Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) a0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calciumactivated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.

Recurrent herpes simplex virus type 1 (HSV-1) disease usually results from reactivation of latent virus in sensory neurons and transmission to peripheral sites. Therefore, defining the mechanisms that maintain HSV-1 in a latent state in sensory neurons may provide new approaches to reducing susceptibility to recurrent herpetic disease. After primary HSV-1 corneal infection, CD8+T cells infiltrate the trigeminal ganglia (TGs) of mice, and are retained in latently infected ganglia. Here we demonstrate that CD8 1 T cells that are present in the TGs at the time of excision can maintain HSV-1 in a latent state in sensory neurons in ex vivo TG cultures. Latently infected neurons expressed viral genome and some expressed HSV-1 immediate early and early proteins, but did not produce HSV-1 late proteins or infectious virions. Addition of anti-CD8 a monoclonal antibody 5 d after culture initiation induced HSV-1 reactivation, as demonstrated by production of viral late proteins and infectious virions. Thus, CD8+T cells can prevent HSV-1 reactivation without destroying the infected neurons. We propose that when the intrinsic capacity of neurons to inhibit HSV-1 reactivation from latency is compromised, production of HSV-1 immediate early and early proteins might activate CD8+T cells aborting virion production.

Our previous studies have shown that the enzymatic activities of Neu-1, an endogenous sialidase encoded in the murine MHC, are involved in promoting IL-4 synthesis by naive CD4+T cells. Our present studies have characterized responsible sialoconjugate targets of Neu-1 and questioned possible biochemical mechanisms responsible for their regulatory influences on IL-4 gene expression. These studies determined that treatment of T cells with the naturally occurring ganglioside GM3 inhibited the production of IL-4 without affecting the production of IL-2. An analysis of IL-4-primed CD4+ T cells further demonstrated that GM3 treatment specifically inhibited the restimulated production of IL-4, IL-5 and IL-13, without inhibiting the production of IL-2 and IFN-. The inhibitory effects of GM3 could be overcome by treatment with thapsigargin or ionomycin, suggesting ganglioside regulation occurs upstream of activationinduced calcium mobilization. GM3 treatment attenuated the level of calcium influx following CD3 crosslinking, and CD4+T cells from Neu-1-deficient B10.SM strain mice (neu-1a and IL-4-deficient) expressed reduced levels of intracellular calcium following activation. Our results indicate that activities by membrane gangliosides can influence the cytokine programs in CD4+T cells, possibly through the modulation of calcium responses induced by T cell activation.

IL-4 is important in controlling the development of immune responses. Following activation with anti-CD3e under serum-free conditions, splenocytes from most normal (neu-7') mouse strains directly produced IL-4 and other T cell cytokines. However, splenic T cells from SM/J and B1O.SM (H-21; neu-1") strain mice, deficient in neu-7 sialidase activity, failed to produce IL-4 but produced normal levels of IL-2 following activation. Moreover, sialidase-deficient mice produced markedly less IgE and lgGl Abs following immunization with protein Ags than did mouse strains with normal neu-7 sialidase activity. Enriched T cells from newla mice failed to be effectively primed with exogenous murine 11-4 to become IL-4-producing cells. Treatment of splenocytes or enriched T cells from neu-V mice with bacterial sialidase prior to activation or IL-4 priming promoted their subsequent capacity to produce IL-4. In contrast, activation of T cells from neu-7' mice in the presence of a sialidase inhibitor almost completely blocked subsequent IL-4 production. The presence of IL-4 during priming enhanced T cell expression of neu-7-specific sialidase activity and increased the membrane expression of asialo-G,, compared with T cells activated without 1L-4. These results suggest that T cell-associated neu-1 sialidase is required for early IL-4 production by splenic T cells and is involved in the 11-4 priming process of conventional T cells to become active IL-4 producers.