After cell entry, the release of the pVI structural protein and the disruption of endosomes impair capsid integrity, leading to partial capsid disassembly (Wiethoff et al. 2005). As shown with our live cell-imaging data (Figure 24), partially disassembled capsids expose their genomes before their total release. Total genome-capsid dissociation is blocked by LMB, suggesting a role for CRM1 during this process. Moreover, such effects of LMB suggest the requirement for a CRM1-NES interaction for the total dismantling of the Ad5 capsid. Little is known about Ad5 proteins harbouring NESs. E1A, E1B-55K and E4orf6 are AdV proteins known to contain an NES (Jiang et al. 2006; Kindsmüller et al. 2007; Weigel and Dobbelstein 2000). However, these proteins are expressed in late stages of AdV cycle, after nuclear genome import, therefore not present in newly infectious particles.
To explain the inhibition of genome release upon LMB treatment, we hypothesized that within the partially disassembled state of Ad5 capsid upon entry, a virally encoded and virion associated NES might be sufficiently exposed to be recognised by CRM1. This NES could be found either on a structural protein (capsid protein) or directly on a core protein. Interestingly, we found a predicted NES in the TP, a core protein of Ad5 that is covalently attached to each end of the viral genome (Rekosh et al. 1977). We focused our next analysis on the study of this predicted NES to investigate if this intricate link to the viral genome could explain the role of CRM1 in capsid disassembly and genome release.
III.1 Terminal Protein interacts with chromatin
TP is found in two copies per virion, covalently bound to both extremities of the Ad5 genome.
TP protects Ad5 genome from exonuclease degradation and promotes vDNA replication by stabilizing the replication complex and anchoring the genome to the nuclear matrix (Rekosh et al. 1977; Schaack et al. 1990; Komatsu et al. 2018). Synthetized as a precursor (pTP), this protein is cleaved by the AdV protease before the release of newly synthetized virions. The site of cleavage in pTP has been mapped in its N-terminal part (Webster et al. 1994). pTP contains an NLS and the function of this sequence in nuclear import of the AdV polymerase has been discussed (Zhao and Padmanabhan 1988). The site of cleavage in pTP is placed upstream of this NLS (Webster et al. 1994). Therefore, this NLS is conserved in the mature form of TP. When GFP-TP construct was transfected in U2OS cells, it showed a clear interaction with chromatin, in interphase and mitotic cells (Figure 40). Mutation of this Chromatin Binding Site (CBS) domain led to the loss of interaction with cellular chromatin and to the homogeneous redistribution of TP within the cell.
Results
128 Figure 40. The chromatin binding site targets Ad5 Terminal Protein to chromatin. U2OS cells were transfected with a construct coding for GFP-TP, TP wild type (upper panel) or TP mutated for its CBS (lower panel). Mitotic cells (lower row) were synchronised with colcemid treatment for 14 to 16 h prior to fixation. GFP-TP signals (green) and DAPI (grey) staining for chromatin visualisation were used. Cells were imaged by confocal microscopy and maximal projection images of cells are shown. (Scale bars, 20 µm).
Due to the strong affinity of this CBS with the chromatin, we performed our following analyses with the GFP-TP constructs mutated for the CBS.
Results
129 III.2 Terminal Protein is sensitive to LMB treatment
To test the possible interaction of CRM1 with TP, we transfected cells with a construct coding for GFP-TP (mutated for CBS) and analysed TP localization after LMB treatment. Upon addition of LMB, the homogeneous distribution of GFP-TP was impaired, in favour of a nuclear retention of GFP-TP (Figure 41). Such phenotype reminds the nuclear retention of RanBP1 upon LMB treatment (see Figure 14). TP is sensitive to LMB effect. This result suggests that TP could be an interacting partner for CRM1 and a CRM1 export cargo.
Figure 41. Ad5 Terminal Protein is sensitive to LMB treatment. U2OS cells were transfected with a construct coding for GFP-TP mutated for its chromatin binding site. 24 h post-transfection, cells were treated (+ LMB) or not (- LMB) with LMB for 45 min. Cells were fixed and stained with DAPI (grey) for chromatin staining. GFP-TP signals are depicted in green. Confocal images of transfected cells imaged by confocal microscopy. Maximal projection images of cells are shown. (Scale bars, 20 µm).
III.3 NES of Terminal Protein is functional
CRM1 recognises its cargo via the binding of a consensus NES. These sequences contain a set of five spaced hydrophobic amino acids. The nature of these hydrophobic residues and the spacing between them define the affinity of the sequence for CRM1. Consensus NES have been redefined as Φ0Φ1-(x)2–3-Φ2-(x)2–3-Φ3-x-Φ4 (Güttler et al. 2010). The predicted NES of TP is as followed: LIRLLEEELTV (with the critical hydrophobic residues underlined). Spacing between hydrophobic residues deviates from the consensus (no spacing between Φ1 and Φ2).
Results
130 To test the functionality of the TP-NES, we introduced point mutations in this sequence:
LARLLEEEATA. Transfection of this mutant led to a higher GFP nuclear signal, showing a defect in the export of this construct (Figure 42). Inactivation of this NES did not impair the NLS, which explains the nuclear localization of TP. The effect observed upon mutation of the NES was the same than what we observed upon transfection of the wild-type construct in cells treated with LMB, as just shown before (Figure 41), it also induced the nuclear retention of TP.
This result confirms the functionality of the TP-NES. TP could indeed be a partner of CRM1, via the binding with this NES sequence.
Figure 42. The Nuclear Export Signal of Ad5 Terminal Protein is functional. U2OS cells were transfected with constructs coding for GFP-TP mutated for its chromatin binding site, with (NES mutation) or without a mutation in the NES domain. 24 h post-transfection, cells were fixed and stained with DAPI (grey) for chromatin staining. GFP-TP signals are depicted in green. Confocal images of transfected cells imaged by confocal microscopy. Maximal projection images of cells are shown. (Scale bars, 20 µm).
The presence of an NES in the mature TP has never been studied. The functionality of this NES and the nuclear retention of TP upon LMB treatment are in favour of a CRM1-TP interaction. Further biochemical analyses need to be performed to study in details CRM1-TP interaction, but we developed some tools to confirm these preliminary data (see Appendix).
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DISCUSSION
Discussion
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