Herpes simplex virus mutants lacking the vhs protein are severely attenuated in animal models of pathogenesis and exhibit reduced growth in primary cell culture. As a result of these properties, viruses with vhs deleted have been proposed as live-attenuated vaccines. Despite these findings and their implications for vaccines, the mechanisms by which vhs promotes infection in cell culture and in vivo are not understood. In this study we demonstrate that vhs-deficient viruses replicate to reduced levels in interferon (IFN)-primed cells and that this deficit has both IFN-dependent and IFN-independent components. Furthermore, vhs-defective viruses induce increased and physiologically active levels of IFN, increased amounts of IFN-stimulated transcripts, and more phosphorylated eIF2␣. In addition, we demonstrate greater accumulation of viral RNAs following infection with a vhs-deficient virus. This leads to the hypothesis that attenuation of viruses lacking vhs may be attributed to increased levels of double-stranded RNA, a potent pathogen-associated molecular pattern. Together these data show that vhs likely functions to reduce innate immune responses and thereby acts as a critical determinant of viral pathogenesis.Herpes simplex virus type (HSV) is a ubiquitous human pathogen responsible for a variety of conditions, including cold sores, genital sores, and keratitis in immunocompetent hosts. In neonates, or in hosts lacking a competent immune system, HSV can become disseminated and cause severe and lifethreatening diseases, including hepatitis and encephalitis. The outcome of infection is determined largely by the status of the host's immune response and its interaction with the many viral genes that act to counter it. Of these, the virion host shutoff protein (vhs), while being nonessential for replication cell in culture, is a crucial factor for both HSV type 1 (HSV-1) and HSV-2 replication, virulence, and pathogenesis in vivo (35, 39). Viruses lacking vhs have an increased susceptibility to interferon (IFN), an increased ability to activate dendritic cells, and a decreased ability to down-regulate major histocompatibility complex class I in cell culture (31,40,43). These observations highlight the multifactorial impact of vhs on the innate and adaptive immune system (32). In addition, the reduced replication of vhs-deficient viruses relative to wild-type virus is observed rapidly, within 24 h of infection in the cornea or vaginal epithelium (35, 39), suggesting that an inability to counter innate immunity, rather than adaptive immunity, contributes most significantly to the attenuated phenotype. In addition, vhs of HSV-2 plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-mediated antiviral response (5). It has been proposed that HSVs with vhs deleted might be useful as live attenuated vaccines in both prophylactic and therapeutic settings and as such have proved effective in animal models (11,15,44,45). Together these data point to a critical role for vhs in the counteraction...

The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC’s) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC’s in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB’s generated with either HCMEC/D3 or primary HBMEC’s revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC’s. Leukocyte migration responses and inflammatory cytokine production were statistically indistinguishable between both endothelial cell types, and both cell types responded similarly to astrocyte coculture, stimulation of leukocytes with phorbol myristate acetate (PMA) and ionomycin, and inflammatory cytokine treatment. This report is the first to validate the HCMEC/D3 cell line in a neuroimmunological experimental system via direct comparison to primary HBMEC’s, demonstrating remarkable fidelity in terms of barrier resistance, immune migration profiles, and responsiveness to inflammatory cytokines. Moreover, we report novel findings demonstrating that interaction effects between immune cells and resident CNS cells are preserved in HCMEC/D3, suggesting that important characteristics of neuroimmune interactions during CNS inflammation are preserved in systems utilizing this cell line. Together, these findings demonstrate that HCMEC/D3 is a valid and powerful tool for less expensive and higher throughput in vitro investigations of immune migration at the BBB.

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Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH؉gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.Varicella-zoster virus (VZV) is a highly fusogenic virus, but the degree of fusion is dependent on the cell substrate in which the virus is propagated (20). In human fibroblast cells, fusion formation is limited to a small number of nuclei per syncytium. In contrast, in human melanoma cells, all VZV strains examined to date exhibit fusion formation in which the entire monolayer is eventually involved. Polykaryon formation also occurs during primary VZV infection in human epidermal cells. Therefore, fusion formation appears to be related to cells of ectodermal origin (20). The question of which glycoproteins are involved in VZV-induced fusion was addressed in two earlier reports in which transfection studies were carried out with the VZV gH and gL genes (13,14). Transfection with gH alone caused little or no syncytium formation. In contrast, cotransfection with gH and gL genes led to multiple foci of fusion within the monolayer, where syncytia from 6 to 25 nuclei were easily detected. This set of experiments also documented the utility of confocal microscopy as an instrument to detect syncytium formation and glycoprotein expression within a polykaryon.Of interest, the VZV results differed markedly from the herpes simplex virus type 1 (HSV-1) data, which showed that cotransfection with four glycoprotein genes, namely those of gH, gL, gB, and gD, was required for syncytium formation (42,55). In the latter case, each syncytium often included 10 to 20 ...

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