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The effects of interferon gamma (IFN gamma) on early steps of herpes simplex virus (Type 1; HSV-1) replication in primary cultures of splenic mouse macrophages were analyzed and compared to IFN alpha/beta. Pretreatment of macrophages with recombinant murine IFN gamma led to a dose-dependent reduction in the yield of progeny virus. Inhibition of protein synthesis was observed for HSV-1 alpha, beta and gamma-proteins. Expression of the "immediate early" (IE) gene IE3 (ICP4) was investigated in detail. Steady-state level of the RNA and transcriptional activity of the gene in IFN gamma-treated cells were comparable to control-infected cells except for a delay in their kinetics. This is in contrast to IFN alpha/beta, which leads to a stable decrease in IE3 transcripts. Since IFN gamma causes a stable decrease in the IE3 gene product ICP4, our data suggest a translational inhibition of HSV-1 IE gene expression in IFN gamma-treated macrophages. Thus, IFN gamma and IFN alpha/beta inhibit HSV-1 replication by different mechanisms which may lead to a synergistic enhancement of inhibition after combined treatment.

There is increasing evidence for the therapeutic effectiveness of Interferon-alpha (IFN-alpha) in malignant diseases. However, the antitumor mechanisms of IFN-alpha are not known. Using two examples, hairy Cell leukemia (HCL) and renal cell cancer (RCC), it is shown that the requirements for successful IFN-alpha therapy of HCL and RCC are different. In HCL low doses of IFN-alpha are sufficient to treat the disease. The reduction of hairy cells in peripheral blood is detectable within the first week of treatment. The endogenous IFN-alpha production in these patients is impaired as demonstrated by the lack of IFN-alpha induction and by low levels of 2-5 oligoadenylate synthetase in peripheral blood mononuclear cells. A possible reason for deficient endogenous IFN-alpha production is the lack of monocytes in HCL patients. It is likely that therapy with low doses of IFN-alpha substitutes for the endogenous IFN-alpha deficiency. In RCC comparatively high doses of IFN-alpha are necessary for a clinical response. There may be differences between the effectiveness of natural and recombinant alpha interferons. High doses given within a week seem to be more important than high single doses, which therefore suggests the need of daily treatment. Responses of RCC to IFN-alpha therapy are usually seen several months after the beginning of therapy. These differences in the effectiveness of IFN-alpha therapy for HCL and RCC suggest that IFN-alpha acts differently in the treatment of each disease.

Pretreatment of primary cultures of splenic mouse macrophages with murine IFN-alpha/beta leads to a stable inhibition of herpes simplex virus type 1. Analysis of viral DNA, RNA, and protein synthesis identifies expression of "immediate-early" genes as a major target of IFN-mediated inhibition. Determination of viral DNA in the nuclei early after infection, i.e., before onset of DNA replication, suggests that virus uptake, transport to the nucleus, and DNA stability are not decreased in IFN-pretreated macrophages. Nuclear runoff transcription analysis shows a significant reduction of immediate-early transcription rates following IFN treatment. End-specific probes for the ICP4 gene locate the inhibition to the onset of transcription. Northern blot analysis reveals a decrease in ICP4 transcripts in accordance with the observed inhibition of transcription. The observed inhibition of early gene transcription may be a consequence of decreased immediate-early gene expression.

Tumor necrosis factor (TNF) induces an antiviral state in various cell lines. This antiviral state is quite similar to that established by interferon (IFN), e.g., TNF treatment of HEp-2 cells induces 2',5'-oligoadenylate synthetase activity. Both antiviral activity and synthetase induction are greatly reduced when TNF treatment occurs in the presence of a beta interferon subtype 1 (IFN-beta 1)-neutralizing antiserum. However, no one has yet directly demonstrated IFN-beta 1 induction, either as an antiviral activity in supernatants from TNF-treated cells or as IFN-specific mRNA by Northern (RNA) blot analysis. We have adopted a recently described in vitro DNA amplification protocol for the detection of specific RNAs. By applying this method to RNA from HEp-2 cells, we could demonstrate increased levels of IFN-beta 1-specific transcripts after TNF treatment. Dose response and kinetics of IFN-beta 1 induction coincided with the TNF-induced antiviral state. Nuclear run-on analysis showed enhanced transcriptional activity of the IFN-beta 1 gene in TNF-treated cells. Our data substantiate a role of IFN-beta 1 as mediator of the biological activity of TNF in HEp-2 cells.

A murine cosmid clone harboring the single-copy interferon-beta 1 (IFN-beta 1) gene and extended flanking sequences was isolated. The functional IFN-beta 1 promoter is contained within a 170-bp DNA fragment located 5' of the coding sequence. This was shown by fusion of this fragment to a heterologous reporter gene and transient as well as stable expression in mouse L and monkey CV-1 cells. With the help of these functional assays, it could be demonstrated that the 5'-flanking sequences are the target for the typical regulatory action of common type I IFN activators. DNA sequencing reveals a considerable homology to the human IFN-beta 1 promoter within the 280 upstream base pairs. The homology is particularly pronounced within the DNA region containing the virus responsive element (VRE). This phenomenon may explain the similarity of both genes in the mode of regulation. The mouse promoter fragment compared with the human equivalent was shown to be several times more efficient in transcriptional activation in murine and primate cells.