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ABSTRACTThe induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-a/ß or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-ß induction pathway have been analyzed. The transcription factor NF-?B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-?B by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

ABSTRACTHuman herpesvirus 8 (HHV-8) is the probable viral etiologic agent for Kaposi’s sarcoma. The HHV-8 genome encodes viral interferon regulatory factor (vIRF), a gene product that has homology to the IRF family of transcription factors. We demonstrate that vIRF inhibits responses to type I and type II interferons and blocks IRF-1-mediated transcription. vIRF does not compete with IRF-1 for binding to DNA or complex directly with IRF-1. The ability of vIRF to block IRF-1-mediated transcription is independent of the DNA binding domains of both vIRF and IRF-1. These data suggest that vIRF may contribute to viral pathogenesis and cellular transformation by interfering with interferon- and IRF-1-mediated gene expression through a novel mechanism.

Apoptosis involves the activation of a cascade of interleukin-1β converting enzyme-like proteases (caspases), a group of cysteine proteases related to the prototype interleukin-1β-converting enzyme (caspase-1). These proteases cleave specific intracellular targets such as poly(ADP-ribose) polymerase, DNA-dependent protein kinase, and nuclear lamins. We show here that apoptosis can be induced by double-stranded RNA. The induction of apoptosis by double-stranded RNA and other agents leads to the cleavage by a caspase of the signal transducer and activator of transcription factor, STAT1 which is pivotal in the signal transduction pathways of the interferons and many other cytokines and growth factors. The product of this cleavage is no longer able to mediate interferon-activated signal transduction and the cleavage event may play a role in regulating the apoptosis response itself.

Treatment of cells with virus or synthetic double-stranded RNA (dsRNA) leads to the transient transcriptional activation of the β-interferon gene. Genetic analysis has revealed that the 5' regulatory sequence responsible for this induction contains multiple positive and negative elements. One of these, Positive Regulatory Domain I (PRD I), has been shown to bind the positively-acting transcription factor IRF-1. In this study we show that this element is inducible under conditions where IRF-1 cannot be detected, suggesting that additional cellular factors are involved in the Induction process. To investigate the existence of such factors we have analysed the range and properties of PRD l-binding activities present In HeLa cells. In addition to the repressor protein IRF-2, several novel factors can bind to PRD I in uninduced cells: two of these have properties consistent with a role in negative regulation; levels of two others increase upon priming, and may be alternative candidates for activators. Upon induction we also observe a novel factor whose appearance does not depend upon de novo protein synthesis, and which appears to be a truncated form of IRF-2. The potential involvement of these factors in regulating the β-interferon gene is discussed.

We describe a novel procedure for the construction of deletion mutants. Existing exonuclease-based protocols are efficient at producing randomly positioned deletions over large regions of DNA, but are of limited use in targetted mutagenesis due to their inherent sequence-specificity. We have taken advantage of the Exonuclease Ill-resistant nature of α-thlo-dNTPs, incorporated into the target DNA template by a primer extension reaction, to generate base-specific α-thio-dNTP terminated products. Following removal of the 5′ overhanging strands, the products can be cloned to generate a nested set of deletions with single base-pair increments. We demonstrate the utility of this technique by isolating multiple deletions over a 40bp region of the human β-interferon promoter.

We have examined the behaviour of the β-interferon promoter in mouse F9 embryonal carcinoma cells. In undifferentiated cells, the β-interferon promoter is not responsive to dsRNA or to Sendai virus. In cells stimulated to differentiate into parietal endoderm by treatment with retinoic acid, the β-interferon promoter responds to both inducers, but only Sendai virus can activate the transcription factor NF-ϰB previously thought to be essential for β-interferon induction. Differentiated F9 cells therefore present an unprecedented situation in which induction of the β- interferon gene does not require NF-ϰB. In addition to these differences, induction by dsRNA, but not by Sendai virus, is significantly enhanced by a pretreatment with interferon (priming). These observations suggest that paramyxo-viruses can participate in β- interferon induction in a manner that is distinct from a simple generator of dsRNA. Analysis of the promoter requirements for induction in differentiated F9 cells suggests that induction is brought about by a novel mechanism using the currently identified regulatory domains.

We have studied the role of protein turnover in the induction of NF-ϰB DNA binding activity. Treatment of cells with tumour necrosis factor (TNF), doublestranded RNA (dsRNA), or phorbol esters is shown to be associated with an increase in the rate of p105 to p50 processing, and the loss of immunologically detectable MAD3⊟;lxBα. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (Na-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (Na-p-Tosyl-LPhenylalanine Chloromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-ϰB activation. These data suggest a model for NF-ϰB activation in which phosphorylation destabilises the NF-ϰxB−MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.