adenoviruses with amino acid substitutions or a deletion in the ribonucleoprotein motif the interaction of E1B 55kDa with RNA was found to correlate with timely and efficient viral DNA replication and viral late mRNA accumulation and splicing.
Characterization of the 55K adenovirus type 5 E1B product and related proteins
Journal of General Virology, 1994
In addition to major proteins of 19K and 55K (176 and 496 residues, 176R and 496R, respectively), early region 1B (E1B) of human adenovirus type 5 (Ad5) is predicted to encode at least three other polypeptides of 156R, 93R and 84R that share 79 amino-terminal residues with 496R. We have used a series of specific antipeptide sera to identify and partially characterize these proteins. 84R was produced in large amounts, 156R somewhat less, and 93R at very low levels. Synthesis of 176R, 496R, as well as the E2A 72K DNA-binding protein commenced shortly after that of E1A proteins in Ad5-infected KB cells. Production of 156R, 93R and 84R began somewhat later, but prior to the synthesis of the late structural protein IX and hexon. 156R, which is composed of the 79 amino-terminal and 77 carboxy-terminal amino acids of 496R, migrated on SDS-PAGE as two species which appeared to differ by their degree of phosphorylation. 156R and 496R yielded identical tryptic phosphopeptides that contained both phosphoserine and phosphothreonine, and one of these was immunoprecipitated by a serum specific for the carboxy terminus. These results suggested that Ser-490 and/or Ser-491 as well as Thr-495 are major sites of phosphorylation in these proteins.
Journal of Virology
We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the s...
Journal of Virology, 2005
The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76: 4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of norma...
Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein
Journal of Virology, 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. ...