Herpes simplex virus 1 induces egress channels through marginalized host chromatin

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.


Figure S3. SXT and CFM analysis of nuclear organization of VRC in infected cells.
Ortho-slices, or virtual sections, of an X-ray tomographic reconstruction of the infected cell at 24 h p.i. A CFM image of the distribution of viral EYFP-ICP4 (yellow). Scale bar is 3 µm.

SUPPLEMENTAL TEXT
Text S1. Materials and Methods

Infection of B cells by HSV-1.
Mouse B cells were infected with a herpes simplex virus type 1 (HSV-1, strain 17+), using a virus stock with a titer of 1 x 10 10 plaque-forming units (pfu)/ml. These cells were infected at a density of 1 x 10 6 /ml for 1 hr at a multiplicity of infection (MOI) of 0.5 or 5 plaqueforming units (pfu)/cell. There after the medium and the unbound virus were removed and the cells were washed with a sterile PBS. After infection the cells were maintained in 24-well plates in RPMI1640 (Gibco) supplemented with a 10 % fetal calf serum (PromoCell, Heidelberg, Germany), glutamine and gentamycin.

Infection of B cells by HSV-1
HSV-1 is a human pathogen with a broad host range in terms of both species and cell type (Spear and Longnecker, 2003;Eling, 2000). For SXT image acquisition, cells were placed in a (cylindrical) capillary tube of a diameter up to 15 µm. B cells were small enough to fit into these tubes.
In the nucleus, lytic infection by HSV-1 proceeds in a regulated cascade in which three temporal classes of viral genes are expressed: immediate-early, early and late (Honess and Roizman, 1974). The immediate-early gene products exert regulatory functions essential for the expression of both early and late genes, and immune-evasion functions (Allen and Everett, 1997;de Bruyn Kops et al., 1998;Quinlan et al., 1984;Watson et al., 1979). In order to characterize the HSV-1 replication in B cells, the infectivity of cells was analyzed by cytometry based on intracellular staining of immediate-early and late viral proteins ( Fig.   S1A). At 24 h post infection (p.i.), this analysis indicated that viral glycoproteins C (gC) and D (gD) were detectable in 1.0 ± 0.6 % and 2.2 ± 0.5 % of the cells, respectively, infected at MOI 0.5 pfu/cell, and in 4.5 ± 2.1 %, and 10.6 ± 1.0 % of the cells when infected at MOI 5.
Moreover, the percentages of ICP0-expressing cells were 1.6 ± 0.5% at MOI 0.5, and 7.2 ± 1.6% at MOI 5. These data indicated that ICP0 and viral glycoproteins were expressed in infected B cells at 24 h p.i. in an MOI-dependent manner.
In conclusion, we detected the presence of viral proteins, as well as the expression of viral lytic genes of all three phases of the replication cycle, and substantial production of the progeny virus from the B cells. This suggests that HSV-1 not only could enter and infect B cells but that its replication cycle was completed. Based on these findings, we decided to use an MOI of 5 and the time point 24h in all our subsequent experiments of this study.