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. 2004 Mar 12;279(11):10136-41.
doi: 10.1074/jbc.M306124200. Epub 2003 Dec 29.

Coronavirus replication complex formation utilizes components of cellular autophagy

Erik Prentice  1 , W Gray Jerome, Tamotsu Yoshimori, Noboru Mizushima, Mark R Denison
Affiliations

Coronavirus replication complex formation utilizes components of cellular autophagy

Erik Prentice et al. J Biol Chem. .

Abstract

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells. In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

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Figures

F<sc>ig</sc>. 1
Fig. 1
Replicase proteins co-localize with markers for autophagy.A, DBT cells were infected with MHV for 6 h and then fixed and processed for immunofluorescence. Cells were probed with antibodies against LC3, Apg12, β-tubulin, N, p22, and M. The localization of LC3 and β-tubulin (red, all panels) was compared with N, p22, and M (green, as labeled in panels), with yellow pixels indicating areas of co-localization. B, quantitation of fluorescence co-localization. The percent colocalization of the indicated proteins was determined as described under “Experimental Procedures.”
F<sc>ig</sc>. 2
Fig. 2
The hel and N proteins translocate away from LC3-positive membranes to sites of assembly over the course of infection. MHV-infected DBT cells were harvested from 5 to 9 h p.i. Quantitative analysis of co-localization was performed for the indicated protein pairs. P22 and LC3 (triangles) were 85% co-localized throughout infection. Hel and N (stars) were 85% co-localized throughout infection. N and LC3 co-localization (diamonds) decreased over the course of infection from 75% at 5 and 6 h p.i. to 18% at 8 and 9 h p.i. Hel and M co-localization (squares) increased from 12% at 5 h p.i. to 60% by 8h p.i. M and LC3 (circles) were <18% co-localized throughout infection.
F<sc>ig</sc>. 3
Fig. 3
Long-lived protein degradation assay in DBT cells. DBT cells were treated as labeled. Data represent the amount of protein degraded over an 8-h time course. The amount of long-lived protein degradation was determined as described under “Experimental Procedures.” Asterisks denote statistically significant values.
F<sc>ig</sc>. 4
Fig. 4
Long-lived protein degradation assay in APG5+/+ and APG5–/–cells. R1 (APG5+/+) and A11 (APG5–/–) cells were treated as labeled, and long-lived protein degradation levels were determined as described under “Experimental Procedures.” Data represent the amount of protein degraded over an 8-h time course.
F<sc>ig</sc>. 5
Fig. 5
MHV growth is decreased in autophagy-deficient cells.A, Western blot analysis of R1 (APG5+/+), A11 (APG5–/–), B22 (APG5–/–), and WT13 (APG5–/–+ APG5 plasmid) cells with Apg12 antibody. B, Western blot analysis of R1 (APG5+/+), A11 (APG5–/–), B22 (APG5–/–), and WT13 (APG5–/–with APG5 plasmid) cells with Apg5 antibody. C, the indicated cells were infected with MHV, and virus growth was determined by plaque assay.
F<sc>ig</sc>. 6
Fig. 6
Electron micrographs of infected R1 cells and infected and mock-infected A11 cells. Infected R1 cells (APG5+/+) do not show a swollen ER phenotype (white arrow) but instead are filled with viral replication complexes (white arrowheads, top left panel). Mock-infected A11 cells (top right panel) appeared morphologically normal, with minor swelling of the rough ER (white arrow) occasionally detected. MHV-infected A11 cells contained extremely swollen interconnected areas of RER that appear to surround cytoplasmic regions to form vesicles (bottom left panel). The lower right panel shows a higher magnification view of swollen RER (black arrow points to same vesicle in both panels). The black arrowheads indicate an area of membrane confluence with the outer nuclear membrane (bottom right panel). Mi, mitochondrion; Nuc, nucleus.
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