The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R3W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR3GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex ϳ21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for ϳ21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.RNA interference (RNAi) is a posttranscriptional RNA silencing process that is evolutionary conserved across eukaryotic kingdoms to regulate the levels of specific RNAs during development. RNAi also functions as a host defense system against virus infections, as was first shown for plant viruses (1). As a countermeasure, viruses have evolved suppressors of gene silencing (15,26,27,32,38). One of the biochemically best characterized suppressors is a protein of 19 kDa (P19) encoded by Tomato bushy stunt virus (TBSV) and related tombusviruses that is functional as a suppressor in several model systems (4,12,13,40,41).During TBSV replication in plants, abundant levels of highly structured genomic single-stranded RNA (ssRNA) and doublestranded RNAs (dsRNAs) accumulate that potentially form excellent substrates for one of the first steps in RNAi: Dicer-mediated cleavage of dsRNAs into duplex short interfering RNAs (siRNAs) (2,19,20,43). In a subsequent step of RNAi, these ds-siRNAs unwind to donate one of the siRNA strands to the RNA-induced silencing complex (RISC). This forms the catalytic entity for RNA degradation that uses the incorporated siRNA to specifically target complementary RNAs (7-9, 14, 16, 17). However, suppressors of RNAi, like P19, can interfere with this process (10, 33).The X-ray crystallographic structure of the P19-siRNA com...
Background and purpose: Chronic morphine administration produces tolerance in vivo and attenuation of m opioid receptor (MOR)-mediated G-protein activation measured in vitro, but the relationship between these adaptations is not clear. The present study examined MOR-mediated G-protein activation in the CNS of mice with different levels of morphine tolerance. Experimental approach: Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR-mediated behaviours. MOR function was measured using agonist-stimulatedand receptor binding throughout the CNS. Key results: Morphine pellet implantation produced 6-12-fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED 50 values between morphine-treated and control groups. Pellet implantation plus supplemental injections produced 25-50-fold tolerance in these tests. In morphine pellet-implanted mice, MOR-stimulated [ 35 S]GTPgS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet-implanted mice. In contrast, MOR-stimulated [35 S]GTPgS binding was significantly decreased in most regions examined in morphine pellet þ morphine injected mice, including nucleus accumbens, caudate-putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord. Conclusions and implications: Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR-mediated G-protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS-mediated effects of morphine.
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