Replication-competent herpes simplex virus type 1 (HSV-1) vectors have recently been developed for selective destruction of cancer cells. These vectors kill the cancer cells either by replicating in them or by eliciting an immune response against them. Lytic HSV-1 infection induces major structural changes in host chromatin including chromosomal damage and compaction but also major chromatin marginalization in the nuclear periphery.
The compacted layer of host chromatin constitutes a potential bottleneck of intranuclear transit of progeny capsids that poses a challenge both for replication of the virus and for activation of the immune response. In this study, we will examine the mechanisms of virus-induced changes in chromatin structure and dynamics and their effect on the dynamics of viral capsids within the chromatin network.
Specifically, we are interested in understanding the role of viral nucleases and whether their manipulation affects the emergence interchromatin channels and enlarged interchromatin domains. Key to achieving our goals, will be an approach that combines FM-SXM with other imaging techniques, advanced image analysis, and biophysical modelling. This work will build upon related SXM work performed at ALS-NCXT (Aho 2017, 2019; Myllys 2016, Figure 1).
Figure 1. Herpesvirus-induced compaction and re-organization of the host chromatin. (A) Soft x-ray tomography orthoslices and (B) 3D views of segmented and color-coded nuclei reveal the spatial organization and density of chromatin in heerpes simplex virus type 1 (HSV-1) infected cells (Aho et al. 2017). (C) Distribution of low-density channels in close proximity to the nuclear envelope (Myllys et al. 2016).
Since intranuclear chromatin reorganization is potentially crucial for nuclear egress of capsids, it is essential to understand how the infection induces changes in the chromatin. This is important from the angle of basic research of virus-cell interactions but also for the improvement of oncolytic HSV-1-mediated virotherapy, which requires effective cellular release and transduction of newly formed viruses to the neighbouring cells.
Little is currently known about mechanisms of virus -induced change in chromatin architecture and mobility of viral capsids in chromatin. Here we will employ a strategy integrating 3D soft X-ray imaging, fluorescent microscopy, advanced data analysis and modelling in order to study in more detail the HSV-1-induced changes in molecular organization of the host chromatin.
Aim 1: To elucidate virus-induced temporal changes in chromatin structure.
Time dependent change of chromatin distribution in herpes simplex virus type 1 (HSV-1) infected cells at various time points after infection will be investigated by a combination of microscopy techniques (laser scanning confocal microscopy, LSCM; expansion microscopy, ExM; electron microscopy, EM) and image analyses. Furthermore, chromatin distribution, compaction and specifically presence of low-density regions or channels in wild type (WT) HSV-1 infected cells will be studied by SXM imaging and analyses both in adherent human and monkey cells placed on grids, and in suspension human B cells placed on glass capillaries.
Aim 2: To study the effect viral nuclease activities have on chromatin organization:
To study the effect of viral nucleases on chromatin structure, we will use both wt and exo- and endonuclease activity deleted fluorescent viral nucleases and viruses. Chromatin distribution in cells infected or transfected by WT or mutant nucleases and viruses will be investigated by a combination of microscopy techniques (LSCM, ExM, SIM) and advanced data analyses. To study the detailed distribution of capsids with respect to chromatin and their association with nucleases we will use nanogold EM, tomography with DRAQ5 staining of chromatin (ChromEMT), and correlative light-electron microscopy (CLEM).
Finally, chromatin distribution, compaction and presence of low-density regions or channels, and distribution of viral nucleases and fluorescent capsids will be investigated by correlative FM-SXM. For SXM image acquisition, the cells will be placed on either grids or class capillarie.
The image analysis will be performed using the automatic segmentation and analysis pipeline created in this project.
Aim 3: To determine the effect of chromatin reorganization on nuclear mobility of viral capsids:
One of the first roadblocks on the viral egress pathway is the chromatin network that restricts the intranuclear movement of capsid-sized particles (Aho et. al., 2017, 2019; Bosse et. al., 2015, Fig. 2).:
Figure 2. Simulated random walk tracks of herpesviral capsids in the chromatin reconstruction of an expansion microscopy. The magnified pictures show the paths of two capsids reaching the NE through the interchromatin space (Aho et al. 2019). Scale bar, 5 µm.
The mobility of single capsids and its correlation with chromatin distribution will be studied by 3D image correlation analyses of LSCM/SDM data. These analyses will be performed to analyse diffusion of capsids in the interchromatin areas. Finally, nuclear mobility of capsids will be modelled by random walk modelling in 3D reconstructions (Aho et. al., 2017, Myllys et. al., 201)of chromatin created from SXM images using automatic segmentation and analysis pipeline created in the project.
Myllys, M., Ruokolainen, V., Aho, V. et al. Herpes simplex virus 1 induces egress channels through marginalized host chromatin. Sci Rep 6, 28844 (2016).
Aho, V., Myllys, M., Ruokolainen, V. et al. Chromatin organization regulates viral egress dynamics. Sci Rep 7, 3692 (2017).
Bosse JB, Hogue IB, Feric M, Thiberge SY, Sodeik B, Brangwynne CP, Enquist LW. 2015. Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion. PNAS 112(42): E5725–E5733.
Aho, V.; Mäntylä, E.; Ekman, A.; Hakanen, S.; Mattola, S.; Chen, J.-H.; Weinhardt, V.; Ruokolainen, V.; Sodeik, B.; Larabell, C.; Vihinen-Ranta, M. Quantitative Microscopy Reveals Stepwise Alteration of Chromatin Structure during Herpesvirus Infection. Viruses 2019, 11, 935.