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Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria.

ABSTRACT: The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus micro1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that micro1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic alpha-helices within a carboxyl-terminal portion of micro1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by micro1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated when micro1 was coexpressed with sigma3, with which it is known to coassemble. We propose that micro1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of micro1 to associate with intracellular membranes. Moreover, during reovirus infection, association with sigma3 may regulate apoptosis induction by micro1.

SUBMITTER: Coffey CM

PROVIDER: S-EPMC1563861 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria.

Coffey Caroline M CM Sheh Alexander A Kim Irene S IS Chandran Kartik K Nibert Max L ML Parker John S L JS

Journal of virology 20060901 17

The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus micro1 protein, encoded by the M2 gene, was sufficient to induce a ...[more]

PMID: 16912293

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Project description:Cell apoptosis is a biochemical and molecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (H2O2) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of H2O2 levels in different compartments is seldom explored, particularly in two important organelles: mitochondria and endoplasmic reticulum (ER). To solve this problem, we developed two new organelle-specific fluorescent probes termed MI-H2O2 and ER-H2O2 that can detect H2O2 in mitochondria and ER, respectively or simultaneously. Experimental results demonstrated that MI-H2O2 and ER-H2O2 display distinguishable excitation and emission spectra, as well as excellent organelle targeting capabilities. Therefore, we used MI-H2O2 and ER-H2O2 to successfully image exogenous or endogenous hydrogen peroxide in the mitochondria and ER. Interestingly, during diverse apoptotic stimuli, dual-color fluorescence imaging results revealed that the changes of H2O2 levels in mitochondria and ER are different. The H2O2 levels are enhanced in both the mitochondria and ER during the l-buthionine sulfoximine (BSO)-treated cell apoptosis process. During mitochondria-oriented apoptosis induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or rotenone, H2O2 levels prominently and continuously increase in the mitochondria, whereas the ER H2O2 levels were found to rise subsequently after a delay. Moreover, during ER-oriented apoptosis induced by tunicamycin, ER is the major site for overproduction of H2O2, and delayed elevation of the H2O2 levels was found in the mitochondria. Altogether, this dual-probe and multicolor imaging approach may offer a proven methodology for studying molecular communication events on H2O2-related apoptosis and also other physiological and pathological processes within different subcellular organelles.

Project description:Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein sigma3 is rapidly removed from virus particles and then protein mu1C is cleaved. Initial cleavage of mu1C has been well described and generates an amino (N)-terminal delta peptide and a carboxyl (C)-terminal phi peptide. However, cleavage and removal of sigma3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS-PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut sigma3 within a "hypersensitive" region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of mu1C, followed by a peptide that corresponded to amino acids 214-236 of the sigma3 protein. Other regions of mu1C were not observed until protein sigma3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of sigma3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different sigma3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of sigma3 is acetylated.

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Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria.

Publications

Reovirus outer capsid protein micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria.

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