In order to analyse the role of CRM1 in Ad5 genome delivery, we first analysed the impact of point mutations on CRM1 in Ad5 capsid disassembly in mitotic cells. We performed a screening of several mutants of CRM1, all insensitive to LMB (to inhibit the endogenous CRM1) but holding additional point mutations. We found an interesting candidate, with two point mutations in its N-terminal domain, CRM1 W142 P143 C528S-HA. We infected mitotic cells upon expression of this mutant, and we observed that this mutant was not able to rescue the Ad5 genome release. We then monitored genome import and gene expression, upon expression of this triple mutant. During the first 2 h of infection, we observed an accumulation of Ad5 at the MTOC, same phenotype that was observed in control cells treated with LMB, without expression of CRM1 constructs. However, at 2 h pi, nuclear Ad5 genomes started to be detected, showing a delay in genome import. Same results were obtained in the analysis of Ad5 gene expression by RNAscope, the expression of E1A mRNA were delayed compare to cells expressing the simple mutant CRM1 C528S-HA. Interestingly, 6 days pi, no difference between CRM1 528S and the triple mutant were observed. This mutant was showing a striking defect only during the first steps of Ad5 infection.
The point mutations W142A P143A introduced in CRM1 are located close to the binding site of Nup214 FG-repeats (Port et al. 2015). To better explain the defects observed upon Ad5 infections, we performed several in vitro and in vivo biochemical assays. We generated U2OS cells constitutively expressing this mutant and we monitored the export of several known cargoes of CRM1. Our results show a functional export of CRM1, able to rescue the LMB effect. We then purified recombinant CRM1 proteins, and we performed in vitro export assays.
Our results confirmed a functional export mediated by this mutant. However, slight defects in the kinetics of export, as well as in the binding with the PKI-NES were observed.
Discussion
145 The Kd was measured three times higher compared to CRM1 wild type, showing a defect in the affinity of CRM1 W142A 143A C528S for the PKI-NES.
In conclusion, our results showed that we generated a mutant of CRM1 functional for export, but inducing a clear defect in Ad5 genome delivery. Ad5 are not physiological cargoes for the cell. Viruses highjack cellular pathways to perform they infection cycle. CRM1 export cargoes form the nucleus to the cytoplasm (Fornerod, et al. 1997b; Fornerod,Kehlenbach et al. 1998;
Fukuda et al. 1997) but upon Ad5 infection, CRM1 promotes upstream events of genome import, such as NE translocation (Strunze et al. 2005) or capsid release, as we just discussed.
The clear differences observed in the triple mutant of CRM1, between the physiological export function of CRM1 and its role in Ad5 genome delivery raise some question about its role, its structure and potential new interacting partners.
W142A P143A point mutations are located in the CRIME domain of CRM1, site of binding with RanGTP (Fornerod et al. 1997a). Formation of a trimeric complex between RanGTP-CRM1 and an NES occurs in a cooperative manner, inducing structural conformation changes in CRM1 (Fornerod et al. 1997b; Monecke et al. 2013). A lower affinity of CRM1 for RanGTP or NES could thus impair binding of the second partner. We showed a lower affinity of CRM1 W142A P143A for PKI-NES, compared to CRM1 wild type. This result could be explained by a lower affinity of CRM1 for the NES itself, but we cannot exclude a lower affinity for RanGTP, thus impairing binding with PKI-NES.
Moreover, we observed degradation products of the CRM1 mutant during its purification.
Mutations introduced in the N-terminal part of CRM1 could destabilize the protein, leading to a misfolded recombinant product. The expression level of CRM1 mutant in the newly generated U2OS cell lines are comparable to those in control cells. Recombinant CRM1 is also correctly expressed in bacteria. Thus, the higher yield of degradation product in newly synthetized CRM1 may be explained by less stable proteins, due to a misfolded structure. Slight defects observed in export kinetics and NES binding assays could thus be due to conformational conflicts during the binding of RanGTP and NES. Further structural studies are required to characterize effect of these point mutations.
CRM1 has a centrosomal location, driven by its CRIME domain (Liu et al. 2009). Point mutations W142A P143A are part on this domain. We cannot exclude that these mutations impair the centrosomal localization of CRM1, by decreasing the portion of CRM1 at the MTOC.
Therefore, the amount of CRM1 W142 P143A C528S would be less available for the Ad5, explaining the blocking of Ad5 capsids at the MTOC, observed in CRM1 triple mutant cells.
Discussion
146 However, the CRM1 mutant was available in mitotic U2OS CRM1-HA expressing cells but capsid disassembly was still impaired.
We generated a tool to help to understand CRM1 function and the nature of Ad5-CRM1 interaction during Ad5 infection. Despite the functionality of this mutant, the observations of accumulation of Ad5 the MTOC in CRM1 mutant cells are in favour of a requirement for the virus to pass by the MTOC. Delay in capsid disassembly despite a functional export are also in favour of a direct interaction of CRM1 with Ad5, rather than an interaction mediated by an exported cargo. Finally, our results point the importance of the structural organisation of CRM1 to form a stable ternary complex with RanGTP and the requirement of a proper folding to maintain the strength of interactions.
Discussion
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