The Ubiquitin−Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells

Abstract

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

Pyrrolidine dithiocarbamate inhibits enterovirus 71 replication by down-regulating ubiquitin–proteasome system

“…Accumulating evidence has shown that UPS is involved in numerous cellular activities, such as cell cycle control, apoptosis, and immune responses (Choudhury et al, 2013;Rastogi and Mishra, 2012;Tu et al, 2012). It has been demonstrated that UPS is important for the replication of positive-stranded RNA viruses such as CVB3, hepatitis E virus, and dengue virus (Kanlaya et al, 2010;Karpe and Meng, 2012;Si et al, 2007). In the present study, we demonstrated that EV71 infection promoted ubiquitination, while the activity of proteasome remained unchanged.…”

Dynamic Interactions between Bombyx mori Nucleopolyhedrovirus and Its Host Cells Revealed by Transcriptome Analysis

“…The ubiquitin-proteasome pathway is a major regulator of intracellular protein levels and plays an important role in viral infections through the avoidance of host immune surveillance, viral progeny release and budding, transcriptional regulation of the virus, and suppression of apoptosis (17,25). We observed that genes related to the ubiquitin-proteasome pathway were upregulated throughout the course of the infection (Table 2; see also Table S5 in the supplemental material).…”

Endoplasmic Reticulum Protein SCAP Inhibits Dengue Virus NS2B3 Protease by Suppressing Its K27-Linked Polyubiquitylation

“…The ubiquitin ligase CBLL1 and the ubiquitin/proteasome pathway are thought to be essential for West Nile virus replication (38,39), whereas ubiquitylation of JEV NS3 and NS5 results in viral protein degradation (21). Previously, it was observed that inhibitors of the cellular ubiquitylation could dramatically decrease DENV replication (40). However, the target(s) of ubiquitylation and the underlying molecular mechanisms of why inhibition of ubiquitylation affects DENV replication were largely unknown.…”