scholarly journals Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells

2010 ◽  
Vol 84 (14) ◽  
pp. 7029-7038 ◽  
Author(s):  
Sabrina Schreiner ◽  
Peter Wimmer ◽  
Hüseyin Sirma ◽  
Roger D. Everett ◽  
Paola Blanchette ◽  
...  
Keyword(s):  
Viral Protein ◽  
Protein A ◽  
Human Adenovirus ◽  
Nuclear Bodies ◽  
Virus Growth ◽  
Reading Frame ◽  
Early Region ◽  
Early Protein ◽  
Antiviral Activities ◽  
Infected Cells

ABSTRACT The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin α3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level.

scholarly journals Proteomic Analysis of Ubiquitin-Like Posttranslational Modifications Induced by the Adenovirus E4-ORF3 Protein

2014 ◽  
Vol 89 (3) ◽  
pp. 1744-1755 ◽  
Author(s):  
Sook-Young Sohn ◽  
Rebecca G. Bridges ◽  
Patrick Hearing
Keyword(s):  
Structural Changes ◽  
Human Adenovirus ◽  
Cellular Protein ◽  
Interferon Response ◽  
Global Pattern ◽  
Diverse Range ◽  
Orf3 Protein ◽  
Antiviral Responses ◽  
Early Protein ◽  
Infected Cells

ABSTRACTViruses interact with and regulate many host metabolic pathways in order to advance the viral life cycle and counteract intrinsic and extrinsic antiviral responses. The human adenovirus (Ad) early protein E4-ORF3 forms a unique scaffold throughout the nuclei of infected cells and inhibits multiple antiviral defenses, including a DNA damage response (DDR) and an interferon response. We previously reported that the Ad5 E4-ORF3 protein induces sumoylation of Mre11 and Nbs1, which are essential for the DDR, and their relocalization into E4-ORF3-induced nuclear inclusions is required for this modification to occur. In this study, we sought to analyze a global change in ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, by the Ad5 E4-ORF3 protein and to identify new substrates with these modifications. By a comparative proteome-wide approach utilizing immunoprecipitation/mass spectrometry, we found that Ubl modifications of 166 statistically significant lysine sites in 51 proteins are affected by E4-ORF3, and the proteome of modifications spans a diverse range of cellular functions. Ubl modifications of 92% of these identified sites were increased by E4-ORF3. We further analyzed SUMO3 conjugation of several identified proteins. Our findings demonstrated a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and revealed new SUMO3 substrates induced by E4-ORF3.IMPORTANCEThe adenovirus E4-ORF3 protein induces dynamic structural changes in the nuclei of infected cells and counteracts host antiviral responses. One of the mechanisms that accounts for this process is the relocalization and sequestration of cellular proteins into an E4-ORF3 nuclear scaffold, but little is known about how this small viral protein affects diverse cellular responses. In this study, we analyzed for the first time the global pattern of ubiquitin-like (Ubl) modifications, with particular focus on SUMO3, altered by E4-ORF3 expression. The results suggest a role for the Ad5 E4-ORF3 protein as a regulator of Ubl modifications and reveal new SUMO3 substrates targeted by E4-ORF3. Our findings propose Ubl modifications as a new mechanism by which E4-ORF3 may modulate cellular protein functions in addition to subnuclear relocalization.

scholarly journals Analysis of Synthesis, Stability, Phosphorylation, and Interacting Polypeptides of the 34-Kilodalton Product of Open Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus Type 5

1999 ◽  
Vol 73 (2) ◽  
pp. 1245-1253 ◽  
Author(s):  
Dominique Boivin ◽  
Megan R. Morrison ◽  
Richard C. Marcellus ◽  
Emmanuelle Querido ◽  
Philip E. Branton
Keyword(s):  
Protein Synthesis ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Open Reading Frame ◽  
Cellular Proteins ◽  
Cell Protein ◽  
Reading Frame ◽  
Early Region ◽  
Early Region 4 ◽  
Adenovirus Type 5

ABSTRACT The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

scholarly journals Functional Cooperation between Human Adenovirus Type 5 Early Region 4, Open Reading Frame 6 Protein, and Cellular Homeobox Protein HoxB7

2012 ◽  
Vol 86 (15) ◽  
pp. 8296-8308 ◽  
Author(s):  
D. Muller ◽  
S. Schreiner ◽  
M. Schmid ◽  
P. Groitl ◽  
M. Winkler ◽  
...  
Keyword(s):  
Human Adenovirus ◽  
Adenovirus Type ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Early Region ◽  
Homeobox Protein ◽  
Early Region 4 ◽  
Adenovirus Type 5

scholarly journals Human Cytomegalovirus UL84 Localizes to the Cell Nucleus via a Nuclear Localization Signal and Is a Component of Viral Replication Compartments

2002 ◽  
Vol 76 (17) ◽  
pp. 8931-8938 ◽  
Author(s):  
Yiyang Xu ◽  
Kelly S. Colletti ◽  
Gregory S. Pari
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Human Fibroblasts ◽  
Reading Frame ◽  
Early Protein ◽  
Localization Signal ◽  
Infected Cells

ABSTRACT The UL84 open reading frame encodes a protein that is required for origin-dependent DNA replication and interacts with the immediate-early protein IE2 in lytically infected cells. Transfection of UL84 expression constructs showed that UL84 localized to the nucleus of transfected cells in the absence of any other viral proteins and displayed a punctate speckled fluorescent staining pattern. Cotransfection of all the human cytomegalovirus replication proteins and oriLyt, along with pUL84-EGFP, showed that UL84 colocalized with UL44 (polymerase accessory protein) in replication compartments. Experiments using infected human fibroblasts demonstrated that UL84 also colocalized with UL44 and IE2 in viral replication compartments in infected cells. A nuclear localization signal was identified using plasmid constructs expressing truncation mutants of the UL84 protein in transient transfection assays. Transfection assays showed that UL84 failed to localize to the nucleus when 200 amino acids of the N terminus were deleted. Inspection of the UL84 amino acid sequence revealed a consensus putative nuclear localization signal between amino acids 160 and 171 (PEKKKEKQEKK) of the UL84 protein.

scholarly journals A 49-Kilodalton Isoform of the Adenovirus Type 5 Early Region 1B 55-Kilodalton Protein Is Sufficient To Support Virus Replication

2009 ◽  
Vol 83 (18) ◽  
pp. 9045-9056 ◽  
Author(s):  
Kathrin Kindsmüller ◽  
Sabrina Schreiner ◽  
Florian Leinenkugel ◽  
Peter Groitl ◽  
Elisabeth Kremmer ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Tumor ◽  
Adenovirus Type ◽  
Productive Infection ◽  
Virus Growth ◽  
Reading Frame ◽  
Amino Terminal ◽  
Early Region ◽  
Low Levels ◽  
Adenovirus Type 5

ABSTRACT The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.

Identification of envelope protein ORF10 of channel catfish herpesvirus

2012 ◽  
Vol 58 (3) ◽  
pp. 271-277
Author(s):  
Yulin Liu ◽  
Junfa Yuan ◽  
Weimin Wang ◽  
Xiaoxuan Chen ◽  
Rong Tang ◽  
...  
Keyword(s):  
Channel Catfish ◽  
Envelope Protein ◽  
Viral Protein ◽  
Protein A ◽  
Neutralization Assay ◽  
Open Reading Frame ◽  
Viral Pathogen ◽  
Ovary Cells ◽  
Viral Neutralization ◽  
Reading Frame

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.

scholarly journals The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses

1985 ◽  
Vol 225 (3) ◽  
pp. 649-655 ◽  
Author(s):  
M E Blair Zajdel ◽  
M D Barker ◽  
S C Dixon ◽  
G E Blair
Keyword(s):  
Monoclonal Antibodies ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Human Cells ◽  
Early Region ◽  
Antibody Staining ◽  
Free Translation ◽  
Infected Cells ◽  
Nuclear Location

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3. Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.

scholarly journals Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses

2021 ◽  
Vol 22 (18) ◽  
pp. 10075
Author(s):  
Kemal Mese ◽  
Oskar Bunz ◽  
Wolfram Volkwein ◽  
Sahithya P. B. Vemulapalli ◽  
Wenli Zhang ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Human Adenovirus ◽  
Antiviral Effect ◽  
Virus Growth ◽  
Antiviral Function ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

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