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

Abstract

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCEAutophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus. Dengue virus (DENV) is a member of the family Flaviviridae and is responsible for one of the most common infections transmitted to humans by mosquitoes. DENV is a positive-st...

Flavivirus Infection Uncouples Translation Suppression from Cellular Stress Responses

“…DENV infection interferes with cellular processes such as autophagy (31, 32) and host innate immune responses (33–35); however, the regulation of host cell translation during DENV infection is not well characterized. Paradoxically, DENV induces host stress responses but seems to repress them almost simultaneously (36–39), supposedly to avoid repression of host cell translation, which could also affect translation of the viral genome.…”

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis

“…This included the previously identified interactions between NS1 and complement component C4A (Avirutnan et al, 2010), EI24 and the TRiC chaperone complex (Hafirassou et al, 2017), NS2B3 interacting with members of the CTLH complex (Kobayashi et al, 2007), and NS5 with the PAF1 complex PAF1C (Krogan et al, 2002). Interactions between DENV proteins and p62 (SQSTM1), TTC37, and TRIM26 were also detected, all of which were previously implicated in DENV replication (Marceau et al, 2016; Metz et al, 2015). …”

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

“…15,[17][18][19][20][21] Autophagy is a degradative process that is activated in response to a variety of cellular stresses, including viral infection, to maintain cellular homeostasis. This results in a high turnover of cytoplasmic proteins and organelles, which would be hypothesized to be detrimental to the expression of viral proteins required for replication and assembly.…”

“…[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).…”