Inhibiting Rotavirus Infection by Membrane-Impermeant Thiol/Disulfide Exchange Blockers and Antibodies against Protein Disulfide Isomerase

Abstract

Objectives: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. Methods: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. Results: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. Conclusions: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

Inhibition of rotavirus ECwt infection in ICR suckling mice by N-acetylcysteine, peroxisome proliferator-activated receptor gamma agonists and cyclooxygenase-2 inhibitors

“…In addition, there is some previous knowledge about the implication of certain cellular molecules in the rotavirus infection process (Lopez & Arias 2006, Calderon et al 2012). To get insight into the mechanisms supporting the antiviral action of AA, IBF, DCF and NAC, their impact on the expression of COX-2, ERp57, Hsc70, NF-κB, Hsp70, PDI or PPARγ was evaluated using immunochemistry, indirect immunofluorescence, ELISA, WB and confocal microscopy.…”

“…ECwt infection of villus cells induced the expression of host-encoded proteins COX-2, ERp57, Hsc70, NF-κB, Hsp70, PDI and PPARγ as detected in cross-sections from intestinal villi. It deserves to be mentioned that Hsc70 and PDI have been identified as rotavirus-interacting proteins during the infection of MA104 cells (Guerrero et al 2002, Calderon et al 2012), whereas COX-2 has been found to be required and induced by rotavirus infection of Caco-2 cells (Rossen et al 2004). On the other hand, the influenza virus-induced ER stress has been associated with increased levels of ER chaperone ERp57 (Roberson et al 2012), whilst recruitment of Hsp70 chaperones has been found to be an important component of viral survival strategies (Mayer 2005).…”

“…Rotavirus infectivity has recently been shown to be inhibited by the treatment of cells with membrane impermeant thiol/disulfide exchange inhibitors and antibodies (Abs) against protein disulphide isomerase (PDI), which suggested that infectivity was dependent on cell surface oxidation-reduction reactions (Calderon et al 2012). Some findings have suggested that the rotavirus structural protein (SP) VP7 is a substrate for the endoplasmic reticulum (ER)-associated oxidant PDI (Svensson et al 1994, Mirazimi & Svensson 1998, Maruri-Avidal et al 2008).…”

Protein disulfide isomerase a multifunctional protein with multiple physiological roles

“…Also in MA104 cells thiol blockers and PDI inhibitors decreased the entry of rotavirus, indicating the involvement of thiols for infectivity (Calderon et al, 2012). …”

Inflammatory and oxidative stress in rotavirus infection

“…The inner layer is composed of the core shell (VP2), which encloses VP1, VP3 and the genomic RNA [4,5] . Rotavirus entry into the host cell seems to be mediated by the sequential interaction of virions with various cell surface molecules including sialic acid (SA) [6] , heat shock cognate protein 70 (Hsc70) [7,8] , integrins [9][10][11] and protein disulfide isomerase (PDI) [12] . Virion penetration into the host cell involves the loss of VP4 and VP7 converting the TLP into a double-layered particle (DLP), which becomes transcriptionally active by generating positive-strand RNAs (mRNAs) [13] .…”