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.
Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5′ nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.
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.
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