RNase-dependent inhibition of extracellular, but not intracellular, dsRNA-induced interferon synthesis by Erns of pestiviruses

Abstract

Recombinant pestivirus envelope glycoprotein E rns has been shown to interfere with dsRNAinduced interferon (IFN-a/b) synthesis. This study demonstrated that authentic, enzymically active E rns produced in mammalian cells prevented a dsRNA-induced IFN response when present in the supernatant of bovine cells. Strikingly, IFN synthesis of cells expressing E rns was eliminated after extracellular addition, but not transfection, of dsRNA. Importantly, the same applied to cells infected with bovine viral diarrhea virus (BVDV) expressing E rns but lacking the N-terminal protease N pro . Free E rns concentrations circulating in the blood of animals persistently infected with BVDV were determined to be approximately 50 ng ml "1 , i.e. at a similar order of magnitude as that displaying an effect on dsRNA-induced IFN expression in vitro. Whilst N pro blocks interferon regulatory factor-3-dependent IFN induction in infected cells, E rns may prevent constant IFN induction in uninfected cells by dsRNA that could originate from pestivirus-infected cells. This probably contributes to the survival of persistently BVDV-infected animals and maintains viral persistence in the host population.Bovine viral diarrhea virus (BVDV), a member of the family Flaviviridae and a major ubiquitous cattle pathogen, causes two fundamentally different types of infection. Animals infected post-natally are transiently infected, mostly without showing obvious disease signs such as diarrhoea and coughing (Corapi et al., 1990;Meyers & Thiel, 1996;Ridpath et al., 2000;Thiel et al., 1996). When infected in utero as a result of maternal infection, fetuses may develop and be born normally. They remain infected for life and display a highly specific immunotolerance towards the infecting viral strain (Brownlie, 1990). Immunotolerance of persistently infected (PI) animals is explained by the early time point of infection between approximately 40 and 120 days of intrauterine development. In contrast to the adaptive immune response, innate immune defence mechanisms are operative even in the early stages of intrauterine development when the fetus is invaded by BVDV, and there is broad but indirect evidence that evasion of the host's interferon (IFN) defence is a crucial prerequisite for the establishment and maintenance of persistent infection (Adler et al., 1997;Charleston et al., 2001;Schweizer & Peterhans, 2001).Over the past few years, it has become apparent that virtually all viruses express proteins that target the host's IFN defence mechanisms. Collectively, these proteins target either the induction pathways or the mechanism of IFN action (for a review, see Randall & Goodbourn, 2008). Induction of IFN is initiated by extracellular or intracellular pattern recognition receptors that sense molecular patterns that indicate viral infection (Beutler et al., 2007). In the case of positive-and negative-sense RNA viruses, dsRNA, a by-product of viral RNA replication, and 59-triphosphorylated RNA, respectively, are believed to be the most important IFN inducers ...

Mutation of Cysteine 171 of Pestivirus ErnsRNase Prevents Homodimer Formation and Leads to Attenuation of Classical Swine Fever Virus

“…Both in vitro analyses and experiments with BVDV-infected fetuses showed that E rns is involved in pestiviral interference with the type I IFN response to virus infection (21,31,33). It was therefore interesting to investigate whether differences in the type I IFN levels could be detected in the blood of infected pigs from groups 1, 2, and 3.…”

“…It has been put forward that secreted E rns could be responsible for blocking an IFN response to extracellular dsRNA. Evidence for this hypothesis was found in in vitro experiments, and it was shown that the RNase activity of E rns is essential for this function (21,31). However, the putative origin of this so-far hypothetical extracellular RNA is obscure, and it might be that E rns (also) serves other functions.…”

“…A role of E rns and its RNase in the interaction of the virus with the immune system of the host or the host cell has been proposed (21,32,35). E rns was shown to interfere with the type I IFN response of cells to dsRNA, and this activity was dependent on the RNase activity and its capacity to bind dsRNA (21,31). Recently, we provided compelling evidence for BVDV that the RNase activity of E rns and the N pro protein are both involved in establishment and maintenance of persistent infections (33).…”

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

“…The nonstructural protein N pro targets the transcription factor IRF3 for proteasomal degradation (5), thus antagonizing interferon induction, e.g., by double-stranded RNA (dsRNA), in virus-infected cells (6,7). E rns harbors an RNase active domain belonging to the T2 RNase superfamily (8), and this enzymatic activity is essential for its ability to block the induction of IFN-␣/␤ (9)(10)(11). Together with viral glycoproteins E1 and E2, E rns forms the envelope of the virus, but a significant portion of the E rns protein is also secreted into the extracellular space (8).…”

Bovine Viral Diarrhea Virus Strain- and Cell Type-Specific Inhibition of Type I Interferon Pathways

“…As differences in viral replication did not explain differences in IFN-␣/␤ production, we next assessed the potential effects of both BVDVncp strains on interferon response factors (IRFs). BVDVncp is known to inhibit IFN-␣/␤ production through viral proteins N pro (2,3,10,14) and E rns (16,18,19), which block IFN-␣/␤ production by targeting IRF-3 for degradation by polyubiquitination (7,14) or by degrading viral RNA (19). Translocation of IRF-3 occurs during infection but not binding to DNA (2).…”