Final Report Summary - LENTIVIRAL ENTRY (Understanding the role of nuclear envelope proteins in the regulation of entiviral infectivity in non-dividing cells.)
In lentiviruses, emerin is phosphorylated as soon as 4 hours post-infection and this phosphorylation is required for proviral integration. The aim of this study was to compare oncoretroviruses to lentiviruses in their requirements for proviral integration. In 2013-2014, we started looking at possible post-transcriptional modifications of nuclear lamina proteins in the context of oncoretroviruses infection. In addition to MLV, we added another prototypical oncoretrovirus, the feline leukaemia virus, FeLV, which was available in the laboratory. Chronically infected cells as well as freshly infected cells were compared to non-infected cells. There was no indication of post translational modifications found on Emerin, BAF or LAP2 proteins in infected cells after 4 and 6 hours of infection. In addition, we also looked at other proteins of the nuclear lamina such as SUN2 or lamins A/C and B to see whether oncoretroviruses used a different set of proteins for nuclear access. No modifications were found on these proteins either as shown in the figure 1. These sets of experiments were therefore non-conclusive.
As opposed to HIV or FIV, MLV and FeLV are simple retroviruses and may therefore not come with a kinase that would phosphorylate proteins of the nuclear lamina. During the cell cycle, emerin is naturally phosphorylated prior to mitosis and it is possible that those viruses take advantage of this feature to integrate properly. Since oncoretroviruses infect only dividing cells at the point of mitosis when the nuclear membrane is disassembled, we had to develop a new approach to test this hypothesis. Target cells for MLV and FeLV are cell lines that divide at different time points while growing. To assess this possibility, it is crucial to have cells that are all in the same point of the cell cycle. We had to develop a system to synchronize cells. One simple way to do this is serum starvation of the cells. We tested this approach without much success as cells did not support serum starvation for too long, especially the NIH3T3 cells which died from it. In FEA cells we were able to assess if starvation by looking at emerin phosphorylation but we were not able to see such phosphorylation in the cell cycle therefore rendering this protocol unusable for the question asked. The figure 2 explains the protocol used and shows no phosphorylation of emerin.
We developed another approach which consists of three successive thymidine shocks that block the cells in the S phase. This approach has already been successfully used in HSV-infection studies. It is a much heavier approach as it takes two weeks to synchronize cells prior to infection. This approach has been working and we have assessed its efficacy by looking at differential expression of cyclin B during the cell cycle. As expected shortly after release cells entered the M phase and cyclin B was degraded while no differences were observed in the phosphorylation of EMD or the expression of lamins A/C. However EMD was degraded as expected when cells entered the M phase again at 15 hours post release as shown in Figure 3..
At last we performed infection experiments in which 3T3 murine cells and FEA feline cells were synchronised, released and infected for 4 hours prior to analysis. Again no modifications were found on EMD as stated in the previous studies nor on another of the structural proteins, TREM43 as shown in Figure 4.
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Finally, we also planned to develop an approach to look at DNA integration for MLV and FeLV. While well established for HIV, this quantitative PCR approach is not trivial for MLV and FeLV since repeat sequences in the cell genome are not as numerous in mice and feline cells than in human cells. This technique would have allowed us to determine whether integration is dependent on emerin phosphorylation or not. Nonetheless in view to the lack in finding any modification on proteins, we decided to quit the project.
Note: Figures are given in the attached file
As opposed to HIV or FIV, MLV and FeLV are simple retroviruses and may therefore not come with a kinase that would phosphorylate proteins of the nuclear lamina. During the cell cycle, emerin is naturally phosphorylated prior to mitosis and it is possible that those viruses take advantage of this feature to integrate properly. Since oncoretroviruses infect only dividing cells at the point of mitosis when the nuclear membrane is disassembled, we had to develop a new approach to test this hypothesis. Target cells for MLV and FeLV are cell lines that divide at different time points while growing. To assess this possibility, it is crucial to have cells that are all in the same point of the cell cycle. We had to develop a system to synchronize cells. One simple way to do this is serum starvation of the cells. We tested this approach without much success as cells did not support serum starvation for too long, especially the NIH3T3 cells which died from it. In FEA cells we were able to assess if starvation by looking at emerin phosphorylation but we were not able to see such phosphorylation in the cell cycle therefore rendering this protocol unusable for the question asked. The figure 2 explains the protocol used and shows no phosphorylation of emerin.
We developed another approach which consists of three successive thymidine shocks that block the cells in the S phase. This approach has already been successfully used in HSV-infection studies. It is a much heavier approach as it takes two weeks to synchronize cells prior to infection. This approach has been working and we have assessed its efficacy by looking at differential expression of cyclin B during the cell cycle. As expected shortly after release cells entered the M phase and cyclin B was degraded while no differences were observed in the phosphorylation of EMD or the expression of lamins A/C. However EMD was degraded as expected when cells entered the M phase again at 15 hours post release as shown in Figure 3..
At last we performed infection experiments in which 3T3 murine cells and FEA feline cells were synchronised, released and infected for 4 hours prior to analysis. Again no modifications were found on EMD as stated in the previous studies nor on another of the structural proteins, TREM43 as shown in Figure 4.
.
Finally, we also planned to develop an approach to look at DNA integration for MLV and FeLV. While well established for HIV, this quantitative PCR approach is not trivial for MLV and FeLV since repeat sequences in the cell genome are not as numerous in mice and feline cells than in human cells. This technique would have allowed us to determine whether integration is dependent on emerin phosphorylation or not. Nonetheless in view to the lack in finding any modification on proteins, we decided to quit the project.
Note: Figures are given in the attached file
