A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C 3 HC 4 ) at their carboxylterminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118 -128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.

TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn2+-chelating ability of its amino-terminal RING finger domain abolished TRAC-1’s ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.

The early events that occur after treatment of the highly interferon a (IFN-a)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-a have been examined. IFN-a treatment ofDaudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-a treatment results in a selective, time-dependent activation of the Ca2+-independent E isoform of protein kinase C (PKC), while the a isoform is unaffected by IFN-a treatment. In contrast, IFN-a treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-e. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-a-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-a against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-a involves diacylglycerol production and activation of PKC-E in Daudi cells.-,411, and -y) and Ca2+-independent (PKC-8, -E, -s, and -ti) enzymes. Furthermore, PKC-E has been shown to have Ca2+-independent phorbol ester binding activities and exhibits substrate specificity distinct from other characterized PKC isoforms (10, 11).We have investigated the early events that occur upon treatment of human Daudi lymphoblastoid cells with IFN-a in order to identify the biochemical pathways of transmembrane signaling. IFN-a treatment of these cell lines results in cessation of cell growth, protection against viral infection, and the rapid transcriptional activation of ISGs (12, 13). We report that the outstanding features of the signal transduction pathway of IFN-a in Daudi cells are the generation of DAG in the absence of inositol phospholipid turnover or Ca2+ elevation and the activation of the calcium-independent E isoform of PKC. Furthermore, staurosporine, a potent inhibitor of PKC activity blocks the transcriptional activation of ISGs, the cytoplasmic accumulation of ISG mRNA, and the induction of antiviral activity against vesicular stomatitis virus (VSV) in Daudi cells by IFN-a.