I. At the MTOC, CRM1 is required for genome delivery
I.2. e CRM1 promotes the total Ad5 genome release from the capsid
Our capsid disassembly analyses were based on antibody detection of pVII in fixed cells.
Fixation of cells can impair or hide some epitopes and the sensitivity of detection relies on the accessibility of these epitopes for antibodies. Moreover, single particle track analysis require live cell imaging experiments. To bypass these issues, our group had developed another indirect way of Ad5 genome detection, applicable to living cells (Komatsu et al. 2015). This system involves again pVII detection, but this time, via the detection of TAF-I. TAF-I is a cellular factor known to form ternary complexes with pVII on incoming genomes (Haruki et al. 2003).
Binding of TAF-I molecules to pVII upon genome exposure can then be monitored by fluorescence microscopy using U2OS TAF-I GFP expressing cell lines, generated in our lab by Dr. T. Komatsu.
Upon infection of these cells with Ad5, we clearly observed nuclear TAF-I GFP dots and all of them corresponded to pVII dots, as shown by the merge between TAF-I GFP and pVII channels (Figure 23, upper row). This system is specific, as TAF-I GFP dots were not detectable upon LMB treatment (Figure 23, lower row).
Results
99 Figure 23. TAF-I staining can be used for pVII detection. (previous page) U2OS cells stably transfected with a construct coding for TAF-I GFP were treated with (+ LMB) or without LMB (- LMB) for 45 min. Infection with Alexa 594 labelled Ad5-GFP particles was performed in the presence (+
LMB) or absence (- LMB) of LMB for 1 h. Cells were fixed and stained with anti-pVII (magenta) antibodies and with DAPI (grey) for chromatin staining. TAF-I was detected by GFP signal. Cells were imaged by confocal microscopy and maximal projection images are shown. (Scale bars, 20 µm).
We used TAF-I GFP U2OS cells in order to study the role of CRM1 in capsid disassembly in living cells. In this assay, the dynamic of capsid disassembly is resulting in pVII exposure and is monitored via the detection of TAF-I GFP dots overtime, by live cell imaging microscopy.
Cells were transfected with a construct coding for tagged Histone2B-tdiRFP to stain chromatin.
Cells were synchronised via colcemid treatment (as shown in section I.2.d CRM1 affects Ad5 capsid disassembly in mitotic cells), and infected with Alexa-594 labelled Ad5-GFP particles.
Infections were performed in the presence or absence of LMB. Mitotic cells were identified according to their chromatin staining (condensed chromosomes) and overall round shape.
Single cells were selected and followed overtime. Colocalization events between TAF-I and Ad5 capsid signals were considered as partial disassembled capsids. Under these conditions pVII (i.e Ad5 genome) was enough exposed to interact with TAF-I GFP, but the capsid remained partially intact to be detected via Alexa-594 labelling fluorophore. On the other hand, free TAF-I GFP dots were considered as completely released genomes, separated from capsids.
In non LMB treated control cells, approximatively 1 h 30 to 2 h pi, green TAF-I dots were detectable (Figure 24 A). Within the cell population several TAF-I dots were free from capsids (highlighted with filled white arrows), whereas some dots remained associated with capsids (highlighted with empty white arrows). The number of TAF-I dots free from capsid increased overtime (Figure 24 B). Moreover, virtually all free TAF-I dots and some TAF-I Ad5 associated dots were observed with a restricted mobility associated to cellular chromatin, implying that the genomes became stably anchored to the chromatin (zoom Figure 24 A).
In cells treated with LMB, TAF-I dots were also detectable. However, these TAF-I were exclusively associated with capsids (yellow dots, Figure 24 C), and the number of accumulating free TAF-I dots overtime was strongly decreased compared to control cells (Figure 24 D).
However, in the presence of LMB, chromatin targeting of partially disassembled capsids was also observed (zoom Figure 24 C).
Results
100
Results
101 Figure 24. Functional CRM1 is required for total Ad5 capsid disassembly in mitotic cells. (Fig A, B and C previous page) U2OS TAF-I GFP expressing cells were transfected with H2B-tdiRFP construct (blue) to stain chromatin. After 24 h of transfection, cells were treated with colcemid for 14 to16 h to be synchronised in mitosis. Cells were treated with or without LMB for 45 min in the presence of colcemid. Infection with Alexa-594 labelled Ad5-GFP particles was performed without colcemid but in the absence or presence of LMB. Mitotic cells were identified according to their chromatin staining (blue) and Ad5 capsids (red) as Ad5 genomes (TAF-I GFP dots; green) are depicted on the pictures.
Cells were imaged by spinning disk confocal microscopy. Maximal projection images are shown. (A) and (C) Mitotic U2OS TAF-I GFP cell treated without (A) or with (C) LMB. Maximal projection of signals detected in each channel at 130 min pi. TAF-I GFP dots free from Ad5 colocalization are shown with filled white arrows whereas TAF-I GFP dots colocalizing with Ad5 are shown with empty white arrows. (B) and (D) Overlay of TAF-I GFP (green) and Ad5 capsids (red) signals in one single cell in absence (B) or presence (D) of LMB overtime. From the top left corner (120 min) to the bottom right (137 min) each frame is separated by 1 min. TAF-I GFP dots free from Ad5 colocalization are shown with white arrows.
These results showed that capsid disassembly and genome separation in mitotic cells require functional CRM1. The strong reduction of free TAF-I dots observed upon LMB treatment suggests that inhibition of CRM1 impairs Ad5 genome capsid-release. In contrast, in mitotic cells, partially disassembled capsids were targeted to the chromatin, even in the presence of LMB. One hypothesis is that a partially exposed core-genome is sufficient to target the genome to chromatin, dragging the attached capsid with it. Complete genome release and capsid disassembly, however, would need functional CRM1. In fixed mitotic cells, antibody detection of pVII in LMB treated cells did not give any signal (Figure 22), whereas pVII could be detected using the TAF-I GFP system. The TAF-I GFP pVII detection system appears thus more sensitive and does not rely on epitope recognition. However, we have not tested pVII detection in TAF-I GFP U2OS mitotic fixed cells.
Results
102 Our analyses in U2OS cells confirmed previous studies about the role of CRM1 in efficient nuclear genome import. During the first steps of infection, neither CRM1 nor other nuclear factors are required for Ad5 trafficking to the MTOC. Ad5-MTOC interaction is not well characterized but our data are in favour of an interaction independent of the integrity of the microtubule network. However, functional CRM1 is needed to mediate Ad5-MTOC removal for NPC translocation. Inhibition of CRM1 with LMB impairs nuclear genome import, leading to a defect in Ad5 gene expression. Our model of mitotic infected cells gave us more insights into the role of CRM1. Total genome release from Ad5 capsid requires functional CRM1 and it seems to directly involve CRM1 and none of its cargoes.