RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES).Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5 untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA i Met . Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.For the vast majority of eukaryotic cell mRNAs the initiation of protein synthesis is achieved after the recognition of the cap structure (m 7 GpppN), located at the 5Ј terminus of all cytoplasmic mRNAs, by the cap-binding complex eIF4F (reviewed in reference 12). This translation initiation factor is a heterotrimer comprising eIF4E (which binds to the cap), eIF4A (which has RNA helicase activity), and eIF4G (a scaffold protein). The eIF4F complex acts as a bridge between the mRNA and the small ribosomal subunit. The eIF4G subunit has binding sites for a number of proteins, as well as eIF4E and eIF4A (two independent sites); these proteins include eIF3 (associated with the 40S ribosome subunit), poly(A)-binding protein, and Mnk-1 (an eIF4E-kinase). The importance of eIF4G is demonstrated by the observation that cleavage of this protein occurs within cells infected by many different picornaviruses (e.g., poliovirus and foot-and-mouth disease virus [FMDV]) and results in the inhibition of cellular protein synthesis (for a review, see reference 2). The translation of picornavirus RNA is not inhibited under these conditions (except for hepatitis A virus [HAV] RNA) (3).Picornavirus RNAs are uncapped and the initiation of translation on picornavirus RNA occurs up to 1,300 nucleotides (nt) away from the 5Ј terminus. This process is directed by an internal ribosome entry site (IRES). The picornavirus IRES elements are generally about 450 nt in length (2). Two major classes of picornavirus IRES element have been defined. The entero-and rhinovirus IRES elements constitute one class, whereas the cardio-and aphthovirus IRESs (including the encephalomyocarditis virus [EMCV] and FMDV elements) make up a second group of elements. The HAV IRES (see reference 6) has different characteristics from these two major groups; for example, it requires the int...

Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy. However, potentially lethal toxicity associated with systemic administration of IL-12 precludes its clinical application. Here we redesign the molecule in such a way that its anti-tumor efficacy is not compromised, but toxic effects are eliminated. Deletion of the N-terminal signal peptide of IL-12 can effect such a change by preventing IL-12 secretion from cells. We use a newly designed tumor-targeted oncolytic adenovirus (Ad-TD) to deliver non-secreting (ns) IL-12 to tumor cells and examine the therapeutic and toxic effects in Syrian hamster models of pancreatic cancer (PaCa). Strikingly, intraperitoneal delivery of Ad-TD-nsIL-12 significantly enhanced survival of animals with orthotopic PaCa and cured peritoneally disseminated PaCa with no toxic side effects, in contrast to the treatment with Ad-TD expressing unmodified IL-12. These findings offer renewed hope for development of IL-12-based treatments for cancer.

dVaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. Since the eradication of smallpox, vaccinia virus (VACV) has been developed as a vector for vaccines against infectious diseases and immunotherapies for cancer (1-4), including oncolytic virotherapies (5-8). The renewed interest in VACV has driven a number of vaccine and therapeutic candidates to clinical trials, showing especially encouraging results for cancer treatment (5, 7). To improve VACV as a vector for vaccine or cancer therapy, a flexible system is required to delete viral genes or arm the VACV with therapeutic genes. Such a system would also expedite discoveries in cell biology, such as dissection of the signaling pathways used by VACV for its actin-based motility (9). Several strategies have been developed to construct VACV vectors (10-12); the current method for modification of VACV is based on homologous recombination in mammalian cells, but only 1% to 5% of the recombinant plaques contain the inserted DNA (13).The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system is a natural microbial immune mechanism against invading viruses and other genetic elements (14-16). The CRISPR-Cas9 system, consisting of the RNA-guided Cas9 endonuclease (from Streptococcus pyogenes), a single guide RNA (sgRNA), and the trans-activating CRISPR RNA (tracrRNA), has been adapted for genome editing in eukaryotic cells (17,18). The system has been used successfully for efficient generation of genetically modified cells and animal models (17,(19)(20)(21). Recently, the genomes of adenovirus and type I herpes simplex virus were edited using the gRNA-guided Cas9 system (22). We hypothesized that the CRISPR-Cas9 system could specifically generate doublestrand breaks (DSBs) in the target DNA sites of VACV, increasing the efficiency of editing VACV genomes and constructing new VACV vectors expressing therapeutic genes.Given that VACV replicates in the cytoplasm of infected cells, the Cas9 gene without the nuclear localization signal (NLS) was cloned into the pST1374 vector (Fig. 1A). The expression of Cas9 was confirmed (Fig. 1A). Given the important role of the N1L gene in virulence and in regulating the host immune response to VACV (23-25), we chose this as an example to validate the application of CRISPR-Cas9 for editing VACV. Two individual sgRNAs targeting the VACV N1L (LO24) gene were cloned into this guide RNA (gRNA) cloning vector (Fig. 1B) and designated gRNA N1 and N2. To prove that the CRISPR-Cas9 system could improve the efficiency of constructing a VACV vector expressing a therapeuti...