Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

PNAS 109 (50): 3405-3413

Copyright © 2012 by the National Academy of Sciences.

Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

Sue E. Crawford, Joseph M. Hyser, Budi Utama, and Mary K. Estes1

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030


Figure 01
View larger version (25K):
[in this window]
[in a new window]

 
Fig. 1.. Inhibition of autophagy reduces the yield of rotavirus. (A) MA104 cells were treated with 0, 10, or 25 mM 3-MA for 3 h before rotavirus infection [multiplicity of infection (moi) 1]. Cells and media were harvested at the indicated times, and infectious rotavirus was assayed by fluorescent focus assay. *P ≤ 0.01 compared with cells without 3-MA treatment. Error bars represent SD. (B) MA104 cells were treated with 0, 10, or 25 mM 3-MA for 3 h before rotavirus infection (moi 10). At 6 hpi, cells were analyzed by Western blot for NSP4 and for GAPDH as a loading control. Quantification of NSP4 normalized to GAPDH (n = 3) is shown below the blots. Mouse parental (Atg3+/+, gray bars) and Atg3 KO (Atg3–/–, white bars) embryonic fibroblast (MEF) cells (C) or parental (Atg5+/+, black bars) and Atg5 KO (Atg5–/–, white bars) MEF cells, Atg5–/– MEF cells transfected with WT Atg5-EGFP (dark gray bars), or Atg5–/– cells transfected with the mutant Atg5-GFP K130R (light gray bars) (D) were infected with rotavirus (moi 10) and then harvested at the indicated times. Cells and media were assayed for infectious rotavirus by fluorescent focus assay. Data shown represent one of three separate experiments performed in quadruplicate. *P ≤ 0.01 compared with parental cells. Error bars represent SD. (E) Rotavirus infection of Atg5+/+ MEFs or Atg5–/– MEFs transfected with a plasmid expressing WT Atg5-EGFP (WT) induces LC3 II. Atg5+/+, Atg5–/–, or Atg5–/– cells transfected with plasmids expressing WT Atg5-EGFP (WT) or Atg5-EGFP K130R were infected with rotavirus and harvested 18 hpi. The lysates were analyzed by Western blot using antibodies against LC3 and GAPDH.

 

Figure 02
View larger version (28K):
[in this window]
[in a new window]

 
Fig. 2.. Rotavirus (RV) infection leads to LC3 lipidation and insertion into autophagic membranes. Mock-infected (–) or rotavirus-infected (+) MA104 cell lysates harvested at the indicated time points postinfection were analyzed by Western blot. Proteins on the blots were detected with antibody against LC3 to detect cytoplasmic, endogenous LC3 I and the lipidated and membrane-inserted LC3 II and GAPDH (A) or NSP4 (B).

 

Figure 03
View larger version (87K):
[in this window]
[in a new window]

 
Fig. 3.. Localization of NSP4, LC3, and viroplasms following rotavirus infection. Rotavirus-infected MA104 cells were fixed; permeabilized at 3 (A), 4 (B), 5 (C), and 6 (D) hpi; and stained with antibody against NSP5 to detect viroplasms (blue), endogenous LC3 (green), or NSP4 (red). (Scale bars: A and B,10 μm; C and D, 5 μm.)

 

Figure 04
View larger version (36K):
[in this window]
[in a new window]

 
Fig. 4.. Rotavirus infection suppresses autophagy maturation. (A) Mock- or rotavirus (RV)-infected cells were cultured in the absence (–) or presence (+) of 100 nM bafilomycin A1 (Baf) added at 1 hpi. The cells were harvested at 8 hpi and analyzed by Western blot for cytoplasmic LC3 I, the lipidated membrane-inserted LC3 II, and NSP4, and for GAPDH as a loading control. Quantification of LC3 II or NSP4 normalized to GAPDH (n = 3) is shown below the respective blots. (B) Monomeric (m) RFP-GFP-LC3–expressing rotavirus-infected MA104 cells were fixed at 20 hpi, stained with antirotavirus antibody (blue), and imaged by confocal microscopy. Both RFP and GFP fluorescence are observed in the same puncta in rotavirus-infected cells, indicating that rotavirus infection suppresses autophagosome maturation. (Scale bars: 10 μm.)

 

Figure 05
View larger version (51K):
[in this window]
[in a new window]

 
Fig. 5.. NSP4-mediated increase in [Ca2+]cyto activates the CaMKK-β pathway to induce NSP4/LC3 puncta formation. (A) MA104 cells were transfected with WT NSP4-EGFP (Top) or NSP4-ASDASA-EGFP (Middle and Bottom). NSP4-ASDASA-EGFP–expressing cells were cultured in the absence (Middle) or presence (Bottom) of TG. At 24 hpi, the cells were fixed, stained with antibody against LC3, and imaged by confocal microscopy. (Scale bars: Top, 10 μm; Middle and Bottom, 5 μm.) (B) Quantitation of the number of cells imaged by confocal microscopy in A that contain NSP4 and LC3 that colocalize in puncta. (C) MA104 cells were transfected with WT NSP4-EGFP and cultured in media (Vehicle), 50 μM BAPTA-AM, or 50 μM STO-609. At 24 hpi, the cells were fixed, stained with antibody against LC3, and imaged by confocal microscopy, and the number of cells containing only NSP4 puncta (white bars) or puncta containing both NSP4 and LC3 that colocalize (black bars) were quantitated. *P < 0.001 compared with WT NSP4-EGFP–expressing cells.

 

Figure 06
View larger version (21K):
[in this window]
[in a new window]

 
Fig. 6.. Chelation of [Ca2+]cyto and inhibition of CaMKK-β by STO-609 abrogate rotavirus-induced autophagy and decrease rotavirus yield. (A) Rotavirus-infected MA104 cells were cultured in the absence (–) or presence (+) of STO-609 and harvested at 4 hpi (Left) or 6 hpi (Right). Cell lysates were analyzed by Western blot to detect P-AMPK, LC3, and NSP4, as well as GAPDH as a loading control. Quantification of NSP4 normalized to GAPDH (n = 3) is shown below the blot detected with antibody against NSP4. (B) MA104 cells transfected with NSP4-specific siRNA (G10) or Scr control and either rotavirus (RV)- or mock-infected. Cell lysates were harvested 6 hpi and analyzed by Western blot to detect LC3, and NSP4, as well as GAPDH as a loading control. Quantification of LC3 II/LC3 I ratios and NSP4 normalized to GAPDH is shown below the respective blots (n = 3). MA104 cells were infected with rotavirus [multiplicity of infection (moi) 1], and medium (black bars) or medium containing STO-609 (C; 25 μM, gray bars; 50 μM, white bars) or BAPTA-AM (D; 50 μM, white bars) was then added at 1 hpi. The cells and media were harvested at the indicated times and assayed for infectious rotavirus by fluorescent focus assay. Data shown represent one of three separate experiments performed in quadruplicate. *P ≤ 0.01 compared with cells without STO-609 or BAPTA-AM treatment. Error bars represent SD. Cell viability was assessed by trypan blue exclusion (98% and 96%, respectively, in mock-infected or STO-609– or BAPTA-AM–treated cells).

 

Figure 07
View larger version (38K):
[in this window]
[in a new window]

 
Fig. 7.. Inhibition of autophagy by STO-609 hinders NSP4 and VP7 trafficking to viroplasms. Rotavirus-infected MA104 cells were cultured in the absence [(–)STO-609, Upper] or presence [(+)STO-609, Lower] of 50 μM STO-609. At 7 hpi, cells were fixed, permeabilized, and stained with antibody against NSP5 to detect viroplasms (teal), VP7 (green), or NSP4 (red). The nuclei were detected by DAPI. (Scale bar: 5 μm.)

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882