INTRODUCTION
All positive-strand RNA viruses, including picornaviruses, such as poliovirus, rhinovirus, and hepatitis A virus, and flaviviruses, such as dengue virus and hepatitis C virus (HCV), rely heavily on cellular membranes at numerous stages of their infectious cycles. For example, RNA replication complexes must assemble on the topologically cytoplasmic surfaces of intracellular membranes. In some cases, such as poliovirus and hepatitis A virus, these RNA replication complexes are on the convex outer surfaces of discrete vesicles (
1). In others, such as dengue virus, RNA replication complexes are assembled on invaginated membrane surfaces that are connected to the cytosol only via narrow openings (
2,
3). For dengue virus, newly synthesized viral RNA exits the invaginated cytoplasm and interacts with core protein, which encapsidates the viral RNA and decorates the surfaces of nearby lipid droplets via the high-affinity binding of its N-terminal domain (
4,
5). For HCV, a similar interaction of the core protein with lipid droplets has been described and seems to play a critical role in the assembly of viral particles (
6–9). During dengue virus infection, formation of the nucleocapsid, subsequent interaction with envelope proteins, and budding into the ER lumen are likely to occur in close proximity (
2). In the
cis-Golgi, the virion undergoes a conformational change, and the viral prM (prematrix) protein is cleaved by the cellular furin protease into the mature M (matrix) protein and a peptide (pr) (
10,
11). Upon cleavage, the pr peptide dissociates from the virion, resulting in the formation of mature progeny viruses that are highly infectious. This finely tuned interplay between cellular membrane remodeling, cellular lipid storage, and viral assembly is not only a fascinating cell biological puzzle, but also provides exciting opportunities for the development of antiviral compounds.
The cellular autophagy pathway, originally discovered as a response to starvation, provides a route for cytoplasmic constituents to be targeted to the lysosomal machinery for degradation (reviewed in reference
12). Nascent autophagosomes comprise cellular cytoplasm surrounded by two lipid bilayers that fuse from a C-shaped structure (
12–14). These nascent autophagosomes contain a modified form of the autophagy protein LC3. Originally characterized as microtubule-associated protein light chain 3 (MAP-LC-3), the LC3 protein is cleaved after synthesis by the cellular protease Atg4, generating an 18-kDa species termed LC3-I. When autophagy is activated, a series of covalent transfers links LC3 to the Atg7 protein, then to the Atg3 protein, and finally to phosphatidylethanolamine, generating a lipidated species termed LC3-II (
15–17). This modification allows LC3 to become membrane associated, preferentially marking the developing and newly formed autophagosomes (
18). The ability of the autophagy pathway to target cytoplasmic constituents to the lysosomal pathway allows turnover of damaged mitochondria and other organelles; clearance of aggregated proteins; maintenance of a favorable cellular energetic balance by degrading proteins, lipids, and glycogen; and the destruction of certain intracellular pathogens (
19,
20). Autophagy has thus been hailed as a branch of the innate immune response. However, some viruses have been shown to benefit from the cellular autophagy pathway. Our laboratory (
1,
21–23) and others (
24) have shown that the membranous vesicles induced during poliovirus infection display several hallmarks of cellular autophagosomes: double-membrane morphology, cytoplasmic contents, and the presence of LC3-II and LAMP-1. Recent reports have shown evidence that members of the family
Flaviviridae, such as HCV and dengue virus, also utilize components of the cellular autophagy pathway for their own growth benefit (
25–29).
Assessing the importance of autophagy in the growth of any these pathogens has been hampered by the lack of selective small-molecule inhibitors of the pathway. Until recently, the only available small-molecule inhibitor of the pathway was 3-methyladenine (3-MA), which has a working concentration in the millimolar range and inhibits multiple forms of phosphatidylinositol 3-kinases (PI3Ks). However, a high-throughput, image-based approach has recently been employed to screen a chemical library of compounds known to be bioactive for the ability of any of them to regulate the autophagy pathway. Several inducers were discovered in the screen, including nicardipine, used in the present study (
30). In addition, one inhibitor, MBCQ [4-((3,4-methylenedioxybenzyl)amino)-6-chloroquinazoline], was identified and subsequently derivatized into an effective and selective autophagy inhibitor termed spautin-1 (
specific and
potent
autophagy
inhibitor 1) (
31). Spautin-1 was shown to inhibit the deubiquitination activities of USP10 and USP13, which normally function to reverse the ubiquitination and subsequent degradation of the beclin-Vps34-Atg14 complex, which is essential in the early stages of autophagy. The degradation of beclin-Vps34-Atg14 complexes promoted by spautin-1 treatment also decreases the intracellular concentrations of phosphatidylinositol 3-phosphate (PI3P), an essential lipid component of the membranes required for the initiation of autophagosome formation (
31).
The high selectivity of spautin-1 in inhibiting the autophagy pathway (
31) prompted us to reevaluate the role of this cellular process in dengue virus growth. We also investigated the effect of inducing the pathway in tissue culture and in AG129 mice, a well-established model of infection that reproduces several of the major pathologies of human infection (
32–34). Stimulation of autophagy generated larger amounts of infectious virus both in cells and in animals that correlated with increased pathogenicity in the mice. Inhibition of autophagy, on the other hand, generated drastically reduced amounts of infectious virions, instead producing an abundance of noninfectious particles with apparent maturation defects. Our results show that components of the autophagy pathway are not only involved in viral RNA synthesis, but, more importantly, play a critical role in the generation of infectious viral particles.
MATERIALS AND METHODS
Cells and viruses.
Baby hamster kidney cells (BHK-21, clone 15) and human HeLa cells were cultured as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) calf serum, 100 units of penicillin/ml, 100 μg of streptomycin/ml, and 10 mM HEPES at 37°C and 5% CO2. Human Huh7.A.1 cells were cultured in a similar fashion with 10 mM sodium pyruvate, nonessential amino acids, and 10% (vol/vol) fetal bovine serum (FBS). Huh7.A.1-green fluorescent protein (GFP)-LC3 cells were generated after three rounds of cloning and amplification of individual Huh7.A.1 colonies that stably expressed GFP-LC3. Huh7.A.1-GFP-LC3 culture medium was identical to that of Huh7.A.1 cells but also contained 1 mg/ml of Geneticin as a selectable marker. Aedes albopictus C6/36 cells were cultured as monolayers in Leibovitz's L-15 medium supplemented with 10 mM HEPES, 100 units of penicillin/ml, 100 μg streptomycin/ml, and 10% (vol/vol) fetal bovine serum at 30°C.
Dengue virus type 2 (DENV2) 16681 was propagated from an infectious cDNA clone (pD2/IC, a gift from Eva Harris, University of California [UC], Berkeley). DENV2 PL046 was also generated from infectious cDNA (a gift from Sujan Shresta, La Jolla Institute for Allergy and Immunology). All viruses were grown in C6/36 cells, and their titers were determined in BHK-21 cells. For mouse experiments, virus was concentrated at 53,000 × g for 2 h at 4°C and resuspended in cold, endotoxin-free phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum.
Hepatitis C virus JFH1 serotype 2a was generated after electroporation of susceptible Huh7.5 cells with an infectious cDNA clone synthesized to correspond to the sequence of JFH1 (
35). Concentrated virus stocks were prepared by filtration of supernatants from infected Huh7.5 cells through a Centricon Plus-70 filter (Millipore, Billerica, MA).
Poliovirus type 1 Mahoney was propagated from an infectious cDNA plasmid as previously described (
36).
Antibodies.
Anti-dengue virus antibodies against all four serotypes of dengue virus or against prM were purchased from Abcam (Cambridge, MA). Anti-LC3 antibody was purchased from Sigma (St. Louis, MO). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids and RNA transcription.
The region of the pD2/IC plasmid containing the DENV2 16681 genome was cut into three fragments and subcloned into a pUC18 backbone for easier manipulation (SacI and SphI sites for subclone 1, SphI and KpnI sites for subclone 2, and KpnI and XbaI sites for subclone 3). Mutagenesis of the viral genome was performed in the appropriate subclone plasmid by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). Each amplified DNA segment was sequenced in its entirety to ensure that no adventitious mutations were introduced and was subcloned back into the infectious cDNA backbone to generate infectious RNA. Infectious RNAs were generated by in vitro transcription with the MEGAscript T7 kit (Ambion) with the following modifications to the manufacturer's protocol: 5 mM each GTP, CTP, and UTP; 1 mM ATP; and 5 mM 7mG(5′)ppp(5′)A cap analog incubated for 4 h at 30°C with the addition of 2 mM ATP after 30 min. Free nucleotides were removed by gel filtration chromatography on a Micro Bio-Spin P-30 Tris column (Bio-Rad Laboratories, Hercules, CA). All DNA templates were generated by digestion with XbaI, phenol-chloroform extracted, and ethanol precipitated using standard procedures.
Protein extraction and immunoblotting.
Protein extraction from cultured cells or mouse tissues has been described elsewhere (
37). Briefly, tissues harvested from mice were resuspended in PBS in the presence of complete EDTA-free protein inhibitors (Roche Applied Bioscience, Indianapolis, IN). Cell lysates were separated by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on a 15% acrylamide gel and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) for 60 min at 100 V in a Miniprotean III transfer tank (Bio-Rad, Hercules, CA). Viral particles from total supernatants were collected by centrifugation at 53,000 ×
g for 2 h at 4°C and resuspended in TNE buffer (12 mM Tris, pH 8, 120 mM NaCl, and 1 mM EDTA). Immunoblots were incubated with anti-LC3 antibody (Sigma) or anti-prM antibody (Abcam) at a dilution of 1/1,000 (or 1/5,000 for anti-GAPDH antibody), followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit (LC3), rabbit anti-goat (GAPDH), or goat anti-mouse (prM) immunoglobulin (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1/10,000. The immunoblots were imaged on a phosporimager (Bio-Rad), and band quantitation was conducted with ImageQuant software (Bio-Rad).
RNA transfections.
BHK-21 cells were seeded in 24-well plates, grown to 80% confluence, and transfected with 1 to 2 μg of RNA using Lipofectamine 2000 (Invitrogen, Grand Island, NY), followed by a 4-h incubation at 37°C in 5% CO2. At that point, the RNA was removed, and the cells were washed three times in 1 ml culture medium and then incubated for a total of 48 to 72 h. The supernatants were collected after a quick clarification of cell debris, and the viral titer was assessed by plaque assay.
qRT-PCR.
Extracellular RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA). Intracellular RNA from infected cells was isolated in a similar way after three freeze-thaw cycles. Viral RNA from sucrose fractions was precipitated after phenol-chloroform extraction. Briefly, 200 μl of sample was treated with 200 μg/ml proteinase K and 0.1% (wt/vol) SDS for 1 h at 37°C. Samples were then phenol-chloroform extracted twice, ethanol precipitated, and resuspended in nuclease-free water. Quantitative reverse transcription (qRT)-PCR of viral RNA was performed on an Applied Biosystems 7300 machine using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) with anti-DENV2 NS4B primers. Primer sequences are available upon request.
Infectivity assays.
Virus titrations were conducted on BHK-21 cells. Briefly, BHK-21 monolayers were grown to 70% confluence in 24-well plates and incubated with serially diluted virus supernatants for 1 h at 37°C in 5% CO2. The wells were subsequently overlaid with Dulbecco's modified Eagle's medium, 1% SeaPlaque low-melting-point agarose (Cambrex, Rockland, ME), and 5% FBS; incubated for 3 days; and fixed with 10% formaldehyde. The cells were then permeabilized with methanol for 10 min, washed with PBS, and immunostained using a rabbit anti-DENV1, -2, -3, -4 antibody (Abcam) and a horseradish peroxidase (HRP)-goat anti-rabbit secondary antibody. After incubation of the substrate (3-amino-9-ethyl carbazole) for 10 to 20 min at 37°C, foci were counted under the microscope, and focus-forming units (FFU) per ml were calculated.
Velocity centrifugation.
Gradients were generated using a gradient station (model 153; Biocomp, Fredericton, NB, Canada) following the manufacturer's instructions. Viral supernatants were loaded onto 5 to 50% (wt/wt) sucrose gradients and centrifuged for 16 h at 100,000 × g at 4°C, after which they were fractionated into 1-ml aliquots. The amount of virus in each aliquot was directly quantitated in BHK-21 cells; viral RNA was extracted as described above.
Mouse infections.
AG129 mice (129/Sv mice lacking alpha/beta interferon [IFN-α/β] and IFN-γ receptors), obtained from Harry Greenberg (Stanford University School of Medicine), were bred and housed under specific-pathogen-free conditions at the Stanford University animal care facility, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, Int. The AG129 mouse colony was monitored for adventitious viral, bacterial, and parasitic pathogens by a sentinel dirty-bedding program. Sentinels are euthanized and screened for pathogens every 4 months. The sentinel mice were found to be free of mouse hepatitis virus, mouse rotavirus (EDIM), mouse parvovirus, minute virus of mice, ectromelia virus, Sendai virus, mouse parvovirus, pneumonia virus of mice, respiratory enteric virus III (Reo3), Theiler's murine encephalomyelitis virus, lymphocytic choriomeningitis virus, mouse adenovirus types 1 and 2, Mycoplasma pulmonis, fur mites, and pinworms. All experiments were approved by Stanford's Institutional Animal Care and Use Committee (Administrative Panel of Laboratory Animal Care). Age- and sex-matched mice at 8 to 11 weeks of age were used for all experiments. Virus (107 FFU) in 10% FBS in PBS was injected retro-orbitally in a volume of 100 μl unless otherwise indicated; mice not receiving virus received an equal volume of 10% FBS in PBS injected similarly. Drug treatments were performed intraperitoneally. For survival studies, mice were euthanized when moribund or upon initial signs of paresis/paralysis.
Confocal microscopy.
Quantitation was performed by averaging 10 random fields, containing approximately 100 cells, for each condition. Images were taken on a Zeiss LSM510 Meta inverted confocal microscope.
Autophagy stimulation and inhibition.
Stocks of rapamycin (Cell Signaling Technology, Danvers, MA, and LC laboratories, Woburn, MA) and nicardipine (Sigma-Aldrich) were prepared in dimethyl sulfoxide (DMSO) and used as autophagy stimulators in tissue culture and in mice. Spautin-1, used as an autophagy inhibitor, was also dissolved in DMSO. For tissue culture treatments, 7 × 106 BHK cells on 60-mm dishes were pretreated with either drug (or DMSO as a negative control) for 30 min at 37°C and infected at a multiplicity of infection (MOI) of 0.1 FFU per cell. Infections were always performed in duplicate. Supernatants were harvested after 24 h, and titers were determined on fresh BHK-21 cells as described above. For mouse experiments, the drugs were delivered intraperitoneally in a solution containing the drug in DMSO (5%) combined with Solutol HS15 (Sigma-Aldrich) (25%) and saline (70%). For rapamycin and nicardipine, mice were treated every 12 h for 48 h, after which spleens, leg muscles, and stomachs were collected.
Statistical analysis.
Data were analyzed with Prism software (GraphPad Software, Inc.). For survival studies, Kaplan-Meier survival curves were analyzed by the log-rank test. Statistical significance was determined by using the two-tailed paired t test for in vitro experiments and the Mann-Whitney test for mouse experiments, as well as for viral RNA per FFU comparisons.
DISCUSSION
The present report describes an unprecedented role for autophagy in the infectious cycle of dengue virus: the assembly of infectious particles. Inhibiting autophagy at the stage of beclin-Vps34-Atg14 complex accumulation caused a modest decrease in viral RNA accumulation and a much larger reduction in the production of infectious virions. The smaller amounts of RNA in our experiments are consistent with previous reports (
26) but are not sufficient to explain the large decrease in the viral titer that we observed in the presence of spautin-1. We propose a direct effect of constituents of the autophagy pathway on dengue virus assembly for several reasons. First, the inhibition of infectious dengue virus particle formation by spautin-1 is not reversible by nutritional supplementation. Second, dengue virus RNA-containing particles are still secreted in the presence of spautin-1. Finally, the stimulation of autophagy had the opposite effect from its inhibition: dengue viral particle specific infectivity was greatly increased.
The dependence of dengue virus on cellular autophagy could be direct, such as a requirement for viral RNA packaging on lipidated LC3-II, or indirect, such as the autophagic degradation of some constituent that would otherwise be antiviral or the autophagic provision of some constituent required by the virus. Work by Heaton and Randall established a relationship between autophagy-mediated lipid droplet degradation and dengue virus RNA replication. The role of autophagy was hypothesized to be to provide free fatty acids as an energy source for RNA replication when the pathway was inhibited by 3-MA-reduced RNA levels and titers were rescued by external addition of oleic acid to the cells. However, this is not the case for the defect in virion production observed here, which is not reversed by the provision of oleic acid. Blocking the autophagy pathway with compounds like 3-MA, which inhibits many cellular PI3Ks, renders interpretation of these treatments difficult. Spautin-1 is a potent and selective inhibitor of cellular ubiquitinases USP10 and USP13. The specific destabilization of the beclin-Vps34-Atg14 complex is predicted to inhibit the autophagy pathway at a point upstream of ATG5 or ATG7 knockdowns or knockouts (
Fig. 1C) and may therefore affect even autophagy processes that are Atg5 independent (
41).
Here, data are presented showing that components or sequelae of cellular autophagy play a critical role in dengue virus assembly. Although without precedent for flaviviruses, recent work by Li et al. has shown that hepatitis B virus, a DNA virus, requires the autophagy pathway for correct viral envelopment, with little or no effect on genome replication or protein translation upon autophagy inhibition (
42). For dengue virus, we have shown that the inhibition of autophagy leads to the formation of deranged particles whose prM protein is successfully cleaved but not released to allow the virion to mature. The original defect that prevents this release could be at the level of the formation of the lipid droplets known to be critical for the interactions between dengue virus RNA and core protein, budding of these core-RNA complexes into the endoplasmic reticulum (ER), or any other step at which the structure of the virion is altered so that release of the pr peptide does not occur (
Fig. 7). That a similar defect occurs during murine infection is supported by the increase in specific infectivity of virus particles prepared from mice infected in the presence of stimulators of autophagy.
Inhibition of autophagy is currently being considered as a possible therapeutic strategy for diseases, including acute neurological injuries and cancers (
19), and could be an attractive target for antivirals, as well. The selection of spautin-1-resistant viruses has proven difficult thus far. One of the advantages of targeting a cellular pathway for antiviral treatments is that it can make the appearance of drug-resistant viruses less likely. Although a functional pathway is likely to be important long term to prevent neurodegeneration (
12), its short-term inhibition for acute infections by dengue virus and other autophagy-dependent microbes is promising.