Opposite Effects of Apoptotic and Necroptotic Cellular Pathways on Rotavirus Replication

Soliman, Mahmoud; Seo, Ja-Young; Baek, Yeong-Bin; Park, Jun-Gyu; Kang, Mun-Il; Cho, Kyoung-Oh; Park, Sang-Ik

doi:10.1128/jvi.01222-21

Cited by 9 publications

(19 citation statements)

References 92 publications

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“…The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the cytotoxic effects of the chemicals and their solvents as well as their effect on cell viability following PSaV Cowden strain infection, as previously described [46,47]. Briefly, monolayers of LLC-PK cells grown in 96-well plates were incubated for 24 h with a medium containing different concentrations of each chemical inhibitor.…”

Section: Cytotoxicity and Cell Viability Assaysmentioning

confidence: 99%

“…LLC-PK cells grown in 6-or 96-well plates were washed twice with phosphate-buffered saline (PBS, pH 7.4). Cells were either infected with the PSaV Cowden strain or left mock-infected and treated with the inhibitory chemicals for 1 h at 37˚C or vehicle at the following concentrations: RIPK1 (Nec-1; 20 μM), RIPK3 (GSK'872; 10 μM), MLKL (NSA; 10 μM), and pan-caspase (Z-VAD-FMK; 20 μM) were added to the fresh cell culture medium for the indicated time periods as described elsewhere [47,48]. Cells were then used to measure protein expression by western blotting, viral titer by TCID 50 assay, cell viability by MTT assay, and the percentage of apoptotic and necroptotic cells by flow cytometry assay.…”

Section: Treatment Of Llc-pk Cells With Chemical Inhibitorsmentioning

confidence: 99%

“…LLC-PK cells were cultured in 6-well culture plates at 70-80% confluence and transfected with scrambled siRNA or siRNAs against RIPK1, RIPK3, or MLKL [46,47] using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer's instructions. The knockdown efficiency of each siRNA was optimized by a second transfection 24 h after the first transfection.…”

Section: Transfections Of Sirnasmentioning

confidence: 99%

“…Western blot analysis was performed to determine viral and host target proteins as described elsewhere [47]. In brief, confluent LLC-PK monolayers grown in 6-well plates were infected with or without PSaV Cowden strain, treated with or without various inhibitors, or transfected with scrambled siRNA or siRNAs against target proteins as described above.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…The percentage of apoptotic cells was determined by TUNEL assay [47,49] using an in situ Cell death detection kit (Roche) according to the manufacturer's protocol. Briefly, LLC-PK cells were infected with or without PSaV Cowden strain (MOI = 1 TCID 50 ), harvested at different time points, fixed, and permeabilized as described above.…”

Section: Terminal Deoxynucleotidyl Transferase Dutp Nick End Labeling...mentioning

confidence: 99%

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Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

et al. 2023

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Sapoviruses belonging to the genus Sapovirus within the family Caliciviridae are commonly responsible for severe acute gastroenteritis in both humans and animals. Caliciviruses are known to induce intrinsic apoptosis in vitro and in vivo, however, calicivirus-induced necroptosis remains to be fully elucidated. Here, we demonstrate that infection of porcine kidney LLC-PK cells with porcine sapovirus (PSaV) Cowden strain as a representative of caliciviruses induces receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis and acts as proviral compared to the antiviral function of PSaV-induced apoptosis. Infection of LLC-PK cells with PSaV Cowden strain showed that the interaction of phosphorylated RIPK1 (pRIPK1) with RIPK3 (pRIPK3), mixed lineage kinase domain-like protein (pMLKL) increased in a time-dependent manner, indicating induction of PSaV-induced RIPK1-dependent necroptosis. Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating an antiviral action of PSaV-induced apoptosis. These results suggest that PSaV-induced RIPK1-dependent necroptosis and apoptosis‒which have proviral and antiviral effects, respectively‒counterbalanced each other in virus-infected cells. Our study contributes to understanding the nature of PSaV-induced necroptosis and apoptosis and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.

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Section: Cytotoxicity and Cell Viability Assaysmentioning

confidence: 99%

Section: Treatment Of Llc-pk Cells With Chemical Inhibitorsmentioning

confidence: 99%

Section: Transfections Of Sirnasmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

Section: Terminal Deoxynucleotidyl Transferase Dutp Nick End Labeling...mentioning

confidence: 99%

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Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

et al. 2023

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Approaches to Evaluating Necroptosis in Virus-Infected Cells

Lawson,

Titus,

Koehler

2023

Subcellular Biochemistry

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Identification and characterization of a marine bacterium extract from Mameliella sp. M20D2D8 with antiviral effects against influenza A and B viruses

Kim,

Park,

Moon

et al. 2024

Arch Virol

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Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

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Opposite Effects of Apoptotic and Necroptotic Cellular Pathways on Rotavirus Replication

Cited by 9 publications

References 92 publications

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

Approaches to Evaluating Necroptosis in Virus-Infected Cells

Identification and characterization of a marine bacterium extract from Mameliella sp. M20D2D8 with antiviral effects against influenza A and B viruses

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