Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

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The zinc metalloprotease, endothelin-converting enzyme-1 (ECE-1), which converts the mitogenic peptide endothelin-1 (ET-1) from its biologically inactive precursor big-ET-1, is commonly upregulated in prostate cancer (PC) cells. Consequently, we have sought to suppress ECE-1 expression by using RNAi as a potentially novel therapeutic approach. Therefore, a synthetic 64-nt short-hairpin RNA (shRNA), designed to target the ECE-1 gene, was expressed in an Herpesvirus saimiri (HVS)-based delivery vector. ECE-1 expression in cells transduced with the vector was examined by real-time PCR and Western blotting. The effects of ECE-1 knockdown on PC cell migration and invasion were studied using a scratch assay and Matrigel invasion. These studies, in vitro and ex vivo, demonstrated that the HVS-shRNA viruses could infect and silence ECE-1 expression effectively in human PC cells. Furthermore, it was observed that ECE-1 knockdown in either stromal cells or epithelial cells could significantly reduce invasion of PC-3 cells in coculture by 33 and 31%, respectively. In addition, suppressed migration was also observed in HVS-ECE-1 shRNA-infected PC-3 cells compared to uninfected and HVS-GFP-infected control cell cultures. These findings highlight the potential tumor-suppressing effect of ECE-1 knockdown in cancer cells and novel strategies for future therapeutic developments in advanced PC.Prostate cancer (PC) is the most frequently diagnosed cancer and the second leading cause of cancer death in western males. 1 Although androgen withdrawal can initially give effective control of cancer progression, 2 subsequent transition to the more malignant and metastatic androgen-independent form, which is neuropeptide-driven, requires novel approaches to treatment. In particular, the upregulation of protein components involved in the endothelin-1 (ET-1) signaling axis plays a key role in progression of many types of cancer, including ovarian, colorectal, breast and PC. 3-6 Specifically, plasma levels of ET-1 are abnormally elevated in patients with advanced, androgen-independent PC 7 , and the potent ET receptor subtype A (ET A R) antagonist atrasentan (ABT-627) has been reported to suppress partially prostate tumor growth. 8 Hence, inhibition of ET formation or downstream signaling could provide a novel strategy to treat advanced PC.The final and rate-limiting step in the biosynthesis of ET-1 is catalyzed by endothelin-converting enzyme-1 (ECE-1), a 120-130 kDa integral membrane-associated glycoprotein, belonging to the M13 zinc-metalloprotease family, which also includes neprilysin (NEP), the major ET-1 degradative enzyme. 9 Four isoforms of ECE-1 (ECE-1a-1d) have been identified, which are transcribed from a single gene using alternative promoters. 10,11 They have a common catalytic domain but show distinct subcellular localization because of the differences in their N-terminal tails, with ECE-1a and ECE-1c predominantly being expressed at the plasma membrane, whereas ECE-1b and ECE-1d mainly occur in endosomal membranes. 10,12 Alth...