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. 2023 Feb 13;15(2):523.
doi: 10.3390/v15020523.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication

Chao-Yu Hsu  1   2 , Jing-Wen Huang  3   4 , Wei-Ru Huang  3   4 , I-Chun Chen  2   5 , Ming-Shan Chen  6 , Tsai-Ling Liao  2   7 , Yu-Kang Chang  8   9 , Muhammad Munir  10 , Hung-Jen Liu  2   3   4   11   12
Affiliations

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication

Chao-Yu Hsu et al. Viruses. .

Abstract

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

Keywords: ATeams; HIF-1α; avian reoviruses; c-myc; glut1; glycolysis; oncolytic virus.

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Conflict of interest statement

We declare that we have no competing interests.

Figures

Figure 1
Figure 1
ARV replication in several cancer cell lines. (A) HFL-1, A549, B16-F10, and HeLa cell lines were infected with ARV at an MOI of 10 for 24 h. Plaque assay was performed to analyze the titer of ARV. HFL-1 cell as the negative control for this experiment. All data were obtained in three independent experiments, error bars indicate the mean ± SD. (B) Four cell lines were treated in the same way and examined using Western blots. Signals in all Western blots of β-actin were quantified with Image J.
Figure 2
Figure 2
ARV infection and σA transfection increased of c-myc, HIF-1α, and glut1 in cancer cell lines. (A) A549, B16-F10, and HeLa cancer cell lines were infected with ARV at an MOI of 10. Cell lysates were collected with 2.5× sample buffer dye at 2, 6, 12, and 18 h post-infection for Western blots. (B) Cancer cell lines were transfected with the pCI-neo-σA plasmid at 2, 6, 12, and 18 h. Cell lysates were collected with 2.5× sample buffer dye and examined using Western blots. Signals in all Western blots were quantified with Image J and normalized to β-actin as shown in Supplementary Figure S2.
Figure 3
Figure 3
ARV infection and σA transfection upregulate mRNA levels of c-myc, HIF-1α, and glut1 in cancer cell lines. (A–C) cancer cell lines were infected with ARV or transfection the pCI-neo-σA or σA shRNA vectors for 6 h followed by infection with ARV at an MOI of 10 for 18 h. Cell lysates were collected for examining the mRNA levels of c-myc (A), HIF-1α (B), and glut1 (C) using quantitative real-time RT-PCR. Each value represents mean± SE from three independent experiments, determined using Duncan’s Multiple Range Test. Similar alphabets (a, b, c) denote no significance at p < 0.05.
Figure 4
Figure 4
Knockdown of c-myc or HIF-1α by shRNAs reduced the expression levels of glut1 and virus yields in these cancer cell lines. (A)Three cancer cell lines were transfected with c-myc or HIF-1α shRNAs for the first 6 h, later infected with ARV at an MOI of 10 for 24 h. Plaque assay was performed to analyze virus titers. Each value represents mean± SE from three independent experiments, determined using Duncan’s Multiple Range Test. Similar alphabets (a, b) denote no significance at p < 0.05. (B) Three cancer cell lines co-transfected with the pCI-neo-σA and c-myc or HIF-1α shRNAs for 24 h. Cell lysates were collected and examined using Western blots. Densitometry analysis results for Western blot are expressed as percentages representing levels of c-myc, HIF-1α, and glut1 normalized to β-actin.
Figure 5
Figure 5
Visualization of adenosine triphosphate (ATP) levels inside cancer cell lines infected with ARV. To measure intracellular ATP levels, the cancer cell lines were transfected with the ATeams with or without c-myc, HIF 1α, and glut1 shRNAs for 18 h followed infection with ARV at different time points and MOI of 10 as indicated. The cells were observed and photographed with a fluorescence microscope (scale bar, 20 μm). The fluorescence density was quantitated using Image J software as shown in Supplementary Figure S8A.
Figure 6
Figure 6
Visualization of adenosine triphosphate (ATP) levels inside cancer cell lines transfected with the pCI-neo-σA plasmid. To measure intracellular ATP levels, A549, B16-F10, and HeLa cancer cell lines were co-transfected with ATeams and the pCI-neo-σA plasmid with or without c-myc, HIF 1α, and glut1 shRNAs for 24 h. The cells were observed and photographed with a fluorescence microscope (scale bar, 20 μm). The fluorescence density was quantitated using Image J software as shown in Supplementary Figure S8B.
Figure 7
Figure 7
ARV infection and ARV σA transfection alter levels of LDHA, PKM2, OGDH, and Gls in A549, B16-F10, and HeLa cancer cell lines. (A–D) The cancer cell lines were transfected with or without the pCI-neo-σA or σA shRNA vectors for 6 h followed infection with ARV at an MOI of 10 for 18 h. Cell lysates were collected for examining the mRNA levels of LDHA (A), PKM2 (B), OGDH (C), and Gls (D) using quantitative real-time RT-PCR. Each value represents mean ± SE from three independent experiments, determined using Duncan’s Multiple Range Test. Similar alphabets (a, b, c). (E) Cancer cell lines were transfected with the pCI-neo-σA plasmid for 24 h followed by Western blot assay. Signals in all Western blots were quantified with Image J and normalized to β-actin as shown in Supplementary Figure S9. In this study, all uncropped blots are shown in Supplementary Figure S10.
Figure 8
Figure 8
A model depicting that ARV σA enhances glycolysis and glutaminolysis to produce energy via the HIF-1α/c-myc/glut1 pathway to benefit ARV replication in A549, B16-F10, and HeLa cancer cell lines. →: activation; ⊥: inhibition. GDH: glutamate dehydrogenase [16].
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Grants and funding

This work was financially supported by Ministry of Science and Technology of Taiwan (109-2313-B-005-006-MY3 and 111-2622-B-005-001), The iEGG and Animal Biotechnology Center from The Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (111S0023A), National Chung Hsing University and Taichung Veterans General Hospital (TCVGH-NCHU1117608).

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