AB1431 Sigma-AldrichAnti-Interferon-β Antibody

The ATM kinase plays a critical role in initiating the DNA damage response that is triggered by genotoxic stresses capable of inducing DNA double-strand breaks. Here, we show that ELF4/MEF, a member of the ETS family of transcription factors, contributes to the persistence of γH2AX DNA damage foci and promotes the DNA damage response leading to the induction of apoptosis. Conversely, the absence of ELF4 promotes the faster repair of damaged DNA and more rapid disappearance of γH2AX foci in response to γ-irradiation, leading to a radio-resistant phenotype despite normal ATM phosphorylation. Following γ-irradiation, ATM phosphorylates ELF4, leading to its degradation; a mutant form of ELF4 that cannot be phosphorylated by ATM persists following γ-irradiation, delaying the resolution of γH2AX foci and triggering an excessive DNA damage response. Thus, although ELF4 promotes the phosphorylation of H2AX by ATM, its activity must be dampened by ATM-dependent phosphorylation and degradation to avoid an excessive DNA damage response.

Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI(-)) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI(-) as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI(-)-G3A) was more fusogenic than the parental P/V-CPI(-) mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI(-)-G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI(-) mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI(-)-G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI(-) mutant and the hyperfusogenic P/V-CPI(-)-G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI(-)-G3A mutant was more effective than P/V-CPI(-) at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI(-) were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.

Type I IFNs are crucial components of the innate immune response to viral attack. They are rapidly synthesized and secreted after infection with human cytomegalovirus (CMV) and trigger a signal transduction pathway that involves successive activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1) and STAT2. The activated STATs, together with the IFN regulatory factor 9 protein, form a trimeric transcription complex (IFN-stimulated gene factor 3) that stimulates expression of numerous IFN-responsive genes, many of which exhibit antiviral activity. Here we demonstrate that the viral 72-kDa IE1 protein (IE1-72kDa) confers partial resistance to the antiviral activity of type I IFNs upon CMV. Accordingly, IFN-responsive transcripts accumulate to substantially increased levels after infection with an IE1-deficient mutant as compared with wild-type virus, and ectopic expression of the viral protein in stably transfected cells is sufficient to block their induction. We further show that IE1-72kDa forms a physical complex with STAT1 and STAT2 in nuclei of infected cells and in vitro and prevents association of STAT1, STAT2, and IFN regulatory factor 9 with promoters of IFN-responsive genes in vivo. Our results indicate that the viral protein blocks an intranuclear step after nuclear translocation and before DNA binding of IFN-stimulated gene factor 3, presumably by interfering with the integrity and/or correct subnuclear localization of the protein complex. This study identifies the CMV IE1-72kDa protein as a viral antagonist of the cellular innate immune response, inhibiting IFN-dependent STAT signaling by means of an unprecedented molecular mechanism.

A convenient assay for interferons based on reduction of cytopathic effect was developed. The number of manipulations and the lengths of the various incubation steps were reduced to a minimum. The assay is simple to perform and can be completed within 16 h. Moreover, it can be used with various types of cells and a variety of viruses.