Background: Toll-like receptors have a key role in innate immune response to microbial infection. The toll-like receptor (TLR) family consists of ten identified human TLRs, of which TLR2 and TLR9 have been shown to initiate innate responses to herpes simplex virus type 1 (HSV-1) and TLR3 has been shown to be involved in defence against severe HSV-1 infections of the central nervous system. However, no significant activation of the TLR3 pathways has been observed in wild type HSV-1 infections. In this work, we have studied the TLR responses and effects on TLR gene expression by HSV-1 with Us3 and ICP4 gene deletions, which also subject infected cells to apoptosis in human monocytic (U937) cell cultures.

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.

Measles virus-infected monocytes induce apoptosis in uninfected T cells, suggesting that infected monocytes probably interact via cell-surface molecules with uninfected T cells and induce apoptosis by indirect mechanisms. Apoptosis of the lymphocytes may contribute to the pathogenesis of MV-induced immunosuppression and cell loss.

We have studied cell death and its mechanisms in herpes simplex virus type 1 (HSV-1)-infected monocytic cells. The HSV-1 ICP4 and Us3 deletion mutant, d120 caused both apoptosis and necroptosis in d120-infected monocytic cells. At a late time point of infection the number of apoptotic cells was increased significantly in d120-infected cells when compared with uninfected or parental HSV-1 (KOS)-infected cells. Necroptosis inhibitor treatment increased the number of viable cells among the d120-infected cells, indicating that cell death in d120-infected cells was, in part, because of necroptosis. Moreover, lysosomal membrane permeabilization and cathepsin B and H activities were increased significantly in d120-infected cells. Inhibition of cathepsin B and S activities with specific cathepsin inhibitors led to increased cell viability, and inhibition of cathepsin L activity resulted in a decreased number of apoptotic cells. This indicates that cathepsins B, L and S may act as cell-death mediators in d120-infected monocytic cells. In addition, caspase 3 activity was increased significantly in d120-infected cells. However, the caspase 3 inhibitor treatment did not decrease the number of apoptotic cells. In contrast, inhibition of cathepsin L activity by cathepsin L-specific inhibitor clearly decreased caspase 3 activity and the number of apoptotic cells in d120-infected cells. This might suggest that, in d120-infected monocytic cells, cathepsin L activates caspase 3 and thus mediates d120-induced apoptosis. Taken together, these findings suggest that d120-induced cell death is both apoptotic and necroptotic. INTRODUCTIONHerpes simplex virus type 1 (HSV-1) is a common pathogen that replicates in a variety of cell types during acute infection . After lytic infection HSV-1 remains latent in the neurons of its host for life and can reactivate to cause lesions at or near the initial site of infection. Like other herpesviruses, HSV-1 expresses a large number of enzymes involved in nucleic acid metabolism, DNA synthesis and the processing of proteins. Productive viral infection is accompanied by inevitable cell destruction. HSV-1 has several strategies to combat the responses of the infected host, among them the prevention of the blocking protein kinase R-mediated shut-off of host protein synthesis (He et al., 1997), having a latent form of infection with no protein expression , blocking presentation of antigenic peptides on the cell surface (Fruh et al., 1995;Hill et al., 1995) and blocking apoptosis (Galvan & Roizman, 1998;Leopardi & Roizman, 1996;Leopardi et al., 1997).HSV-1 entry triggers the induction of apoptosis during the early stage of infection (Aubert & Blaho, 1999;Koyama & Adachi, 1997). However, HSV-1 is able to block apoptosis at multiple stages of infection to prevent the host cell from dying prematurely (Aubert & Blaho, 1999;Galvan & Roizman, 1998;Koyama & Miwa, 1997). For example, the late protein kinase Us3 contributes to blocking HSV-1-induced apoptosis (Leopardi et al., 1997;Munger & Roizman, 2001). Other...

The role of cystatins in herpes simplex virus (HSV)-induced apoptosis and viral replication has been studied. Human epithelial (HEp-2) cells infected with wild-type HSV-1 (F), with a deletion virus lacking the anti-apoptotic gene Us3 (R7041) or with a deletion virus lacking the anti-apoptotic genes Us3 and ICP4 (d120) were treated with cystatin A, C or D. Cells and culture media were studied at different time points for replicating HSV-1 and for apoptosis. Cystatins C and D inhibited the yield of replicative HSV-1 significantly in HEp-2 cells. In addition, cystatin D inhibited R7041 and d120 virus-induced apoptosis. Moreover, cystatin A inhibited R7041-induced apoptosis. These inhibitory effects of cystatins on virus replication and apoptosis are likely to be separate functions. Cystatin D treatment decreased cellular cathepsin B activity in HSV-1 infection, suggesting that cathepsin B is involved in virus-induced apoptosis.