RNase L inhibitor (RLI) antisense constructions block partially the down regulation of the 2‐5A/RNase L pathway in encephalomyocarditis‐virus‐(EMCV)‐infected cells

Abstract

The interferon-(IFN)-inducible 2′,5′-oligoadenylate (2-5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2-5A/RNase L pathway appears to be regulated by the cell-growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2-5A/RNase L-pathway-associated antiviral activity.We have recently cloned a cDNA coding for RLI, a RNase-L-specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2-5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independant clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20% for VAS1, 25% for VAS2, 75% for VAS3 and 50% for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90 %) was observed in control cells and in VAS1 while 48% inhibition was observed in VAS4 and 25% in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV-induced down regulation of 2-5A-binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.Keywords : interferon ; ribonuclease L ; ribonuclease L inhibitor ; virus infection; antisense RNA.The 2′,5′-oligoadenylate (2-5A)/endoribonuclease L (RNase cloned a cDNA coding for a 68-kDa polypeptide which behaves as an RNase L inhibitor (RLI) [6]. This cDNA hybridizes with L) pathway is considered as playing a major role in the antiviral and antiproliferative effects of interferons (IFNs) [1, 2]. RNase two mRNAs of 3.5 kb and 2.8 kb, which differ in their 3′ untranslated regions. The expression of RLI in a reticulocyte lysate L is activated by an unusual series of oligomers known as 2-5A in the nanomolar concentration range [3]. RNase L then cleaves prevents 2-5A binding by endogenous RNase L and inhibits its nuclease activity. RLI leads neither to 2-5A degradation nor to mRNAs at the 3′ side of the UpNp sequences [4], leading to the inhibition of protein synthesis. Interestingly, the 2-5A/RNase L an irreversible modification of RNase L. Indeed, RNase L activpathway is one of the best characterized RNA-degradation path-ity is recovered when the two proteins are separated by gel elecways in mammalian cells. The activity of the 2-5A/RNase L trophoresis. Moreover, the two proteins co-immunoprecipitate pathway varies appreciably with cell growth and differentiation with antibodies recognizing RNase L [6]. RNase L inactivation, status, in keeping with a possible role in the catabolism of cellu-thus, seems to result from a direct interactio...

Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells

“…The 2-5A binds to and activates RNase L which leads to degradation of rRNA (Castelli et al, 1998). RNase L activity is also suppressed by RNase-L-specific protein inhibitor (RLI) (Martinand et al, 1998). The mechanism of inhibition of RNase L by RLI is not well defined; however, the ribosomal localization of RLI suggests that it could potentially protect rRNA from cleavage by RNase L (Dong et al, 2004).…”

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNα-induced apoptosis

“…Recombinant RNase L tagged with glutathione Stransferase (GST), produced in baculovirus-infected insect cells 38 was incubated 1 h at room temperature with glutathione-sepharose (Amersham Biosciences, Piscataway, NJ, USA) equilibrated in Tris buffer (10 mM Tris-HCl (pH 7.5), 130 mM NaCl, 1 mM PMSF, 10 mg/ml aprotinin, 150 mg/ml leupeptin). After washing three times with Tris buffer supplemented with 0.1% (v/v) NP-40 and 0.2% (w/v) bovine serum albumin, glutathione-sepharose was incubated with rabbit reticulocyte extract (45 ml), for 1 h at room temperature where human IF2mt was translated in presence of 35 S-methionine according to manufacturer protocol (Promega, Madison, WI, USA).…”

“…Human IF2mt was translated in rabbit reticulocyte extract (45 ml) in the presence of 35 S-methionine according to manufacturer protocol (Promega). After translation, the reaction was incubated 3 h at room temperature under gentle shaking, with polyclonal antibodies against RNase L (80 mg/ml) 38 or with the irrelevant antibody anti-lexA (1/5001) (a generous gift of Didier Fesquet, CRBM, Montpellier, France) in 450 ml of immunoprecipitation buffer (10 mM Tris-HCL (pH 7.5), 130 mM NaCl, 0.1% NP-40, 1 mM PMSF, 10 mg/ml aprotinin, 150 mg/ml leupeptin). After addition of protein A-sepharose (Amersham Biosciences), the reaction was incubated overnight at 41C with gentle shaking.…”

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

“…However, a few viruses may have evolved strategies that specifically counteract the antiviral activity of the OAS/RNase L system. For example, HIV-1 (Martinand et al, 1999) and EMCV (Martinand et al, 1998) apparently downregulate RNase L activity by inducing the expression of the cellular RNase L inhibitor (RLI), which antagonizes 2959-oligoadenylate binding to RNase L and hence prevents its activation. With regards to HIV, potentially the TAR sequence structural element could also bind and activate OAS and PKR, but this is prevented by the virus Tat protein binding the TAR element (Maitra et al, 1994).…”