How Positive-Strand RNA Viruses Benefit from Autophagosome Maturation A Minireview for the

How Positive-Strand RNA Viruses Benefit from Autophagosome Maturation

A Minireview for the Journal of Virology.

Alexsia L. Richards and William T. Jackson

Department of Microbiology and Molecular Genetics

Medical College of Wisconsin

8701 Watertown Plank Road

Milwaukee, WI 53226

414-955-8456

Fax: 414-955-6535

[email protected]

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00460-13 JVI Accepts, published online ahead of print on 12 June 2013

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Abstract. 1

2

The autophagic degradation pathway is a powerful tool in the host cell arsenal against 3

cytosolic pathogens. Contents trapped inside cytosolic vesicles, termed 4

autophagosomes, are delivered to the lysosome for degradation. In spite of the 5

degradative nature of the pathway, some pathogens are able to subvert autophagy for 6

their benefit. In many cases, these pathogens have developed strategies to induce the 7

autophagic signaling pathway while inhibiting the associated degradation activity. One 8

surprising finding from the recent literature is that some viruses do not impede 9

degradation, but instead promote the generation of degradative autolysosomes, which 10

are the endpoint compartments of autophagy. Dengue virus, poliovirus and hepatitis C 11

virus, all positive-stranded RNA viruses, utilize the maturation of autophagosomes into 12

acidic and ultimately degradative compartments to promote their replication. While the 13

benefits that each virus reaps from autophagosome maturation are unique, the parallels 14

between the viruses indicate a complex relationship between cytosolic viruses and host 15

cell degradation vesicles. 16

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Introduction to Autophagy. 17

18

While many viruses avoid or suppress host immune responses, several subvert the host 19

immune machinery to promote their own replication (1). The autophagic pathway is one 20

well-characterized effector mechanism of the innate immune response, resulting in a 21

highly regulated lysosomal degradation mechanism by which a cell degrades its own 22

contents. The pathway has been shown to be essential for cellular clearance of several 23

intracellular pathogens including Mycobacterium tuberculosis, Toxoplasma gondii, and 24

Herpes simplex virus ( HSV-1) (2-4). Despite the role of autophagic signaling in innate 25

immunity, several pathogens are capable of subverting autophagy for their own benefit 26

(5, 6). In particular, the replication cycle of positive-strand RNA viruses, which are the 27

causative agents of many diseases including myocarditis, encephalitis, and hand foot 28

and mouth disease (7-9) can be promoted by some aspect of the autophagic pathway. 29

However, there is a difference between a pathogen benefitting from autophagic 30

signaling or the machinery from the autophagic pathway, and a pathogen benefitting 31

from the endpoint activity of autophagy, which is the degradation of cytosolic contents. 32

Both are of interest, but this review will focus exclusively on relatively new findings that 33

several pathogens can actually benefit from the degradative activity of autophagy. 34

35

Autophagy is a constitutive degradation pathway with important roles in development, 36

differentiation, and stress responses (10). By facilitating the removal of damaged 37

organelles and cytoplasmic protein aggregates, autophagy has proven to be essential 38

for maintaining cellular homeostasis (6). Several signaling pathways induce autophagic 39

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signaling, although the mechanism by which these pathways cooperate to promote 40

vesicle formation remains unknown (11). Inhibition of the Akt / mTOR pathway has long 41

been considered essential for induction of autophagic signaling, although recent reports 42

have demonstrated the existence of mTOR-independent autophagy (12, 13). Induction 43

of an ubiquitin-like conjugation system promotes lipidation of the cytosolic microtubule-44

associated light chain 3 (LC3) protein with phosphotidylethanolamine, generating a 45

membrane-associated species known as LC3-II (14-16). LC3-II is associated with both 46

the inner and outer membrane of the growing autophagosome, and this association is 47

essential for autophagosome formation (14, 17, 18). LC3-II remains the only protein 48

known to stably associate with completed autophagosomes, and as such it is an 49

invaluable marker for monitoring autophagy. The initial events in autophagosome 50

biogenesis have been well described elsewhere (18-20). 51

52

Autophagosomes are unique vesicles composed of two lipid bilayers which, during their 53

formation, engulf cytosolic contents, including long-lived proteins, intracellular 54

pathogens, or damaged organelles. This cargo is then transported to the lysosome for 55

degradation (10). Double-membraned autophagosomes undergo a stepwise maturation 56

process culminating in their fusion with lysosomes to form degradative autolysosomes 57

(Figure 1). Autophagosomes mature into amphisomes, a change primarily characterized 58

by the acidification of the lumen of the vesicle and the acquisition of proteins associated 59

with late endosomes and lysosomes (21). Mature amphisomes then fuse with 60

lysosomes to form autolysosomes, in the process losing one lipid bilayer through an 61

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unknown mechanism (22, 23). The autolysosome is the compartment in which actual 62

autophagy, the degradation of cytosolic contents, takes place. 63

The acidification of the amphisome is believed to be the result of fusion with late 64

endosomes bearing vacuolar ATPases (21, 24). Treatment of cells with inhibitors of 65

vesicular acidification, including bafilomycin A 1, chloroquine, and ammonium chloride, 66

prevents autolysosome formation (24-26). This indicates that acidification of either the 67

autophagosome, the lysosome, or both is a prerequisite for fusion of the 68

autophagosome with the lysosome. Two proteins, the lysosomal-associated membrane 69

protein 2 (LAMP-2) and Rab7, have been reported to be required for autophagosome 70

maturation. Rab7 is a small GTP-binding protein that has functions in late endosomal 71

transport (27, 28). Depletion of endogenous Rab7 or expression of a dominant negative 72

form results in decreased autophagosome maturation (29, 30). LAMP-2, a lysosomal 73

transmembrane protein, is one of the most abundant lysosomal components (31). 74

Depletion of LAMP-2 prevents autophagosome fusion with lysosomes (32-34). These 75

data indicate that multiple steps of the autophagosome formation and maturation 76

pathway are regulated by the cell and may be subverted by pathogens. 77

78

Viral Subversion of the Autophagic Pathway. 79

80

As obligate intracellular parasites, the success of a virus depends on its ability to evade 81

the host cell ’ s antiviral defenses, as well as its ability to regulate cellular processes that 82

facilitate its own replication. Subversion of the autophagic pathway, which aids both of 83

these goals, has been most extensively studied in positive-strand RNA viruses (35, 36). 84

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Optimal production of positive-strand RNA viruses depends on initiation of the 85

autophagic pathway during infection. This is counter-intuitive, because the autophagic 86

pathway promotes degradation of cytosolic contents, and positive-strand RNA viruses 87

replicate in the cytosol. 88

89

The physical hallmark of the autophagic pathway is the formation of cytosolic double-90

membraned vesicles (19, 37). Positive-strand RNA viruses replicate their genome in 91

association with cytosolic membranes (38, 39). Therefore, by inducing autophagy, these 92

viruses may be facilitating the creation of scaffolds for their own replication. However, 93

these vesicles are part of a degradative pathway, and if this pathway is unaltered, the 94

vesicles will fuse with lysosomes and their contents will be degraded. Coxsackievirus B 95

3 (CVB 3), an enterovirus in the Picornaviridae family, appears to have developed a 96

strategy to prevent this. CVB3 relies on autophagosome formation for optimal virus 97

replication (40, 41). However, during both in vitro and in vivo infections, there is 98

evidence that amphisome maturation and autophagic protein degradation are inhibited 99

(40, 41). The mechanism by which CVB3 upregulates autophagosome formation while 100

restricting autophagic degradation is unknown. Treatment of CVB 3- infected cells with 101

inhibitors of autophagosome maturation results in increased virus production, indicating 102

that at least a portion of the virus remains sensitive to autophagic degradation (41). 103

Recently it was also shown that rotavirus induces autophagic signals to promote virus 104

replication, but that the virus blocks protein degradation (42). As with CVB 3, the 105

mechanism by which rotaviruses specifically inhibits autophagosome degradation is not 106

yet known. Further work to identify the specific virus or host cell proteins used by these 107

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viruses to prevent degradation will help our understanding of autophagic regulation in 108

general. 109

110

A similar anti-degradative phenomenon has been observed in bacterial infection 111

models, with the best-characterized being Legionella pneumophila infection, which 112

induces replication vesicles that resemble autophagosomes (43). The vacuoles become 113

acidic; however, the bacteria secrete a factor that delays their fusion with lysosomes 114

(44). A recent report indicates that Legionella can interfere with the formation of LC3-II, 115

although the significance of this to autophagosome maturation is unclear (45). Inhibition 116

of autophagosome maturation has been observed for several other non-viral pathogens 117

(46). 118

119

In the following sections we discuss recent advances in understanding the interaction of 120

three positive-strand RNA viruses with the late stages of the autophagic pathway. 121

Replication of all three viruses is reduced when autophagy is inhibited. Conversely, 122

stimulation of autophagy increases infectious virus production (47-52). To date, this 123

subset of viruses are the only pathogens shown to induce autophagosome formation to 124

promote their own replication while allowing maturation of the vesicles, fusion of 125

amphisomes with lysosomes, and subsequent cargo degradation. For reference, a brief 126

description of assays used to monitor autophagosome maturation and autophagic 127

degradation is provided in Table 1, and more detail is available in (53). 128

129

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Dengue fever virus. 131

132

Dengue virus, a member of the Flaviviridae, is the causative agent of dengue fever, 133

which in a small subset of the population progresses to dengue hemorrhagic fever / 134

dengue shock syndrome (54, 55). Dengue virus (DENV) is comprised of four 135

antigenically related but distinct viruses (DENV-1 to DENV-4) with each virus 136

comprising many distinct genotypes (56). Thus far, only DENV-2 and DENV-3 have 137

been shown to require autophagosome formation for maximum virus replication (52, 54, 138

57). During infection with either virus, viral proteins involved in translation and 139

replication locate to autophagosomes (52, 57). 140

141

While DENV-2 and DENV-3 both subvert autophagosome formation, there are some 142

major differences in the way these viruses interact with the late stages of the autophagic 143

pathway. DENV-3 non-structural proteins primarily co-localize with autolysosomes 144

during infection, whereas DENV-2 proteins are primarily located on immature 145

autophagosomes (52). During DENV-2 virus infection autophagy increases the cells 146

degradative capacity, specifically in regards to cellular lipid droplets (51). The 147

degradation of lipid droplets by autophagy is referred to as lipophagy (58). Increased 148

lipophagy during DENV-2 infection results in both a decrease in triglycerides and an 149

increase in β-oxidation. Inhibition of autophagosome formation reduces infectious virus 150

production, however, when cells are supplied with the products of lipophagy, virus 151

production returns to levels observed in cells capable of autophagy. The authors 152

speculate that the release of free fatty acids during lipophagy increases ATP 153

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generation, which is critical for viral replication (51). A role for lipophagy during DENV-3 154

infection has not yet been reported. 155

156

Hepatitis C Virus. 157

158

Hepatitis C Virus (HCV), another flavivirus, is a major cause of chronic liver disease 159

(59). The HCV RNA-dependent RNA polymerase interacts with the cellular autophagy 160

protein Atg5, and the two proteins co-localize during early time points of infection (60). 161

Additionally, HCV RNA and proteins co-fractionate with LC3-II on a discontinuous 162

sucrose gradient (61). Expression of HCV proteins NS5A and NS4B in isolation is 163

sufficient to induce autophagic signaling (62, 63). While there is agreement that HCV 164

induces autophagic signaling, the specific role of autophagy during HCV infection 165

remains controversial. Autophagy was shown to be essential for translation of the viral 166

genome but dispensable once the infection has begun (49). These data contrast with a 167

report showing knockdown of autophagy genes had no effect on virus translation and 168

RNA replication but instead was essential for HCV particle formation (47). 169

170

HCV-infected cells expressing tandem-tagged GFP-RFP-LC3 (see Table 1) show 171

predominantly red fluorescence indicating maturation of autophagosomes into acidic 172

amphisomes (64, 65). The RFP-positive puncta co-localize with LAMP-1, demonstrating 173

fusion of the autophagosome with either late endosomes or lysosomes (65). However, 174

there is at least one report of an incomplete autophagic response to HCV infection. No 175

change in either p 62 levels or long-lived protein degradation was observed following 176

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transfection with the HCV replicon (66). This discrepancy may result from a difference 177

between transfection of the viral genome and infection with live virus. However, 178

elevated autolysosome formation has been observed following transfection of the HCV 179

replicon, making this an unlikely explanation (67). An alternative hypothesis is that 180

typical autophagosome cargo, such as p 62, is not incorporated into the specialized 181

autophagosomes generated during HCV infection. If this is the case, assays measuring 182

protein degradation levels would not be a reliable measure of autophagosome 183

maturation. 184

185

HCV genome replication is attenuated following depletion of LAMP-2 or Rab7, both of 186

which are essential for autolysosome formation (65). Treatment with either bafilomycin 187

A 1 or chloroquine, both of which inhibit autophagosome maturation, results in reduced 188

viral RNA and protein expression (65, 68). Investigation of the (RIG-I)- like Receptor 189

(RLR) signaling cascade following HCV infection has revealed a role for autophagic 190

degradation during infection in suppressing the innate immune response to infection 191

(65). Activation of the IFN-β promoter by ectopically expressed RIG-I was measured in 192

infected cells in both the presence and absence of autophagic degradation. In control 193

cells HCV was able to inhibit RIG-I mediated IFN-β activation. In the absence of 194

autophagic degradation infected cells showed a significant increase in IFN-activation. 195

The varied reports on the effects of autophagy in HCV production lead us to conclude 196

that the process may play multiple roles in promoting viral replication. 197

198

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Poliovirus. 200

201

Poliovirus (PV), the causative agent of poliomyelitis, is a member of the Picornaviridae 202

family. It is one of the most well-characterized members of this family, in terms of its 203

molecular and cellular biology, biochemistry, structure, life cycle, and pathogenesis, and 204

therefore represents an important model virus (69). By 5 hours post-infection, infected 205

cells exhibit extensive accumulation of autophagic vacuoles throughout the cytoplasm 206

(50, 70). Viral RNA replication proteins localize to the autophagosome membrane 207

during infection (50, 71). LC3 and LAMP-1 also co-localize during infection indicating 208

autophagosome fusion with late endosomes and / or lysosomes (50). Staining infected 209

cells with MDC, a lysosomotropic agent that is concentrated in acidic compartments by 210

an ion-trapping mechanism, reveals that the lumen of autophagosomes acidifies relative 211

to the cytosol (50, 72). Infection promotes autophagic protein degradation; however, 212

unlike Dengue virus and HCV, degradation is not necessary for virus replication since 213

lysosomal protease inhibitors have no impact on intracellular virus titer (73). 214

Our group recently showed that poliovirus utilizes both autophagosome formation and 215

maturation of the autophagic vacuole to promote two separate and distinct steps in the 216

virus life cycle. Inhibitors of autophagosome formation limit viral RNA replication (73). 217

However, if autophagosome formation proceeds normally but vesicle acidification is 218

inhibited, virus production remains attenuated. In the absence of acidic vesicles, viral 219

entry, translation, RNA replication, and genome encapsidation all occur normally. Acidic 220

vesicles are, however, required for the last step in production of an infectious virion, 221

marked by the internal cleavage of capsid protein VP0, which results in the maturation 222

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of a noninfectious provirion to an infectious virion (73). This cleavage step is attenuated 223

in the absence of vesicle acidification, resulting in a decrease in the number of 224

infectious virions produced. How an acidic vesicle can promote the maturation of a 225

presumably cytosolic, non-enveloped virus is a current question of research focus. 226

227

Possible connections among viruses that benefit from autophagosome maturation. 228

We have presented here three examples of how viruses benefit from autophagosome 229

maturation. However, there are indications that some of these benefits may be 230

conserved amongst the viruses. Of the viruses discussed, only HCV has been 231

definitively shown to use autophagic degradation to downregulate immune signaling 232

during infection (65). However, preliminary results indicate that the immune response to 233

Dengue virus infection may also be attenuated by autophagic degradation (65). 234

Recently Japanese encephalitis virus (JEV) has been shown to subvert autophagy as a 235

viral immune evasion strategy. The mechanism may be similar to that used by HCV, 236

since type I interferon (IFN) activation is increased when JEV infects cells deficient in 237

autophagy (74). Infection with JEV increases autolysosome formation in vivo, and this 238

formation is essential for maximum virus production (74). Is it not yet known if 239

autophagic degradation is responsible for restriction of the immune response during 240

JEV infection. Interestingly, autophagy is essential for JEV production even in an IFN-241

defective background, indicating that the virus may have multiple uses for the 242

autophagic pathway during infection. 243

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A recent publication investigated the role of autophagy in lipid metabolism during HCV 245

infection. As with Dengue virus, infection with HCV results in the appearance of 246

autophagosomes filled with lipid cargo (75). Inhibition of autophagosome maturation 247

through bafilomycin A 1 treatment results in an accumulation of cholesterol in both HCV 248

replicon cells and cells infected with HCV strain JFH1 (75). The purpose of increased 249

autophagic breakdown of cholesterol during HCV infection remains elusive. One 250

hypothesis is that the autophagic flux of cholesterol is needed for lipid droplet 251

biogenesis during HCV infection. This would be very intriguing as lipid droplet area is 252

decreased by autophagic degradation during Dengue virus infection (75, 76). The 253

differences in the proposed roles of autophagic degradation of lipids during infection 254

may be a product of the different requirements each virus has for lipid droplets during 255

infection. Unlike Dengue virus, lipid droplets are required for HCV virion assembly (76), 256

therefore increased surface area of these lipid droplets may aid HCV in replication. 257

Conversely, Dengue virus may promote destruction of these lipid pools to provide 258

energy for virus replication occurring at an alternate site in the cell. 259

260

261

The data gathered thus far regarding the role of autophagy during poliovirus and 262

Dengue virus infection is almost exclusively from in vitro systems. Therefore, the effects 263

of autophagic degradation on the host immune response to infection have not been 264

assessed. If it is found that immune signaling during infection is attenuated through 265

autophagic degradation, then autophagic degradation could play multiple pro-viral roles 266

during infection. Recently, impairment of autophagosome formation has been shown to 267

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hamper formation of infectious Dengue virus particles, while having minimal effects on 268

viral RNA replication (77). This suggests that both poliovirus and Dengue virus may be 269

utilizing the environment within a mature autophagosome to promote the final steps in 270

production of viral progeny. It is not yet know if inhibitors of autophagosome maturation 271

have an effect on Dengue virus particle formation. 272

273

Conclusion. 274

275

It is now appreciated that viruses such as poliovirus, Dengue virus, and HCV rely on the 276

degradative activity of the autophagic pathway for efficient replication. While this review 277

focused on the three viruses for which the role of autophagic maturation during infection 278

has been elucidated, the story is far from complete. For example, encephalomyocarditis 279

virus (EMCV) and porcine reproductive and respiratory syndrome virus (PRRSV), both 280

subvert the autophagic pathway for optimal virus production, while promoting 281

autophagic protein degradation (78, 79). Both PRRSV and EMCV replication have been 282

shown to be sensitive to treatments known to restrict autophagosome maturation (80, 283

81). There is currently no model for the role that maturation of the autophagosome is 284

playing during infection with either of these viruses. It will be interesting to learn if either 285

virus shares a mechanism with one of the viruses presented in this review, or if novel 286

roles for autophagosome maturation are discovered. There is also recent evidence that 287

treatment with the Cathepsin inhibitor Pepstatin A results in a decrease in Influenza A 288

production, which the authors of the study have linked to Pepstatin A altering the 289

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regulation of autophagy (82). Together, these data indicate that multiple viruses may 290

utilize autophagic degradation. 291

292

The question remains, how do these viruses thrive in a highly degradative environment? 293

One possibility is that they have evolved to be resistant to degradation within the 294

autolysosome. Alternatively, they may have developed a mechanism to avoid being 295

trapped within the degradative vesicles. Finally, viral replication may occur outside of 296

the degradative autophagosome and thus be unaffected by the degradative 297

environment within the vesicle. 298

299

It is often in the best interest of a virus to maintain the host cell’s integrity until progeny 300

virus production is complete. For example, many viruses have evolved mechanisms to 301

prevent cellular apoptosis, a pathway that is linked to autophagy (83, 84). The way in 302

which a virus interacts with the autophagic pathway may have important implications for 303

cell viability throughout the infection. A number of studies have indicated that autophagy 304

is induced by ER stress (85, 86) In both yeast and mammalian cells autophagy has 305

been shown to have pro-survival effects when the ER is overloaded with misfolded 306

proteins (87-89). Inhibition of the autophagic pathway increases cell death following ER 307

stress. This effect is due to the degradation of protein aggregates and misfolded 308

proteins (87). Both HCV and EMCV have been shown to increase ER stress (65, 66, 309

90-92). By allowing autophagic degradation to proceed unperturbed, these viruses may 310

be minimizing the risk of cell death prior to the completion of the replicative cycle. 311

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Finally, by studying the way in which viruses regulate cellular pathways, a great deal 313

has been learned about the functions of the host cell. The mechanism by which viruses 314

inhibit or promote autophagic degradation will not only improve our understanding of 315

virus replication, but also shed light on the ways in which the late stages of autophagy 316

are regulated. 317

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Figure Legend.

Figure 1. How viruses utilize autophagosome formation and maturation during

infection. As the immature autophagosome forms it captures portions of the cytoplasm.

The lumen of the autophagosome acidifies likely through fusion with late endosomes

carrying vacuolar ATPases, to form the amphisome. The amphisome then fuses with

the lysosome to form the autolysosome. The replication of viruses that subvert the

autophagic pathway is attenuated when autophagosome formation is inhibited. Vesicle

acidification is required for efficient PV virion maturation, while inhibition of degradation

has no effect on the virus. Degradation of cellular triglycerides by autophagy benefits

Dengue virus replication. Autolysosome degradation decreases IFN activation following

HCV infection. Both PPRSV and EMCV require autophagosome maturation; however, it

is not clear if this is due to a requirement for vesicle acidification or autolysosome

degradation.

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Assay Description Read out

Protease sensitivity of LC3-II

LC3-II is degraded by lysosomal proteases following fusion of the autophagosome with the lysosome (93). Lysosomal protease inhibitors, which inhibit LC3-II degradation by the autolysosome, increase the steady-state level of LC3 II (93,53).

Protein degradation by the autophagic pathway

p62 degradation

The p62/SQSTM1 protein directly binds LC3-II on the autophagosome membrane (94). p62 is degraded within the autolysosomes (95). A decrease in the steady state level of p62 following induction of autophagy indicates successful protein degradation through the pathway (95, 53).

Autolysosome formation

LC3-II-lysosome co-localization

The cellular locale of autophagosome-associated GFP-tagged LC3-II can be monitored by fluorescence microscopy (64). During the initial stages of the autophagic pathway co-localization of LC3-II with lysosomal markers is low. As autophagosomes mature and fuse with lysosomes, co-localization with lysosomal markers increases. Cellular lysosomes can be visualized by staining for protein markers such as LAMP-2, LAMP-1 or Cathepsin D (31).

Autolysosome formation

Tandem-tagged GFP-RFP LC3

Tandem tagged RFP-GFP-LC3 localizes to the autophagosome membrane following induction of autophagy (55). Only the signal generated by the GFP protein is sensitive to the acidic and/or proteolytic conditions in the lumen mature autophagosome and lysosomes. Co-localization of GFP and RFP signals is observed on early or immature autophagosomes. As autophagosomes mature the GFP signal is lost leading to only RFP fluorescence.

Autophagosome maturation

Transmission Electron Microscopy

(TEM)

Autophagosomes can by identified by TEM as membrane-bound structures containing cytoplasmic material. Immature autophagosomes (AVi) show a double-membraned visible as two membrane bilayers separated by an electron-lucent cleft. These vacuoles contain cytosol and/or organelles that appear morphologically intact (53). Mature or degradative vesicles (AVd) typically show partial degradation of the enclosed cytoplasmic material, as well as increased electron density in the lumen of the vesicle.

Autophagosome maturation

Table 1. Assays for autophagosome maturation and autophagic degradation. An extensive discussion of known assays for analysis of autophagic signaling, autophagosome formation, and all other aspects of the pathway can be found in reference 53.

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How Positive-Strand RNA Viruses Benefit from Autophagosome Maturation A Minireview for the

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