A role for autophagolysosomes in dengue virus 3 production in HepG2 cells

Abstract

We have recently proposed that amphisomes act as a site for translation and replication of dengue virus (DENV)-2 and that DENV-2 entry and replication are linked through an ongoing association with membranes of an endosomal-autophagosomal lineage. In this report, we present the results of an investigation into the interaction between DENV-3 and the autophagy machinery. Critically, treatment with the lysosomal fusion inhibitor L-asparagine differentiated the interaction of DENV-3 from that of DENV-2. Inhibition of fusion of autophagosomes and amphisomes with lysosomes resulted in decreased DENV-3 production, implying a role for the autophagolysosome in the DENV-3 life cycle. Evidence based upon the co-localization of LC3 and cathepsin D with double stranded RNA and NS1 protein, as assessed by confocal microscopy, support a model in which DENV-3 interacts with both amphisomes and autophagolysosomes. These results demonstrate that the interactions between DENV and the host cell autophagy machinery are complex and may be determined in part by virus-encoded factors.

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

“…Autolysosomes are reported as potential DENV replication sites, at least for DENV serotype 3 (6). Notably, that study revealed a similar increase in LC3-II levels upon DENV-3 infection.…”

“…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.…”

“…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.…”

“…Owing to their morphologies, these rearranged membrane structures have been designated convoluted membranes and vesicle packets (3). Recent studies have demonstrated that DENV replication requires autophagy (4)(5)(6)(7)(8), a process that targets proteins and/or organelles to lysosomes for degradation. Autophagy involves formation of the double-membrane autophagosome, which sequesters target cytosolic content and then fuses with the lysosome to form an autolysosome where sequestered components are degraded (9).…”

MHC presentation via autophagy and how viruses escape from it

“…In addition, inhibition of autophagosome degradation might enhance virus particle exocytosis 58 . Furthermore, the stabilization of autophagosome membranes for viral replication has been proposed for other RNA viruses, like the flaviviruses hepatitic C virus (HCV) and dengue virus [59][60][61][62][63] . Moreover, even RNA viruses that do not use autophagosomal membranes for their replication, seem to benefit from inhibition of autophagosome degradation for their release of infectious viruses from infected cells 64 .…”