School of Medicine and Health Sciences Poster Presentations
Sequential Trafficking and Localization of HCMV Anti-Apoptotic Protein vMIA Alter Mitochondrial Calcium Levels
Poster Number
16
Document Type
Poster
Publication Date
3-2016
Abstract
Congenital infection with the human cytomegalovirus (HCMV) can cause deafness, blindness and microcephaly. During infection, HCMV inhibits host cell apoptosis increasing thereby the production of infectious progeny. One of its immediate early proteins, viral mitochondria-localized inhibitor of apoptosis (vMIA), is anti-apoptotic and essential for HCMV growth in humans. vMIA is synthesized in the endoplasmic reticulum (ER), traffics sequentially through ER-mitochondrial contacts (known as MAM), localizes to MAM lipid rafts, which contain the ER calcium channel, IP3R and chaperones, and to mitochondria. We previously found that HCMV infection significantly increases calcium handling proteins and channels in the MAM proteome, suggesting its regulation of calcium dynamics at the MAM. Consistent with this, vMIA increases ER calcium efflux. Since mitochondrial calcium ([Ca2+]m) uptake from the ER regulates cellular metabolism, analyzing whether vMIA trafficking through the MAM affects [Ca2+]m levels may provide insight into its usurping of mitochondrial metabolic machineries. We examined the effects of vMIA trafficking and localization on [Ca2+]m levels. To that end, we transfected HeLa cells with vectors encoding fluorescently tagged wild-type (WT) vMIA or vMIA mutant (cholesterol binding domain II, CBD-II, and high hydrophobicity B, HHB) proteins and observed their trafficking, localization, and effects on [Ca2+]m levels using live-cell and confocal microscopy. Using pharmacological blocks, our lab had previously observed vMIA localization at mitochondria within 60 minutes. Here, we show that sequential trafficking and location of vMIA play a role in elevating the [Ca2+]m levels by using vMIA mutants that either fail to localize to mitochondria or do not associate with MAM lipid rafts. Our studies show that vMIA increases [Ca2+]m and that association with lipid rafts is not essential for this vMIA function. Thus, vMIA’s trafficking and localization could play an important role in altering [Ca2+]m dynamics at the MAM.
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Open Access
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Sequential Trafficking and Localization of HCMV Anti-Apoptotic Protein vMIA Alter Mitochondrial Calcium Levels
Congenital infection with the human cytomegalovirus (HCMV) can cause deafness, blindness and microcephaly. During infection, HCMV inhibits host cell apoptosis increasing thereby the production of infectious progeny. One of its immediate early proteins, viral mitochondria-localized inhibitor of apoptosis (vMIA), is anti-apoptotic and essential for HCMV growth in humans. vMIA is synthesized in the endoplasmic reticulum (ER), traffics sequentially through ER-mitochondrial contacts (known as MAM), localizes to MAM lipid rafts, which contain the ER calcium channel, IP3R and chaperones, and to mitochondria. We previously found that HCMV infection significantly increases calcium handling proteins and channels in the MAM proteome, suggesting its regulation of calcium dynamics at the MAM. Consistent with this, vMIA increases ER calcium efflux. Since mitochondrial calcium ([Ca2+]m) uptake from the ER regulates cellular metabolism, analyzing whether vMIA trafficking through the MAM affects [Ca2+]m levels may provide insight into its usurping of mitochondrial metabolic machineries. We examined the effects of vMIA trafficking and localization on [Ca2+]m levels. To that end, we transfected HeLa cells with vectors encoding fluorescently tagged wild-type (WT) vMIA or vMIA mutant (cholesterol binding domain II, CBD-II, and high hydrophobicity B, HHB) proteins and observed their trafficking, localization, and effects on [Ca2+]m levels using live-cell and confocal microscopy. Using pharmacological blocks, our lab had previously observed vMIA localization at mitochondria within 60 minutes. Here, we show that sequential trafficking and location of vMIA play a role in elevating the [Ca2+]m levels by using vMIA mutants that either fail to localize to mitochondria or do not associate with MAM lipid rafts. Our studies show that vMIA increases [Ca2+]m and that association with lipid rafts is not essential for this vMIA function. Thus, vMIA’s trafficking and localization could play an important role in altering [Ca2+]m dynamics at the MAM.
Comments
Presented at: GW Research Days 2016