Cells infected with the murine coronavirus, mouse hepatitis virus (MHV), show decreased host protein synthesis concomitant with an increase in viral protein synthesis. We examined the in vitro translation property of the conserved MHV 5'-leader RNA sequence by constructing chimeric mRNAs in which the 72-nt 5'-leader of M protein mRNA (A59 strain) was positioned upstream of the human alpha-globin coding region in a T7 expression vector. Synthetic 5'-capped transcripts of these mRNA constructs were translated in cell-free extracts prepared from uninfected and MHV-infected murine DBT cells. Nonviral mRNAs translated readily in both uninfected and infected cell-free extracts. By contrast, replacement of the human alpha-globin 5'-untranslated region (UR) with the MHV 5'-leader increased translation ca. three- to fourfold in cell-free extracts from MHV-infected cells versus translation in extracts from uninfected cells. Chimeric globin mRNA containing the reverse complementary sequence of the viral leader RNA in the 5'-UR showed no such increase in translation, indicating sequence specificity for the effect. A 13-nt region (-UCUAAUCCAAACA-) immediately proximal to the start codon was found to be important for the increased translation of the MHV leader-containing mRNAs. These data indicate that the apparent down-regulation of host translation is not primarily due to an inhibition of host translation but also involves a significant stimulation of viral translation in cis by a structural feature of the MHV 5'-leader RNA sequence in conjunction with a virus-specified or virus-induced factor.

Similar to the regulation of vasodilation, the balance between NO and superoxide (O2-) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2- is essential for T cell expansion and development of autoimmunity. In mice primed with heat-killed Mycobacterium, a splenocyte population positive for Gr-1 (Ly-6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr-1+ cell subpopulations were separated according to Ly-6G expression. In culture with activated T cells, predominantly monocytic Ly-6G- Gr-1+ cells produced T cell-inhibitory NO but no O2-. However, mostly granulocytic Ly-6G+ cells produced O2- simultaneously but had no measurable effect on proliferation. Recombination of the two purified Gr-1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2- interaction. Coculture of p47phox-/- and inducible NO synthase-/- Gr-1+ cells confirmed this intercellular interaction. These data suggest that bacterial products induce development of distinct Gr-1+ myeloid lineages, which upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2−) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2− production. In contrast, macrophages from p47phox −/− (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2− in this system. To examine the NO-O2− interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2 . On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.Metallothionein (MT) genes offer an attractive system for studying the control of gene expression in animal cells. These genes, which form a multigene family, are expressed in essentially every cell type, and their transcription can be further induced in response to a variety of exogenous stimuli, including heavy-metal ions (e.g., Cd, Zn, and Hg), glucocorticoid hormones, interferon, and interleukin 1. MT gene expression is also stimulated by exposure of cells to tumor promoters, inhibitors of protein and DNA synthesis, and ionizing radiation (see reference 14 for a review). To understand the mechanisms that regulate the basal and induced expression of these genes, it is essential to characterize both the cis-and trans-acting genetic elements involved in MT expression.Previously we have shown that cis elements important for basal and induced expression of the human MT IA (hMT-IA) and hMT-IIA genes are present in their 5'-flanking regions (15,22). Deletion analysis of the hMT-IIA control region defined two distinct regulatory elements that can independently mediate induction of the gene by heavy-metal ions and glucocorticoids. Two copies of the metal-responsive element (MRE) were originally detected, yet a single copy was found to be sufficient for induction. The glucocorticoidresponsive element (GRE) was shown to coincide with the DNA-binding site for the glucocorticoid hormone receptor, a trans regulator of gene expression (16). We also determined the approximate location of an upstream promoter element, centered around nucleotides -80 to -100, which is important for basal-level expression (16,17). Similar elements were found to control both the basal and metal-induced transcription of the mouse MT-I (mMT-I) gene (3,28 The hM-IIA gene is unique in its much higher levels of both basal and induced expression compared with each of the multiple "MT-I-like" genes (11,22). Recently it was found that the high bas...

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