Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production

Abstract

Ubiquitylation is one of the most versatile protein post-translational modifications and is frequently altered during virus infections. Here we employed a functional proteomics screen to identify host proteins that are differentially ubiquitylated upon dengue virus (DENV) infection. Among the several differentially modified proteins identified in infected cells was AUP1, a lipid droplet-localized type-III membrane protein, which exists predominantly in the mono-ubiquitylated form. AUP1 associated with DENV NS4A and relocalized from lipid droplets to autophagosomes upon infection. Virus production was abolished in cells deleted for AUP1 or expressing an AUP1 acyltransferase domain mutant. Ubiquitylation disrupted the AUP1-NS4A interaction, resulting in inhibited acyltransferase activity, defective lipophagy, and attenuated virus production. Our results show that DENV-NS4A exploits the acyltransferase activity of AUP1 to trigger lipophagy, a process regulated by ubiquitylation. This mechanism appears to be a general phenomenon in biogenesis of flaviviruses and underscores the critical role of post-translational modifications in virus infections.

Manipulation of autophagy by (+) RNA viruses

“…AUP1 was regulated by monoubiquitin modification, where infection or a combined expression of viral NS4A and NS4B were necessary and sufficient to generate the unmodified form of AUP1 -a step that was critical in induction of this pathway. Interestingly, loss of AUP1 did not affect viral replication; however, impaired autophagy was accompanied by degradation of viral proteins through the proteasomal pathway resulting in significantly reduced production of progeny virions, supporting a specific role of AUP1-dependent lipophagy in assembly of virus particles [16]. These results were in agreement with an independent report demonstrating expression of Dengue NS4A was sufficient to trigger autophagy and protect against cell death.…”

“…DENV non-structural protein NS1 and dsRNA were reported to co-localise with LC3 and ribosomal proteins [38,39]. Another study described the induction of LC3+ vesicles, which colocalised with NS4A in infected cells, or when transfected with a combination of DENV NS4A and NS4B [16]. This was independently corroborated by ZIKV infection in human fetal neural stem cells where expression of NS4A and NS4B were sufficient to block neurogenesis and promote autophagy, displaying partial colocalisation with LC3+ vesicles [53].…”

“…A link between autophagosomes and virus-induced vesicles was proposed by George Palade by EM imaging of poliovirus containing vesicles that resembled autophagosomal membranes [6]. Over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +RNA RNA viruses, including poliovirus (PV) [7,8], Coxsackievirus B3 (CVB3) [9,10], CVB4 [11], Enterovirus 71 (EV71) [12], Human rhinovirus (HRV) [13], Foot-and-mouth disease virus (FMDV) [14], encephalomyocarditis virus (EMCV) [15], Dengue virus (DENV) [16,17], Zika virus (ZIKV) [18,19], Hepatitis C virus (HCV) [20], Mouse hepatitic virus (MHV), Newcastle disease virus (NDV) [21], Severe and acute respiratory syndrome coronavirus (SARS-CoV) [22], Chikungunya virus (ChikV) [23], and Japanese encephalitis virus (JEV) [24]. In many of the above cases, pharmacological or genetic manipulation of autophagy in vitro confirmed an inhibition in replication and/or spread of these viruses, whereas induction of autophagy resulted in increased production of progeny virions [21,25].…”

“…However, this effect may be viral serotype-specific, since inhibiting lysosome fusion reduced DENV3 production [39]. The mechanisms by which autophagy favors DENV production was recently described where rather than replication, assembly and release of progeny virions was affected by blocking autophagy mediated lipid droplet hydrolysis [16,40,41].…”

Serum lipid profile as a predictor of dengue severity: A systematic review and meta‐analysis

“…To achieve this, viral proteins (especially nonstructural protein 3—NS3) sequester the fatty acid synthase (FASN) and 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HMGCR) to the RC, resulting in increased cholesterol uptake and synthesis of triacylglycerol after viral infection . DENV was also recently related to regulate the degradation of the host lipid droplet; it has been shown that a viral protein (NS4A) exploits the acyltransferase activity of AUP1, a lipid droplet‐localized type‐III membrane protein, to trigger lipophagy . In addition, some cholesterol uptake receptors, such as the low‐density lipoprotein receptor (LDLr), are known to participate during DENV infection, making it possible to identify the changes in lipid metabolism during DENV infection that can be used to develop new biomarkers of disease severity .…”