Effect of blockage of gK interaction with SPP on intercellular transport properties of the HSV-1 in the lysosome and endosome.

<p>RS cells were grown to confluency on chamber slides and transfected with SPP shRNA or scramble shRNA followed by infection with 1 PFU/cell of HSV-1. At 24 hr PI, slides were fixed, blocked and stained with rabbit-anti-EEA1-TRITC (red) for endosome or rabbit-anti-LAMP-TRITC (red) for lysosome. DAPI was used for nuclear staining (blue). Photomicrographs are shown at 40X direct magnification.</p

Blocking HSV-1 replication <i>in vitro</i> by SPP dominant negative mutants.

<p><u>A) Viral Titer is reduced by SPP dominant negatives</u>. RS cells were transfected for 24 hr with either dominant negative SPP D219A, SPP 265A or wild-type SPP and infected with 0.1 PFU/cell of HSV-1 strain McKrae. Titers were measured by standard plaque assay 12, 24 and 48 hr PI. Each point represents the mean ± SEM from 3 independent experiments per time point; and <u>B/C/D) HSV-1 protein expression is reduced by SPP knockdown.</u> RS cells (B) and Vero cells (C) were transfected and infected as in A. At 24 hr PI, cells were stained with anti-HSV-1-gC-FITC (green) and costained with DAPI (blue). Photomicrographs are shown at 10X magnification. D) Quantification of HSV-1 positive cells from (B) and (C).</p

Binding of HSV-1 Glycoprotein K (gK) to Signal Peptide Peptidase (SPP) Is Required for Virus Infectivity

<div><p>Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication <i>in vitro</i>. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.</p></div

HSV-1 induces ER morphology changes which can be blocked by SPP shRNA.

<p>A) RS cells were grown to confluency on chamber slides and transfected with SPP shRNA or scramble shRNA followed by infection with 1 PFU/cell of HSV-1. At 24 hr PI, slides were fixed, blocked and stained with rabbit-anti-calnexin-TRITC (red) and DAPI nuclear stain (blue). Photomicrographs are shown at 40X direct magnification. HSV-1 infection induces condensation of ER while treatment with SPP shRNA restores normal ER morphology. B) RS cells were infected with gKV5DIII and stained for V5 (green) and calnexin (red). Arrow indicates HSV-1 infected cell.</p

Blocking HSV-1 replication <i>in vitro</i> by SPP shRNA.

<p><u>A) Viral Titer is reduced by SPP knockdown</u>. RS cells were transfected for 24 hr with either SPP shRNA or scramble shRNA and infected with 0.1 PFU/cell of HSV-1 strain McKrae. Titers were measured by standard plaque assays at 2.5, 5, 7.5, 10, 20 and 40 hr PI. Each point represents the mean ± SEM from 3 independent experiments per time point; B<u>) HSV-1 gene expression is reduced by SPP knockdown</u>. RS cells were transfected and infected as above. Transfected and infected cells were harvested 2, 4, 6, 8 and 20 hr PI, RNA extracted and cDNA synthesized. Expression of tk, gB and gK were measured using qRT-PCR and each point represents the mean ± SEM from 3 independent experiments; and <u>C) HSV-1 protein expression is reduced by SPP knockdown.</u> RS cells were transfected and infected as in A for 24 hr PI. Cells were stained with anti-HSV-1-gC-FITC (green) and costained with DAPI (blue). Photomicrographs are shown at 10X magnification.</p

Binding of gK to SPP <i>in vitro</i>.

<p>HeLa cells were transfected with c-myc-gK and HA-SPP plasmids at a 1∶1 ratio for 48 hr. <u>A) Expression and pull-down of HA-SPP</u>. Cellular lysates were incubated with anti-HA antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-HA antibody. Lane 1 shows untransfected HeLa cells and no HA-SPP band, while Lane 2 shows HA-SPP correctly immunoprecipitating from transfected lysates. Lane 3 is protein size marker; <u>B) Expression and pull-down of c-myc-gK.</u> Cellular lysates were incubated with anti-c-myc antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-c-myc antibody. Lane 1 shows untransfected HeLa cells no c-myc-gK band, while Lane 2 shows c-myc-gK correctly immunoprecipitating from transfected lysates. Protein sizes are indicated; and <u>C/D) Co-immunoprecipitation of gK and SPP;</u> C) HeLa Cells. Left panel: Cellular lysates were incubated with anti-HA antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-c-myc antibody. Lane 1 shows untransfected HeLa cells and no gK band, while Lane 2 shows a successful pull-down of gK by anti-HA antibody. Right panel: Cellular lysates were incubated with anti-c-myc antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-HA antibody. Lane 1 shows untransfected HeLa cells and no SPP band, while Lane 2 shows a successful pull-down of SPP by c-myc-gK. Protein sizes are indicated; D) Vero Cells. Left panel: Cellular lysates were incubated with anti-HA antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-c-myc antibody. Lane 1 shows untransfected Vero cells and no gK band, while Lane 2 shows a successful pull-down of gK by anti-HA antibody. Right panel: Cellular lysates were incubated with anti-c-myc antibody bound to IgG beads and the resulting IP was subjected to Western blot analysis with anti-HA antibody. Lane 1 shows untransfected Vero cells and no SPP band, while Lane 2 shows a successful pull-down of SPP by c-myc-gK; E) HSV-1 infected lysates subjected to IP with total HSV-1 serum followed by Western blot with total HSV-1 serum pulled down many proteins; F) HSV-1 Infected lysates subjected to IP with total HSV-1 serum followed by Western blot with anti- SPP antibody did not pull down SPP; and G) HSV-1 infected lysates subjected to IP with anti-SPP antibody followed by Western blot with total HSV-1 serum did not pull down any HSV-1 reacting proteins. Protein sizes are indicated.</p

gK colocalizes with SPP <i>in vitro.</i>

<p>HeLa, Vero and RS cells were infected with 100 PFU/cell of each of four different recombinant HSV-1 expressing V5 tagged gK. Infection was allowed to proceed for 24 hr and slides were fixed, blocked and stained with mouse-anti-V5-FITC (green), rabbit-anti-SPP-TRITC (red) and DAPI nuclear stain (blue). Photomicrographs are shown at 40X direct magnification and colocalization was visualized as yellow. Panels: A) HeLa, Vero and RS cells were infected with gKV5DI; B) HeLa, Vero and RS cells were infected with gKV5DII; C) HeLa, Vero and RS cells were infected with gKV5DIII; D) HeLa, Vero and RS cells were infected with gKV5DIV; E) Mock-infected HeLa, Vero and RS cells; F) Qualitative assessment of colocalization of V5-gK and SPP in all cell lines; and G) V5-gK constructs showing the domain location of the V5 tag within the gK protein. Arrows point to less obvious areas of colocalization. In each panel the top cell line is RS cells, the middle panel is HeLa cells and the bottom panel is Vero cells.</p

Role of FoxP3-expressing cells in HSV-induced CNS demyelination.

<p>Female FoxP3^DTR mice were depleted of FoxP3-expressing cells or depleted of both FoxP3-expressing cells and macrophages then ocularly infected with HSV-IL-2 or parental virus as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006401#ppat.1006401.g001" target="_blank">Fig 1</a>. Optic nerve, brain, and spinal cord were collected from euthanized mice on day 14 PI and post-fixed tissue sections were stained with LFB. Representative photomicrographs are shown (Magnification, 20_×; Size bar, 100 μm). Arrows indicate the areas of demyelination. Panels: A) FoxP3 depleted, HSV-IL-2 infected optic nerve; B) FoxP3 depleted, HSV-IL-2 infected brain; C) FoxP3 depleted, HSV-IL-2 infected spinal cord; D) FoxP3 depleted, parental virus infected optic nerve; E) FoxP3 depleted, parental virus infected brain; F) FoxP3 depleted, parental virus infected spinal cord; G) FoxP3 and Macrophage depleted, HSV-IL-2 infected optic nerve; H) FoxP3 and Macrophage depleted, HSV-IL-2 infected brain; I) FoxP3 and Macrophage depleted, HSV-IL-2 infected spinal cord; J) FoxP3 and Macrophage depleted, parental virus infected optic nerve; K) FoxP3 and Macrophage depleted, parental virus infected brain; and L) FoxP3 and Macrophage depleted, parental virus infected spinal cord.</p

Effects of macrophage depletion on the expression of IL-12p35, IL-12p40, CD4, CD8, GFAP, and IFN-γ transcripts in brains and spinal cords of HSV-IL-2 infected mice on day 10 PI.

<p>Female WT mice with or without macrophage depletion were ocularly infected with HSV-IL-2 or parental virus as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006401#ppat.1006401.g004" target="_blank">Fig 4</a>. The brains and spinal cords were isolated on day 10 PI and qRT-PCR was performed using total RNA extracted from the individual brains. GAPDH expression was used to normalize the relative expression of each transcript. The expression of each transcript in the brains and spinal cords of naive WT mice was determined and used to estimate the relative expression of each transcript in the brains and spinal cords of the ocularly infected mice. Each point represents the mean ± SEM from 5 brains. Asterisks (*) indicate p < 0.05 by t-test. Each point represents the mean ± SEM from 5 brains or 5 spinal cords. Asterisks (*) indicate p < 0.05 by t-test. Panels: A) IL12p35 transcript in brain and spinal cord of infected mice; B) IL12p40 transcript in brain and spinal cord of infected mice; C) CD4 transcript in brain and spinal cord of infected mice; D) CD8 transcript in brain and spinal cord of infected mice; E) IFN-γ transcript in brain and spinal cord of infected mice; and F) GFAP transcript in brain and spinal cord of infected mice.</p

Severity of CNS demyelination in macrophage recipient mice.

<p>The LFB-stained sections of the brains, spinal cords, and optic nerves of WT mice that received adoptively transferred macrophages from WT mice and analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006401#ppat.1006401.t002" target="_blank">Table 2</a> were further analyzed in terms of the size of the demyelination plaques in the entire sections of brain, spinal cord, and optic nerves. Data are presented as mean demyelination using a total of 150 sections for brain and spinal cord and 30 sections for optic nerve from 5 mice per group. Arrows indicate no demyelination in the brain, spinal cord and optic nerve of mice received 1 X 10⁷ macrophages infected with HSV-IL-12p70 virus.</p