Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 22:37:101616.
doi: 10.1016/j.bbrep.2023.101616. eCollection 2024 Mar.

Human adenovirus oncolytic properties and the inhibitory role of E4 orf4 and E4 orf6/7 on endogenously activated NF-κB

Anran Wang  1 , Kazuki Uchida  2 , Atsuro Yokoyama  1 , Fumihiro Higashino  3 , Motoaki Yasuda  4
Affiliations

Human adenovirus oncolytic properties and the inhibitory role of E4 orf4 and E4 orf6/7 on endogenously activated NF-κB

Anran Wang et al. Biochem Biophys Rep. .

Abstract

Human adenovirus is a promising tool for cancer therapy as an oncolytic virus. To predict which region of the oncolytic adenovirus E4 gene could be deleted, we investigated the relationship between the E4 proteins and NF-κB. Here, we report that TLR2-dependent NF-κB activation in Ad5-infected cells was significantly inhibited 24 h post-infection. Among the six E4 proteins, E4 orf4 and E4 orf6/7 exhibited notable suppressive effects on NF-κB activation. However, only E4 orf4 was co-immunoprecipitated with the RelA protein, also known as p65. It appears likely that E4 orf6/7 represses NF-κB activation via E2F-dependent pathways. Our results suggest that both E4 orf4 and E4 orf6/7 are novel inhibitors of NF-κB activation. The inhibition of endogenous NF-κB activation by E4 proteins during the late phase of infection also appears to elucidate the previously reported suppression of E1A expression in the late phase of infection. These redundant suppressive effects of E4 orf4 and E4 orf6/7 on NF-κB suggest that these proteins may play a major role in the anticancer properties of oncolytic adenovirus.

Keywords: Adenovirus; E4 orf4; E4 orf6/7; NF-κB; TLR2.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Cell type-specific NF-κB activation upon human adenovirus (Ad) infection. A: The indicated cells were transfected with an NF-κB luciferase reporter plasmid together with a pGL4.72 plasmid, as described in the Materials and Methods section. Transfected cells were infected with Ad wt300 (20 PFU/cell) and incubated for 6 h and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. Error bars represent standard deviations for triplicate wells in a single experiment; data are representative of three independent experiments. B: HEK293 cells were transfected with an NF-κB luciferase reporter plasmid together with a pGL4.72 plasmid and the indicated effector plasmids, followed by infection with the indicated Ad (m.o.i = 10 PFU/cell) and incubated for 6 or 24 h. Error bars represent standard deviations for triplicate wells in a single experiment; data are representative of three independent experiments. C: RPC-C2A cells were transfected with an NF-κB luciferase reporter plasmid together with a pGL4.72 plasmid, followed by infection with the indicated Ad (m.o.i = 10 PFU/cell) for the indicated periods. Luciferase assay was performed as described above. Data are representative of three independent experiments. D: HEK293 and RPC C2A cells were infected with indicated adenovirus for 24 h. Subsequently, the cells were lysed, and the corresponding lysates were subjected to Western blotting using RSA3 antibodies (anti-N-terminal region of both E4 orf6 and orf6/7) and anti-E4 orf4 rabbit serum. The lowest row depicts anti-β Actin staining, indicating that equal volumes of cell extracts were analyzed. Endogenous E1A of HEK293 is sufficient for the expression of E4 orf4, 6, and 6/7 with Ad LacZ.
Fig. 2
Fig. 2
Human adenovirus E4 orfs suppresses the TLR2-dependent NF-κB activation. A: 293TLR2 cells were transfected with an E1 luciferase reporter plasmid or NF-κB luciferase reporter plasmid together with a pGL4.72 plasmid and the indicated effector plasmids, followed by 6 h incubation with FSL-1 (final 10 nM) containing serum free medium. Error bars represent standard deviations for triplicate wells in a single experiment; data are representative of three independent experiments. Comparisons were made using an unpaired Student's t-test. Results are represented as mean ± S.D. Statistical significance is displayed as: *, p < 0.05; * *, p < 0.01. B: HEK293 cells were transfected with the combination of Myc tagged hRelA and indicated HA orfs. Cells were incubated for 48 h and lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-HA antibody. Only HA orf4 was co-immunoprecipitated with over expressed myc-tagged RelA protein. Exogenously expressed myc-tagged RelA has a larger molecular weight than endogenous RelA. The lowest row exhibits anti-β Actin staining.
Fig. 3
Fig. 3
NF-κB repression by loss-of-PP2A-binding and a loss-of-killing mutant E4 orf4 mutant. A: 293TLR2 cells were transfected with either an E1 luciferase reporter plasmid or an NF-κB luciferase reporter plasmid, along with a pGL4.72 plasmid and the specified effector plasmids. The transfected cells were then incubated with FSL-1 (final concentration 10 nM) in serum-free medium for a duration of 6 h. Subsequently, a luciferase assay was conducted as previously described. The lower panel of the figure illustrates the amino acid sequences in E4 orf4 and its mutants. The C-terminal 36 amino acids of E4 orf4CT correspond to a frameshifted E4 orf6. The data presented are representative of three independent experiments. The error bars shown in the graphs indicate standard deviations calculated from triplicate wells within a single experiment. Comparisons were made using an unpaired Student's t-test. Results are represented as mean ± S.D. Statistical significance is displayed as: *, p < 0.05; * *, p < 0.01. These results are consistent across three separate independent experiments. B: HEK293 cells were transfected with the combination of myc-tagged RelA and indicated HA Eorf4s. Cells were incubated for 48 h and lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-HA antibody. Exogenously expressed myc-tagged RelA has a larger molecular weight than endogenous RelA. The lowest row depicts anti-β Actin staining.
Fig. 4
Fig. 4
E4 orf6/7 repress NF-κB activation via E2F pathway. A: The indicated HA-tagged E4 orf6/7s and NF-κB luciferase reporter plasmid or E2A luciferase reporter, along with a pGL4.72 plasmid were transfected into RPC- C2A cells. Twenty-four hours after transfection, cells were incubated with FSL-1 (final concentration 10 nM) in serum-free medium for a duration of 6 h. Subsequently, a luciferase assay was conducted as previously described. The data presented are representative of three independent experiments. The error bars shown in the graphs indicate standard deviations calculated from triplicate wells within a single experiment. These results are consistent across three separate independent experiments. Comparisons were made using an unpaired Student's t-test. Results are represented as mean ± S.D. Statistical significance is displayed as: NS, not significant; *, p < 0.05; * *, p < 0.01. ; ***,p < 0.001. B: Myc-tagged E2F1 and E2F4, along with NF-kB luciferase reporter were transfected into RPC C2A cells. Twenty-four hours after transfection, cells were incubated with FSL-1 (final concentration 10 nM) in serum-free medium for a duration of 6 h. The data presented are representative of three independent experiments. The error bars shown in the graphs indicate standard deviations calculated from triplicate wells within a single experiment. Comparisons were made using an unpaired Student's t-test. Results are represented as mean ± S.D. Statistical significance is displayed as: NS, not significant; * *, p < 0.01. C: HEK293 cells were transfected with the combination of Myc tagged E2Fs and human RelA. Cells were incubated for 48 h and lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-RelA antibody. The lowest row depicts anti-β Actin staining. D: GFP-tagged E2F4, together with DsRed-tagged RelA or DsRed-tagged p50, were co-transfected onto H1299 cells. After 24 h from transfection, cells were fixed with 4 % paraformaldehyde and observed by confocal laser scanning microscope.
Fig. S1
Fig. S1
Fig. S2
Fig. S2
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Lewis J.B., Mathews M.B. Control of adenovirus early gene expression: a class of immediate early products. Cell. 1980;21:303–313. doi: 10.1016/0092-8674(80)90138-5. - DOI - PubMed
    1. Katze M.G., Persson H., Philipson L. Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products. Mol. Cell Biol. 1981;1:807–813. doi: 10.1128/mcb.1.9.807-813.1981. - DOI - PMC - PubMed
    1. Fessler S.P., Young C.S. Control of adenovirus early gene expression during the late phase of infection. J. Virol. 1998;72:4049–4056. doi: 10.1128/JVI.72.5.4049-4056.1998. - DOI - PMC - PubMed
    1. Boivin D., Morrison M.R., Marcellus R.C., Querido E., Branton P.E. 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. J. Virol. 1999;73:1245–1253. doi: 10.1128/JVI.73.2.1245-1253.1999. - DOI - PMC - PubMed
    1. Huang M.M., Hearing P. Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection. J. Virol. 1989;63:2605–2615. doi: 10.1128/JVI.63.6.2605-2615.1989. - DOI - PMC - PubMed

LinkOut - more resources