HCMV infection downregulates GPX4 and stimulates lipid peroxidation but does not induce ferroptosis.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection, including key components in cellular iron homeostasis. Iron is essential to many cellular processes but, if present in excess, drives cell death through ferroptosis. Ferroptosis is a process that is dependent upon the accumulation of oxidatively damaged phospholipids (lipid peroxides); when these lipid peroxides accumulate in membranes, this culminates in plasma membrane rupture and eventual cell lysis. Here, we demonstrate that HCMV infection downregulates the expression of a key modulator of lipid peroxidation, glutathione peroxidase 4 (GPX4). HCMV infection also markedly increased levels of lipid peroxides within infected cells. Despite the marked downregulation of GPX4 by HCMV, further inhibition of GPX4 impaired virus replication. Interestingly, overexpression of GPX4 did not reduce the production of lipid peroxides within infected cells. In contrast, lipid peroxide levels were reduced by treatment with ferrostatin-1, a ferrous iron-dependent scavenger of alkoxyl radicals, indicating a role for iron in the production of lipid peroxides. HCMV-infected cells became less sensitive to GPX4 inhibition as infection progressed, requiring substantially higher levels of GPX4 inhibitors to induce ferroptosis compared to uninfected cells. This observed difference in sensitivity to ferroptosis upon infection correlated with a large increase in lipid production by infected cells. Therefore, the marked stimulation of lipid peroxidation by HCMV likely proceeds through a pathway that is independent of GPX4 regulation, but the ability of lipid peroxides to stimulate ferroptosis by modulating plasma membrane rupture is likely blunted by the massive increase in lipid production during HCMV infection. IMPORTANCE: Human cytomegalovirus (HCMV) infection is intimately linked with countless host cell pathways that are modulated in a coordinated fashion to facilitate infection. Here, we describe HCMV-induced regulation of lipid peroxidation, a precursor of the iron-regulated cell death pathway known as ferroptosis, during human cytomegalovirus infection. These studies reveal hitherto unidentified changes in metabolism mediated by HCMV that decrease sensitivity to ferroptosis, despite increases in lipid peroxidation and transient increases in intracellular iron levels in infected cells.