Metabolic labeling and direct imaging of choline phospholipids in vivo

Jao, Cindy Y.; Roth, Mary R.; Welti, Ruth

doi:10.1073/pnas.0907864106

Cited by 218 publications

(264 citation statements)

References 23 publications

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“…For poliovirus, detection of PC was done by a "click chemistry" method described in Jao et al (36). HeLa cells were infected with an MOI of 10 of poliovirus type I Mahoney.…”

Section: Methodsmentioning

confidence: 99%

“…To monitor the synthesis of PC in poliovirus-infected HeLa cells, we used a choline analog, propargyl-choline, which is efficiently incorporated into PC without affecting the overall PC composition (36). In uninfected cells, propargyl-choline was evenly incorporated into cellular organelles but excluded from the nucleus (Fig.…”

Section: Virus-stimulated and Viral Replication Site-localized Pc Accmentioning

confidence: 99%

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Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

Zhang

Chukkapalli

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

112

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All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.positive-strand RNA viruses | viral replication complexes | virus-host interactions | virus control | phospholipids A ll positive-strand RNA viruses [(+)RNA viruses], which include numerous important human, animal, and plant pathogens, share similar strategies for genomic replication. A highly conserved and indispensable feature of their replication is the proliferation and reorganization of host cellular membranes to assemble viral replication complexes (VRCs). Despite this central importance, it is largely unknown how cellular membranes are rearranged by the viral replication proteins and how cellular lipid metabolism is modulated to accommodate membrane proliferation and remodeling.Brome mosaic virus (BMV) serves as a model for understanding VRC formation of (+)RNA viruses (1). BMV is the type member of the plant virus family Bromoviridae and a representative member of the alphavirus-like superfamily, which includes many human, animal, and plant-infecting viruses (2). BMV encodes two replication proteins, 1a and 2a pol . 2a pol serves as the replicase, whereas 1a has an N-terminal methyltransferase domain (3, 4) and a C-terminal ATPase/helicase-like domain (5). Together, 1a and 2a pol are necessary and sufficient for BMV replication. BMV induces vesicular structures in its surrogate host, the yeast Saccharomyces cerevisiae, and its natural host, barley (6, 7). These structures, termed spherules, have been shown to be the VRCs in yeast as 1a, 2a pol , and nascent viral RNAs reside in the interior of th...

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“…For poliovirus, detection of PC was done by a "click chemistry" method described in Jao et al (36). HeLa cells were infected with an MOI of 10 of poliovirus type I Mahoney.…”

Section: Methodsmentioning

confidence: 99%

Section: Virus-stimulated and Viral Replication Site-localized Pc Accmentioning

confidence: 99%

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

Zhang

Chukkapalli

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

112

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“…approaches have been successfully used. One approach is the introduction of an alkyne moiety to lipids that can be conjugated to a fl uorophore using bio-orthognal chemistry ( 40,44,45 ). Although the alkyne moiety does not greatly alter the biophysical properties of the sterols, it precludes live cell imaging due to the conjugation requirements ( 40,44 ).…”

Section: -Hc Has a Distribution Similar To Cholesterolmentioning

confidence: 99%

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

Iaea

Gale

Bielska

et al. 2015

Journal of Lipid Research

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This article is available online at http://www.jlr.org by coordinated homeostatic mechanisms, primarily involving the regulated proteolysis of the sterol regulatory element-binding protein (SREBP)-2 transcription factor in the Golgi ( 1 ). When ER sterols are abundant, SREBP-2 is retained in the ER in a complex with the cholesterol-sensing protein, SREBP cleavage-activating protein (SCAP), and the ER retention proteins, Insig-1 or -2 ( 2, 3 ). When cholesterol content in the ER is depleted, SCAP undergoes a conformational change and is released from Insig, allowing the SREBP-SCAP complex to translocate to the Golgi apparatus ( 4, 5 ) where it undergoes proteolytic maturation with release of the SREBP transcription factor ( 6 ).Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols ( 1, 7-9 ) Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are generated enzymatically and act as important regulators of cholesterol homeostasis, despite their presence in cells being only at 0.1% the concentration of cholesterol ( 9 ). At the transcriptional level, 25-HC can bind the liver X receptors (LXRs) to active LXR-mediated transcription that results in increased cholesterol effl ux and elimination ( 10, 11 ). 25-HC also inhibits SREBP maturation and subsequent transcription of genes involved in cholesterol biosynthesis and uptake ( 1 ). In contrast to cholesterol, which directly interacts with SCAP, 25-HC enhances the interaction between SCAP and Insig proteins, resulting in retention of the SREBP-2-SCAP complex in the ER ( 12, 13 ).

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“…Data were corrected for chemical or instrumental noise in the samples (Jao et al, 2009). The molar amount of each phospholipid species in the sample was calculated and normalized to the sample dry weights to produce data in the units nmol/mg.…”

Section: Esi-ms/ms Analysis Of Dag and Pamentioning

confidence: 99%

Nonspecific Phospholipase C NPC4 Promotes Responses to Abscisic Acid and Tolerance to Hyperosmotic Stress inArabidopsis

et al. 2010

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Diacyglycerol (DAG) is an important class of cellular lipid messengers, but its function in plants remains elusive. Here, we show that knockout of the Arabidopsis thaliana nonspecific phospholipase C (NPC4) results in a decrease in DAG levels and compromises plant response to abscisic acid (ABA) and hyperosmotic stresses. NPC4 hydrolyzes various phospholipids in a calcium-independent manner, producing DAG and a phosphorylated head group. NPC4 knockout (KO) plants display decreased ABA sensitivity in seed germination, root elongation, and stomatal movement and had decreased tolerance to high salinity and water deficiency. Overexpression of NPC4 renders plants more sensitive to ABA and more tolerant to hyperosmotic stress than wild-type plants. Addition of a short-chain DAG or a short-chain phosphatidic acid (PA) restores the ABA response of NPC4-KO to that of the wild type, but the addition of DAG together with a DAG kinase inhibitor does not result in a wild-type phenotype. These data suggest that NPC4-produced DAG is converted to PA and that NPC4 and its derived lipids positively modulate ABA response and promote plant tolerance to drought and salt stresses.

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Metabolic labeling and direct imaging of choline phospholipids in vivo

Cited by 218 publications

References 23 publications

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol

Nonspecific Phospholipase C NPC4 Promotes Responses to Abscisic Acid and Tolerance to Hyperosmotic Stress inArabidopsis

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