Modification of Cellular Autophagy Protein LC3 by Poliovirus

JOURNAL OF VIROLOGY, Nov. 2007, p. 12543–12553 Vol. 81, No. 22 0022-538X/07/$08.00ϩ0 doi:10.1128/JVI.00755-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Modification of Cellular Autophagy Protein LC3 by Poliovirusᰔ Matthew P. Taylor and Karla Kirkegaard* Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 Received 7 April 2007/Accepted 27 August 2007 Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and pro- Downloaded from viding a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus. jvi.asm.org All positive-strand RNA viruses of eukaryotes replicate their gosomes, double-membraned organelles that become degrada- genomes on cytoplasmic membranes, and infections are often tive upon maturation, breaking down cytoplasmic proteins and at SERIALS CONTROL Lane Medical Library on January 10, 2008 accompanied by dramatic reorganization of those membranes. organelles entrapped within their lumen. Originally identified We and others have argued that one function of membrane as a process induced by cellular starvation, autophagy is now localization of viral RNA replication proteins is to create two- appreciated as a cellular response to a variety of stimuli. For dimensional surfaces to promote the oligomerization of viral example, the estrogen receptor agonist tamoxifen is a potent proteins (6, 31). Yet the detailed cell biology of the targeted inducer of autophagy, indicating a role for the hormonal in- membranes and the mechanisms by which they are altered duction of autophagy (2–4). In Saccharomyces cerevisiae, for- differ greatly from virus to virus (40, 53). mation and maturation of autophagosomes requires the func- Poliovirus infection rearranges intracellular membranes, tion of many genes (reviewed in reference 52). Human creating membranous vesicles, 200 to 400 nm in diameter, that homologs of several yeast autophagy genes have recently been accumulate in the cytoplasm (9, 42, 47). Immunoelectron mi- identified, including LC3, the human homolog of ATG8, and it croscopy revealed that the cytoplasmic surfaces of these mem- is likely that much of the autophagic pathway has been evolu- branous vesicles, not the interior lumen, are the sites of viral tionarily conserved (22, 50). Many recent experiments have RNA replication (1, 7). The specific morphology of these identified the autophagy pathway as a critical component of membranes has proven somewhat controversial, because they the innate immune response of plants and mammals (reviewed are rich in lipids (17, 33) and their apparent morphology is in references 10 and 30). Therefore, we sought to discover the therefore sensitive to the fixation method used in electron similarities and differences between the poliovirus-induced microscopy (43, 46). Using high-pressure cryopreservation membranes and cellular autophagosomes and to investigate methods, we have consistently observed double-membraned the mechanism of formation of the double-membraned vesi- vesicles (42, 47), as did Dales et al. using more conventional cles induced by poliovirus and the role of the autophagy path- fixation techniques (9). Double-membraned vesicular struc- way during viral infection. Previous studies show that reduced tures have also been associated with viral RNA replication function of the autophagy pathway resulted in reduced complexes in cells infected with several other positive-strand amounts of extracellular and intracellular poliovirus (20), ar- RNA viruses, including equine arterivirus (37), murine hepa- guing that the autophagic pathway plays a positive role in the titis virus (16), and sudden acute respiratory syndrome-associ- growth of both these positive-strand RNA viruses. ated virus (15). Therefore, understanding the mechanism(s) by The formation and maturation of autophagosomes involve which viral proteins induce the formation of double-membrane the stepwise acquisition of proteins from disparate cellular vesicles is of interest for understanding the formation of RNA compartments (reviewed in references 28 and 39). Nascent replication complexes of several kinds of RNA viruses. autophagosomes form either de novo or from the endoplasmic Several of the features displayed by the vesicles induced reticulum and comprise cellular cytoplasm surrounded by two during poliovirus infection are shared with cellular autopha- lipid bilayers that fuse from a C-shaped intermediate (14, 32, 36). Although relatively protein poor, they have been shown to contain a modified form of the autophagy protein LC3. Orig- * Corresponding author. Mailing address: Department of Microbi- inally characterized as microtubule-associated protein light ology and Immunology, 299 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305. Phone: (650) 498-7075. Fax: (650) chain 3, or MAP-LC-3, LC3 protein is cleaved after synthesis 498-7147. E-mail: [email protected]. by cellular protease Atg4, generating an 18-kDa species ᰔ Published ahead of print on 5 September 2007. termed LC3-I (see Fig. 1A). When autophagy is activated, a 12543 12544 TAYLOR AND KIRKEGAARD J. VIROL. series of covalent transfers links LC3 to Atg7, then to Atg3, 2 ϫ 105 cells per 10-cm dish, followed by transfection with 2 ␮g of plasmid DNA and finally to phosphatidylethanolamine, generating a lipi- using Effectene reagent (QIAGEN, Valencia, CA) and incubation for 48 h at dated species termed LC3-II (Fig. 1A) (19, 45, 50). This mod- 37°C. Cells were harvested with phosphate-buffered saline (PBS) containing 5 mM EDTA (pH 8.0) and collected by centrifugation at 300 ϫ g for 5 min. Cell ification allows LC3 to become membrane associated, prefer- pellets were washed in the same buffer, pelleted in an Eppendorf microcentrifuge entially associating with the developing and newly formed at 6,000 ϫ g, resuspended in RSB–NP-40 (10 mM Tris–HCl [pH 7.5], 10 mM autophagosomes (23, 24). NaCl, 1.5 mM MgCl2, 1% NP-40) supplemented with Mini-complete EDTA-free The genomes of poliovirus and other picornaviruses consist protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and in- cubated on ice for 15 min. Nuclei and insoluble debris were pelleted by centrif- of a single, positive-stranded RNA. A single encoded polypro- ugation at 6,000 ϫ g for 5 min. Cell extracts were then stored at Ϫ20°C or tein is processed by viral proteases to produce individual capsid subjected immediately to sodium dodecyl sulfate-polyacrylamide gel electro- proteins, the viral RNA-dependent RNA polymerase 3D, and phoresis (SDS-PAGE). For saponin extraction, cell pellets were resuspended in several other multifunctional nonstructural proteins. Of the a solution containing 0.5% saponin, 80 mM PIPES [piperazine-N,NЈ-bis(2-eth- nonstructural proteins, three (2B, 2C, and 3A) associate with anesulfonic acid), pH 6.8], 1 mM CaCl2, and 5 mM EGTA as described else- where (29) and collected by centrifugation prior to the addition of the RSB— membranes when expressed in isolation; mutations in any of NP-40 solution. Cell extracts were mixed with 6ϫ Laemmli loading buffer and these three proteins lead to defects in RNA replication. The heated to 95°C for 5 min prior to electrophoresis at 120 V for 1 to2honeither Downloaded from expression of stable protein precursor 2BC and protein 3A, in 10% or 13.5% SDS-polyacrylamide gels. For immunoblotting, proteins were combination but not separately, was found to induce the for- transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Milli- pore, Billerica, MA) for 75 min at 20 V in a Miniprotean III transfer tank mation of double-membraned vesicles that display biochemical (Bio-Rad, Hercules, CA). Membranes were then air dried before rewetting with markers and fractionate similarly to the vesicles induced dur- 70% methanol followed by blocking with PBS supplemented with 0.1% (vol/vol) ing poliovirus infection (20, 47). Thus, should these mem- Tween-20 and 2% (wt/vol) bovine serum albumin. All primary antibody incuba- tions were done in blocking solution for a minimum of1hatroom temperature branes prove to derive directly from the autophagy pathway, jvi.asm.org these viral proteins will be among the few molecularly charac- followed by washing with PBS-Tween (PBS supplemented with 0.1% Tween). Alkaline phosphatase-conjugated secondary antibodies were diluted 1:10,000 in terized inducers of this process. PBS-Tween,

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