July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Human Cytomegalovirus (HCMV) Does Not Induce Necroptosos-related Reactive Oxygen Species (ROS) Production Early After Infection of ARPE-19 Cells
Author Affiliations & Notes
  • Shauntelle Byfield
    Georgia State University, Atlanta, Georgia, United States
  • Jessica Carter
    Georgia State University, Atlanta, Georgia, United States
    Ophthalmology, Emory University, Georgia, United States
  • Judee Grace Nemeño
    Georgia State University, Atlanta, Georgia, United States
  • Jay Oh
    Georgia State University, Atlanta, Georgia, United States
  • Madeline Welch
    Georgia State University, Atlanta, Georgia, United States
  • Richard D Dix
    Georgia State University, Atlanta, Georgia, United States
    Ophthalmology, Emory University, Georgia, United States
  • Footnotes
    Commercial Relationships   Shauntelle Byfield, None; Jessica Carter, None; Judee Grace Nemeño, None; Jay Oh, None; Madeline Welch, None; Richard Dix, None
  • Footnotes
    Support  NIH/NEI Grant EY010568, NIH/NEI Grant EY024630, NIH/NEI Core Grant P30/EY006360, Emory Eye Center Vision Training Grant NIH/NEI 5T32/EY007092-32, Research to Prevent Blindness, Fight for Sight
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4615. doi:
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      Shauntelle Byfield, Jessica Carter, Judee Grace Nemeño, Jay Oh, Madeline Welch, Richard D Dix; Human Cytomegalovirus (HCMV) Does Not Induce Necroptosos-related Reactive Oxygen Species (ROS) Production Early After Infection of ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4615.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have shown previously that necroptosis may contribute to the pathogenesis of AIDS-related human cytomegalovirus (HCMV) retinitis using a mouse model of murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). A common feature associated with HCMV infection and cell death is production of ROS which can happen within minutes after virus entry into host cells. Because the MAIDS model of MCMV retinitis suggests that RPE play a prominent role in retinal disease development, we performed a pilot study to test the hypothesis that HCMV infection of ARPE-19 cells induces ROS production early in the course of virus replication that ultimately leads to stimulation of necroptosis-related molecules RIP1, RIP3, and MLKL.

Methods : Monolayers of ARPE-19 cells were infected with HCMV (moi = 5) or maintenance medium alone. HCMV-infected and uninfected ARPE-19 cells were also treated or not treated with H2O2 (200 uM) and/or ROS scavenger N-acetyl-L-cysteine (NAC) (30 mM). ROS activity was measured and compared in HCMV-infected and uninfected cells at 1, 2, 4, 6, and 24 hrs postinfection. HCMV-infected and uninfected cells (with or without NAC treatment) were also subjected to western blot analysis for detection of RIP1, RIP3, and MLKL proteins.

Results : Exposure of uninfected ARPE-19 cells to H2O2 for 4 and 6 hrs increased ROS production, an observation that was mitigated by NAS treatment. In contrast, HCMV infection did not lead to increased ROS production at all time points examined, even after exposure to H2O2 to induce ROS production. Cleaved RIP1, RIP3, and MLKL proteins were all expressed in ARPE-19 cells regardless of HCMV infection, although RIP3 protein production was uniquely mitigated by NAC treatment.

Conclusions : HCMV infection of ARPE-19 cells does not result in production of ROS during early infection as expected. A lack of ROS activity in HCMV-infected ARPE-19 cells, even in the presence of H2O2, suggests that HCMV may possess a mechanism to inhibit ROS production in this cell line. Decreased cleaved RIP3 protein production by NAC also suggests that RIP3-mediated pathways may rely on ROS production. While providing proof-of-principle, these findings must be reproduced using primary cultures of human RPE because ARPE-19 cells do not faithfully mimic human RPE in culture.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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