Viral mitochondria-localized inhibitor of apoptosis (UL37 exon 1 protein) does not protect human neural precursor cells from human cytomegalovirus-induced cell death.

Congenital human cytomegalovirus (HCMV) infection can cause severe brain abnormalities. Apoptotic HCMV-infected brain cells have been detected in a congenitally infected infant. In biologically relevant human neural precursor cells (hNPCs), cultured in physiological oxygen tensions, HCMV infection (m.o.i. of 1 or 3) induced cell death within 3 days post-infection (p.i.) and increased thereafter. Surprisingly, its known anti-apoptotic genes, including the potent UL37 exon 1 protein (pUL37x1) or viral mitochondria-localized inhibitor of apoptosis (vMIA), which protects infected human fibroblasts (HFFs) from apoptosis and from caspase-independent, mitochondrial serine protease-mediated cell death, were expressed by 2 days p.i. Consistent with this finding, an HCMV UL37x1 mutant, BADsubstitutionUL37x1 (BADsubUL37x1) induced cell death in hNPCs (m.o.i. = 1) to level which were indistinguishable from parental virus (BADwild-type)-infected hNPCs. Surprisingly, although BADsubUL37x1 is growth defective in permissive HFFs, it produced infectious progeny in hNPCs with similar kinetics and to levels comparable to BADwild-type-infected hNPCs (m.o.i. = 1). While delayed at a lower multiplicity (m.o.i. = 0.3), the BADsubUL37x1 mutant reached similar levels to revertant within 12 days, in contrast to its phenotype in HFFs. The inability of pUL37x1/vMIA to protect hNPCs from HCMV-induced cell death did not result from impaired trafficking as pUL37x1/vMIA trafficked efficiently to mitochondria in transfected hNPCs and in HCMV-infected hNPCs. These results establish that pUL37x1/vMIA, although protective in permissive HFFs, does not protect HCMV-infected hNPCs from cell death under physiologically relevant oxygen tensions. They further suggest that pUL37x1/vMIA is not essential for HCMV growth in hNPCs and has different cell type-specific roles.

Quantitative proteomic analyses of human cytomegalovirus-induced restructuring of endoplasmic reticulum-mitochondrial contacts at late times of infection.

Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.