Structural basis for viral genome tethering to host chromatin – CApTIVE

I- Structural basis for nucleosome interaction by viral proteins
We will use a combination of structural and biochemical approaches. We developed the production of crystallography and cryo-EM grade nucleosome structures. These structures will also be used for interaction experiments by pull down, calorimetry and interferometry.
II- Functional implications of nucleosome capture by viral proteins
In this axis we will use our PFV infection system with particles harboring Gag residues substitutions. We rationally designed Gag substitutions based on the interactions with distinct acidic patch cavities. Using immunofluorescence approaches we will follow the virus trafficking in the cell. We optimized an infection system on synchronized cells to better characterize the kinetic of chromatin capture during mitosis.
III- Modulation of the chromatin structure by the viral proteins
We optimized the production of recombinant DNA and histones proteins necessary for assembling polynucleosomes and chromatin fibers in vitro. These assemblies will be submitted to microcentrifugation assay in the presence of Mg. Increasing Mg concentration has been show to promote polynucleosome oligomerization. Using our material we could reproduce the publised observations and develop further the system to monitor the role of acidic patch viral protein binders in the modulation of chromatin architecture.

Using biochemical approaches we would show that the Gag R540Q and Y537Q substitutions are affected for nucleosome interactions (affinities: wt > Y537Q > R540Q). Also we discovered that polynucleosome compaction selectively altered the binding of the mutated Gag proteins while the wild type retained the interaction. Following the recruitment of a post doc fellow, we could develop infections experiments coupled with imaging. We compared the chromatin capture kinetics of the wild type vs Gag substitutions described above. Using an infection system on synchronized cells we could show that the wild type virus engages the host chromatin as soon as the nuclear envelop breaks down in prophase. R540Q and Y537Q trafficking dynamics are completely altered. In this system we reproduced the published observations on the R540Q. However we described here the unique behavior of the Y537Q substitution. Indeed this virus stalls at the centrosome during the early mitosis phases and captures the chromatin during the telophase. Integration site selection analysis of this viral mutant show also a unique redistribution phenotype.
The differences observed in the tethering chronology of the Y537Q can be explained by the need of a chromatin relaxation as it is occurring in
telophase. Our biochemical data on the increased sensitivity to chromatin compaction strenghten this observation.

The future perspectives are declined in two parts. First, short time perspectives are to finalize the study on the kinetic of PFV chromatin tethering. This will constitute a first manuscript to be sent by the end of the year. Our long term perspectives are to pursue the characterization of the chromatin remodeling induced by viral proteins.Based on preliminary data recently obtained, we will also investigate the propensity of Gag protein to induce liquid-liquid phase separation and its relation with the viral transactions orchestrated in the nucleus.

A mini review is currently under review in the journal mBio
Targeting the nucleosome acidic patch by viral proteins: two birds with one stone? Lagadec et al. mBio

Eukaryotic chromatin harbors a variety of structures and its dynamics is key in many cellular processes like DNA replication, transcription and repair. High order folding of the chromatin is mediated by histone H4 N-terminal tail interaction with the acidic patch of the neighboring nucleosome. Interestingly the acidic patch acts as a docking station for several viral proteins like Kaposi sarcoma herpes virus LANA, cytomegalovirus IE1 or spumaretrovirus Gag. It has been shown that LANA and IE1 can affect in vitro high order structure of chromatin but the functional significance of this phenomenon remains unclear. Recently we solved the structure of retroviral Gag protein bound to the nucleosome acidic patch and showed that this interaction is crucial for optimum viral infectivity. By using a combination of structural, cellular and biophysical approaches this project aims at deciphering the molecular mechanism of the retroviral proteins tethering to the nucleosome and the consequences on both the viral replication and host chromatin architecture. A better understanding of these molecular interplays between the virus and the host is of major interest both in the virology and chromatin field with potential of future medical applications.