Evidence of RedOX Imbalance during Zika Virus Infection Promoting the Formation of Disulfide-Bond-Dependent Oligomers of the Envelope Protein

Abstract

Flaviviruses replicate in membrane factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus viral protein accumulation contribute to ER stress. As a consequence, the host cell exhibits an Unfolded Protein Response (UPR), subsequently stimulating appropriate cellular responses such as adaptation, autophagy or apoptosis. The correct redox conditions of this compartment are essential to forming native disulfide bonds in proteins. Zika virus (ZIKV) has the ability to induce persistent ER stress leading to the activation of UPR pathways. In this study, we wondered whether ZIKV affects the redox balance and consequently the oxidative protein folding in the ER. We found that ZIKV replication influences the redox state, leading to the aggregation of the viral envelope protein as amyloid-like structures in the infected cells.

Keywords: ER stress; Zika virus; amyloid aggregates; disulfide bond; oligomer; unfolded protein response.

Figure 1

ZIKV infection is associated with redox imbalance. (A) ZIKV infection of A549 cells. A549 cells were either mock infected or infected with ZIKV at MOI 5 for 48 h. Cell extracts were reduced using DTT and heat-treated at 95 °C for 5 min. Western blot analysis of cell extracts following infection leads to detection of ZIKV envelope protein (~55 kDa). ZIKV-E protein is detected using rabbit anti-EDIII antibody. This is representative of three independent experiments. (B) ZIKV leads to a decrease in the GSH/GSSG ratio, indicating a loss of glutathione activity in infected A549 cells. 48 h.p.i. glutathione activity was assessed in mock infected vs. infected cells. ***: p < 0.01. h.p.i: h post-infection.

Figure 2

Amyloid-like protein accumulation during ZIKV infection. A549 cells infected with ZIKV at MOI 1 for 48 h were incubated with Thioflavin-T (THT), a green fluorescent ER stress indicator that binds aggregated proteins. Immunodetection of ZIKV envelope protein confirmed the infection of the cells. Scale bar: 5 µm. All images are representative of three independent experiments.

Figure 3

ZIKV infection results in the formation of disulfide-crosslinked E oligomers. (A) ZIKV-E in native conformation contains intermolecular disulfide bridges. After infection of A549 cells with ZIKV in a dose-dependent manner (MOI 0.5, 1 and 5), protein extracts were either treated or not with TCEP before SDS-PAGE. Western blot analysis was performed with mouse 4G2 antibody, which recognizes a conformational epitope of the E protein. (B) ZIKV-E protein forms disulfide-cross-linked oligomers. A549 cells were infected with increased quantities of ZIKV (MOI 0, 0.5, 1 and 5), and the total fraction was treated or not with TCEP before Western blotting. Western blot assay was achieved with a rabbit anti-EDIII antibody. M indicates the 55 kDa monomer, and HMW indicates high molecular weight oligomers of ZIKV-E. These images are representative of three independent experiments.

Figure 4

Disulfide bonds in flavivirus prM/E protein. (A) Alignment of prM/E protein sequence from YFV (POLG_YEFVA), DENV2 (POLG_DEN2N), WNV (POLG_WNV9) and ZIKV (ARB08102.1). The cysteine positions are highlighted in yellow. (B) Crystallography structure of DENV2 prM/E (3C6E), with cysteine positions involved in disulfide bonds formation highlighted in pink. (C) Crystallography structure of ZIKV-E (5JHM), with cysteine positions involved in disulfide bonds formation highlighted in pink.

Figure 5

ZIKV E disulfide-cross linked oligomers are insoluble. A549 cells were infected with ZIKV at MOI 5. 48 h.p.i. cells were lysed in TX100 buffer A and fractionated by centrifugation at 3400× g. An equal volume of total (T), soluble (S) and insoluble (I) fractions were separated under non-reducing conditions and immuno-blotted for ZIKV-E with anti-EDIII or 4G2 antibodies. M indicates the 55 kDa monomer, and HMW indicates high molecular weight oligomers of ZIKV-E. This is representative of three independent experiments.

Figure 6

Graphical overview of the impaired redox cycle and the formation of protein aggregates during ZIKV infection.