Consistent with this, RTT is associated with abnormalities in the Glu metabolism

the nucleoli while SC35 changed from its normal nuclear speckle pattern to a more diffuse pattern in GFP-3C expressing cells. No change in the localisation of any hnRNP proteins was observed by IF after transfection with GFP-3C. Overall, our analysis indicates that 3C’s effects on the nuclear pore lead to the mislocalisation of some but by no means all nuclear proteins. Discussion This is the first study 21560248 to clearly establish that HRV16 3C protease is the likely mediator of Nup153 cleavage, and mislocalisation of SC35 and nucleolin in HRV16 infected cells. In particular, the results from using different approaches indicate that proteolytically active 3C protease is sufficient to effect cleavage of Nup153. This activity appears to be complementary to that of HRV16 2A protease in cleaving Nup62 cleavage, which contributes to hnRNP relocalisation. Our study is the first to Orange Yellow S assess the effect of HRV16 infection on FG- and non-FG-Nups. Interestingly, HRV16 infection resulted in cleavage of all FG-Nups but none of the non-FG-Nups analysed. This finding is significant in the context of the functions attributed to these two classes of Nups; FG-Nups are involved directly in the movement of cargo through the nuclear pore complex while the non-FG Nups have a structural role. Our finding provides the underlying mechanism for the observations in previous reports that the overall structure of the NPC remains intact in picornavirus infection even while nuclear transport is disrupted through a so-called “leaky”pore. Significantly, expression of active GFP-tagged 3C in transfected cells resulted in the mislocalisation of the splicing factor SC35 from nuclear speckles to become diffusely nucleoplasmic, precisely paralleling SC35 localisation in HRV16-infected cells. This has not been observed in previous studies using HRV14 or poliovirus, but may reflect a serotypespecific difference with respect to HRV16. Since SC35 plays a key role in elongation of nascent mRNA transcripts, determination of RNA splice junctions and spliceosome assembly, its mislocalization in HRV infected cells is likely to contribute significantly to host cell shut-down. The hnRNPs A1 and C1/C2 shuttle in a signal dependent fashion between the nucleus and cytoplasm, and are believed to be 18000030 involved in mRNA splicing and export and mRNA splicing and stability, respectively, with both playing a role in translational control of mRNAs that contain IRESs, such as those found on HRV genomes. HnRNP-A1 in particular is reported to bind to the IRES of HRV2, with its nuclear import pathway dependent Active HRV16 3C is Sufficient to Effect Cleavage of FGNups and Mislocalisation of Nuclear Proteins in Transfected Cells To assess the extent to which the above changes in HRV 16 infected cells could be solely attributed to the activity of 3C, we transfected COS-7 cells to express GFP fused HRV16 3C or 3Cinac, a mutant derivative where the active site cysteine has been mutated to alanine to result in a lack of protease activity, and then GFP positive cells FACS sorted, lysed and their levels of Nups/nuclear proteins assessed by Western analysis. Equivalent numbers of lysed, non-transfected COS-7 cells were included as an untreated control. We observed cleavage of Nup153, as well as nucleolin, in cells expressing GFP-3C, but not in those expressing GFP3Cinac or untransfected cells; tubulin levels were not different among the three cell groups, implying the specificity of the results. Quantitative analysis