Complex I Binding by a Virally Encoded RNA Regulates Mitochondria-Induced Cell Death

doi:10.1126/science.1142984

Account Information

Guest Alerts | Access Rights | My Account | Sign In

Site Navigation

Article Views

Article Tools

My Science

More Information

Related Jobs from ScienceCareers

Science 1 June 2007:
Vol. 316. no. 5829, pp. 1345 - 1348
DOI: 10.1126/science.1142984

Prev | Table of Contents | Next

Reports

Complex I Binding by a Virally Encoded RNA Regulates Mitochondria-Induced Cell Death

Matthew B. Reeves¹^*, Andrew A. Davies¹, Brian P. McSharry², Gavin W. Wilkinson² and John H. Sinclair¹

¹ Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
² Section for Infection and Immunity, College of Medicine, University of Wales, Heath Park, Cardiff, CF14 4XX, UK.

Fig. 1. HCMV protects cells from rotenone-induced apoptosis. (A) Percentage of TUNEL-positive cells in mock- (1 and 2), Toledo- (3 and 4), ${Delta}$ ß2.7Tol- (5 and 6), and ${Delta}$ ß2.7Tol-Revertant ( ${Delta}$ ß2.7Tol-Rev)–infected (7 and 8) U373 cells incubated with acetone (1, 3, 5, and 7) or rotenone (ROT) (2, 4, 6, and 8). n = 3 independent experiments. Asterisks denote P < 0.01 with Student's t test. Error bars indicate 1 SEM. (B) Percentage of TUNEL-positive pREP10- (Con) (1) and pREP10-ß 2.7–transfected (2) cells incubated with rotenone. from three independent analyses. Reverse transcription (RT)–PCR for ß2.7 expression in pREP10- (lane 1) and pREP10-ß2.7–transfected (lane 2) cells is shown. [View Larger Version of this Image (17K GIF file)]

Fig. 2. HCMV interacts with and prevents the rotenone-induced relocalization of, GRIM-19 in virally infected cells. (A) GRIM-19 localization (red) in mock- (a and d), Toledo- (b and e), and ${Delta}$ ß2.7Tol-infected (c and f) U373 cells incubated with solvent control (Con) (a, c, and e) or rotenone (b, d, and f) 24 hpi. (B to D) Immunoprecipitation of control immunoglobulin G (lane 2), GRIM-19 (G-19) (lane 3), native complex I (C-1) (lane 4), and native complex V (C-V) (lane 5) from Toledo- (B and C) or ${Delta}$ ß2.7Tol-infected (D) cells, and RT-PCR for ß2.7 [(B) and (D), lanes 1 to 5], IE72 (C), or a ß2.7 PCR with no prior RT [(B), lanes 6 to 10]. Inputs are shown in lane 1. (E) IPs on Toledo-infected cells with anti-sense oligonucleotides to ß2.7 (lane 3) or IE72 (lane 4) or with no oligonucleotide (N) (lane 2). Silver stain of 1% input (INP) (lane 1), the immunoprecipitated proteins (lanes 2 to 4), and known protein loading controls (5 to 500 ng, lanes 5 to 7) is shown. (F) GRIM-19 expression in 10% input (lane 1) and the ß2.7 (lane 2) and IE72 (lane 3) RNA-IP samples. [View Larger Version of this Image (27K GIF file)]

Fig. 3. ß2.7 expression maintains ATP production in infected cells. (A) Percentage of ATP content of mock- (1 and 2), ${Delta}$ ß2.7Tol- (3 and 4), Toledo- (Tol) (5 and 6), AD169-, and AD169-GFP–infected cells with (2,4, 6,8, and 10) or without (1, 3, 5, 7, and 9) rotenone for 2 hours was determined at 48 hpi, as compared to mock (1). (B) Percentage of ATP in mock-(1), ${Delta}$ ß2.7Tol- (2), Toledo- (3), AD169- (4), or AD169-GFP–infected cells (5) 5 dpi. n = 3 independent experiments. Asterisks denote P < 0.01 with Student's t test. Error bars indicate 1 SEM. [View Larger Version of this Image (14K GIF file)]

Fig. 4. Toledo, but not ${Delta}$ ß2.7Tol, grows normally in metabolically stressed cells. (A) Growth of Toledo (triangle), AD169 (diamond), AD169-GFP (square), or ${Delta}$ ß2.7Tol (cross) in human fibroblasts (HFs). PFU, plaque-forming units. (B) Growth of Toledo (diamond and triangle) or ${Delta}$ ß2.7Tol (square and cross) in U373 cells. At 24 hpi, Toledo-infected (triangle) and ${Delta}$ ß2.7Tol-infected (cross) U373 cells were incubated with rotenone (asterisk). (C) Growth of Toledo (square and cross) or ${Delta}$ ß2.7Tol (diamond or triangle) in U373 and HF cells in glucose-depleted media. [View Larger Version of this Image (20K GIF file)]