Prolonged Activity of the Pestiviral RNase E ^rns as an Interferon Antagonist after Uptake by Clathrin-Mediated Endocytosis

Abstract

The RNase activity of the envelope glycoprotein E rns of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E rns is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E rns . Here, we show that the activity of soluble E rns as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E rns protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E rns mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E rns remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E rns acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCEThe pestiviral RNase E rns was previously shown to inhibit viral ssRNA-and dsRNA-induced interferon (IFN) synthesis. However, the localization of E rns at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E rns is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E rns remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7

“…In this study, we investigated for the first time the cell entry process of CSFV particles through dynamin-dependent, clathrin-mediated endocytosis with a pH-dependent step, consistent with the clathrin-mediated endocytosis used by BVDV, another pestivirus (20,59). Additionally, we determined that Rab5 and Rab7 are involved in CSFV entry into PK-15 cells.…”

The Molecular Biology of Pestiviruses

“…Recently, two publications reported on the crystal structure of N pro (Gottipati et al, 2013;Z€ ogg et al, 2013). Both groups used aminoterminally truncated N pro expressed in bacteria for crystallization.…”

“…Both groups used aminoterminally truncated N pro expressed in bacteria for crystallization. The starting sequences were derived from a HoBi-like BVDV-3 strain or CSFV strain Alfort 187, and the N-terminal truncations encompassed residues 2-21 or 1-17 (Gottipati et al, 2013;Z€ ogg et al, 2013). Such N-terminal deletions do not interfere with N pro functions (Hilton et al, 2006;Ruggli et al, 2009;R€ umenapf et al, 1998).…”

Pestivirus N pro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

“…Full-length and ⌬17N N pro have been shown to be functionally identical with respect to their protease and interferon antagonistic activities (20,30,34). Native gel shift assays showed that in the presence of either full-length or ⌬17N N pro , the IRF3 protein band shifted to a higher molecular mass (Fig.…”

“…The secreted form of E rns is released into the extracellular space, where a role of E rns for degradation of extracellular viral dsRNA and single-stranded RNA molecules has been postulated (17)(18)(19). Interestingly, E rns can enter cells by clathrin-dependent endocytosis and digest viral RNA in endolysosomal compartments via its RNase activity before they can trigger type I IFN induction (20). Importantly, E rns is very efficient at preventing activation of plasmacytoid dendritic cells (pDCs) in contact with CSFV-infected cells (21).…”