Oncogenic Neu/ErbB-2 Increases Ets, AP-1, and NF-B-dependent Gene Expression, and Inhibiting Ets Activation Blocks Neu-mediated Cellular Transformation

Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers

Different Effect of Proteasome Inhibition on Vesicular Stomatitis Virus and Poliovirus Replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2α, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection

Inhibition of Encephalomyocarditis Virus and Poliovirus Replication by Quinacrine: Implications for the Design and Discovery of Novel Antiviral Drugs▿

The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections

Different activation of eIF2α phosphorylation by VSV and poliovirus infections.

<p>HeLa cells were infected with VSV for 4 h, infected with poliovirus for 4 h, or treated with 1 µM of thapsigargin for 1 hour. Cytoplasmic protein extracts from these and control cells were analyzed with Abs against eIF2α and phosphorylated form of eIF2α (panel A). Same membrane was analyzed with Abs against VSV P- protein and poliovirus capsid proteins (panel B).</p

The effect of MG132 on VSV replication at different time of infection.

<p>(A) Titration of VSV virus from medium of overnight infected HeLa cells. HeLa cells were infected with VSV MOI = 1. The incubation of the cells with virus lasted one hour with additional washing. 5 µM of MG132 were added to cells at time of infection (15 h), 1 h (14 h), 2 h (13 h), and 3 h (12 h) after VSV infection. Results represent average data of two experiments. (B) VSV mRNA synthesis in MG132 treated cells. Northern blot analysis of 10 µg of total RNA from VSV (MOI = 5) infected for 4 h cells treated with MG132 at a time of infection (4 h), or 1 h after infection (3 h). Hybridization with P³² labeled P-protein cDNA probe. RNA loading was standardized by hybridization with GAPDH-gene probe. The hybridization signal of each band was estimated by ImageJ software to calculate percentage of RNA synthesis inhibition. (C) Immunoblotting with anti P-protein Abs. HeLa cells were infected with VSV (MOI = 1) and treated with 5 µM of MG132 as indicated in panel A. Total protein extracts (5 µg) from these cells were purified and tested by Western blotting with anti-P-protein Abs. Keratin 18 was a protein loading control. (D) Immunoprecipitation of S³⁵-methionine labeled P-protein from VSV infected cells. HeLa cells were infected with VSV (MOI = 5) and treated with 5 µM of MG132 at time of infection (4 h), 1 h after infection (3 h), or 2 h after infection (2 h). After 4 h of infection the cells were incubated with S³⁵-methionine/cysteine for 30 min. Cytoplasmic protein extracts were purified and VSV P-protein was precipitated with anti-P-protein Abs. The efficiency of P-protein synthesis was estimated by electrophoresis and autoradiography.</p