Inhibition of Cellular Autophagy Deranges Dengue Virion Maturation

Abstract

Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatmentresistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.A ll 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...

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

“…Here, we report findings that suggest a biphasic response of autophagy to DENV infection, where DENV infection initially activates and then later on inhibits autophagy. Although it has been reported by several groups that multiple components of the autophagy pathway are required for DENV replication (4,6,7,(27)(28)(29), we demonstrate here that DENV activates autophagy only during the early infection stage. Moreover, we established that, by 24 h after infection with the NGC strain, autophagosome formation is inhibited and lysosomal degradation of AVs is reduced.…”

“…However, 3MA suppresses autophagy induction by nutrient deprivation, whereas under typical growth conditions 3MA activates autophagy (56). Furthermore, inhibition of autophagy with the drug spautin-1 leads to increased production of noninfectious DENV particles (28), suggesting a role of autophagy during infectious particle production and/or release. The block of lysosomal targeting may be required by DENV for furinmediated maturation of viral particles in a post-Golgi compartment.…”

“…Autophagic flux is determined by the capacity of the cell to degrade forming autophagosomes, which occurs within minutes (25), and AV content increases upon disruption to lysosomal activity. It is commonly reported that DENV infection increases AV number as detected by high-resolution microscopy (6,7,24,26,27) and Western blot analysis of lipidated LC3-II levels (5,6,28,29). Notably, in autophagosomes colocalizing with lysosomes, pronounced pH-dependent quenching of green fluorescent protein (GFP)-LC3 fluorescence (30) was not observed (7), suggesting that DENV may reduce lysosomal acidification and so disrupt lysosomal function.…”

Protein composition of the hepatitis A virus quasi-envelope

“…4B), and we have shown previously that RNAi-mediated depletion of beclin 1 has no effect on eHAV release (2). Autophagy-related release of poliovirus is inhibited by beclin-1 knockdown (6) but not by spautin 1, an autophagy inhibitor that blocks deubiquitinases targeting beclin 1 (33,34). Further studies are needed to determine the extent to which eHAV biogenesis may involve beclin 1-independent aspects of secretory autophagy.…”

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B

“…[11][12][13][14] Several flaviviruses, including DENV and ZIKV, potently induce autophagy, which allows for efficient viral replication and virion assembly. [15][16][17][18][19][20][21][22] Interestingly, DENV infection relies on selective autophagy of lipid droplets (lipophagy) to provide energy required for viral replication. 15 A number of other forms of selective autophagy have been described including degradation of ribosomes (ribophagy), peroxisomes (pexophagy), mitochondria (mitophagy), and ER (reticulophagy).…”