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. 2006 Oct;2(10):e102.
doi: 10.1371/journal.ppat.0020102.

COPI activity coupled with fatty acid biosynthesis is required for viral replication

Sara Cherry  1 , Amit Kunte, Hui Wang, Carolyn Coyne, Robert B Rawson, Norbert Perrimon
Affiliations

COPI activity coupled with fatty acid biosynthesis is required for viral replication

Sara Cherry et al. PLoS Pathog. 2006 Oct.

Abstract

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. COPI Coatamer Complex Is Required for Viral Replication
(A) Frequency of encoded functional groups as curated by GO (The FlyBase Consortium) and manually assigned to representative categories for all verified candidates. Categories that are overrepresented with p < 0.05 are indicated. (B) Decreased viral replication post dsRNA treatment with dsRNA against alphaCOP, betaCOP, beta'COP, gammaCOP, deltaCOP, and zetaCOP as compared to dsRNA treatment with GFP or epsilonCOP. Images were quantified as the percentage of infected cells (FITC-anti DCV [green]) divided by (Hoescht 33342 [red]).
Figure 2
Figure 2. COPI Is Specifically Required for DCV Replication Postentry
(A) FACS analysis demonstrates that COPI (betaCOP) and COPII (sec23) are required for Delta secretion. DeltaWTNdeMYC cells were treated with dsRNA and subsequently Delta expression was induced for 2 h. Extracellular Delta expression was monitored by FACS. The bar was set such that 98% of the total uninduced cells were negative for Delta staining. Induction leads to a shift in the population to express surface Delta, such that only 31% of the cells remain negative. Under these conditions, COPI, COPII, and SREBP block surface staining. (B) DCV entry requires endocytosis (Rab5) but not COPI (bCOP) or COPII (sec23) function. Cells were pretreated with dsRNA, infected at 4 °C to allow surface binding, followed by 3 h at 25 °C to release the block to endocytosis and monitor viral trafficking. Viral uptake was measured by determining the percentage of cells (red) that contained virus (green). Green, anti-DCV; red, Alexa-fluor-568-phalloidin.
Figure 3
Figure 3. Ultrastructural Analysis Reveals Virus-Dependent Vesicular Compartment
(A) Uninfected cells with intact Golgi. (B) Vesicles were generated at 10 h postinfection throughout the cytoplasm of cells pretreated with dsRNA against GFP and infected with DCV. (C–E) Cells were pretreated with dsRNA against COPI (bCOP) (C), COPII (sec23) (D), or SREBP (E), infected with DCV, and prepared for electron microscopy. (F) Immunoelectron microscopy of Drosophila cells infected with DCV and the RNA replication machinery was visualized using anti-DCV helicase and a secondary antibody coupled to 10-nm gold particles. The surfaces of cytoplasmic vesicles (arrows) are stained. (G) Higher-magnification view of DCV helicase–labeled vesicle. (H) Immunoelectron microscopy of Drosophila cells infected with DCV and the RNA replication machinery was visualized using anti-DCV helicase and a secondary antibody coupled to 5-nm gold particles. The Golgi was visualized using an anti-Golgi antibody (DG13) and a secondary antibody coupled to 12-nm gold particles.
Figure 4
Figure 4. COPI-Dependent Golgi Disassembly in DCV Infected Cells
Confocal analysis of cells pretreated with the indicated dsRNA and infected with DCV. (A) Golgi morphology of DCV-infected control cells (GFP) reveals that the normal punctate staining in uninfected cells is dispersed during viral replication. (B–F) Loss of COPI (bCOP) (B), SREBP (E), or CG3523 (F) but not COPII (sec23) (C) or Syx5 (D) results in a decrease in viral infection. Note that the Golgi stain is reduced in uninfected COPI, COPII, SREBP, CG2523, and Syx5, but only the loss in COPI, SREBP, or CG3523 results in a decrease in DCV replication. Green, anti-Golgi (DG13); red, anti-DCV; blue, Hoescht 33342.
Figure 5
Figure 5. Attenuation of Fatty Acid Biosynthesis in Animals Is Protective
(A) SREBP-null flies were generated by rescuing the larval lethality using an inducible transgene and have no detectable SREBP protein as adults as measured by immunoblot probed with anti-SREBP. (B) These SREBP-null flies are resistant to viral infection as measured by viral antigen production post infection. Heterozygous or homozygous SREBP mutant flies were challenged with DCV, and viral antigen production was measured as a function of time postinfection. Protein lysates were generated (hours postinfection indicated [Hr p.i.], normalized, and probed with anti-DCV or anti-tubulin for normalization. (C) SREBP-null flies are resistant to viral infection as measured by viral RNA production postinfection. Heterozygous or homozygous SREBP mutant flies were challenged with DCV, and viral RNA production was measured by RT-PCR at the indicated time points postinfection (Hr p.i.).
Figure 6
Figure 6. Attenuation of COPI but Not COPII Protects Human Cells from Poliovirus Infection
(A) Poliovirus infection of Caco-2 cells pretransfected with siRNAs against alphaCOP results in inhibition of viral replication but not control siRNA or siRNA against sec23B as measured by immunofluorescence analysis of infected cells (nuclei [blue] = DAPI, infected cells [green] = FITC-conjugated mouse anti-enterovirus VP1). (B) Percent infection (FITC-positive cells/DAPI * 100) is shown for two independent experiments performed in triplicate where error bars represent one standard deviation. *p < 0.05. (C) Plaque-forming units/mL (pfu/mL) are shown for the experiments performed in (B). *p < 0.05. (D) RT-PCR on Caco-2 cells treated with siRNA against alphaCOP or sec23B demonstrates that treatment with either siRNA leads to a significant depletion of the cellular mRNA and amplification of GAPDH was used as loading control.
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