Helmut Jacobsen

Interferon treatment inhibits onset of herpes simplex virus immediate-early transcription

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.

Beta interferon subtype 1 induction by tumor necrosis factor.

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.

Antiviral activity of tumour necrosis factor. Synergism with interferons and induction of oligo-2',5'-adenylate synthetase

Tumour necrosis factor (TNF) induces antiviral activity in HEp-2 cells. Virus yield reduction assays with vesicular stomatitis virus as challenging virus demonstrated that the antiviral state was more pronounced in confluent cultures under low serum conditions. A significant enhancement of the antiviral state was obtained by combining TNF with low concentrations of either interferon (IFN)-beta 1 or IFN-gamma. The reduction in virus yield was significantly higher than that expected from summation of the independent antiviral activities of either substance alone, i.e. TNF and IFN acted synergistically as antiviral agents. Synergism of TNF with IFN-beta or IFN-gamma appeared to be mediated by different pathways, since different requirements for pretreatment and different effects on oligo-2,5-adenylate synthetase (2-5AS) induction were observed. Induction of 2-5AS by TNF could be shown to be an indirect event that was sensitive to an antiserum against natural IFN-beta 1.

ANTIVIRAL ACTIVITY OF PROSTAGLANDIN-A ON ENCEPHALOMYOCARDITIS VIRUS-INFECTED CELLS - A UNIQUE EFFECT UNRELATED TO INTERFERON

Antiviral effects of prostaglandins of the A series (PGAs) on Sendai, vaccinia and vesicular stomatitis viruses have previously been reported and a relationship between the antiviral actions of PGAs and interferons has been suggested. We have investigated the antiviral activity of PGAs on encephalomyocarditis (EMC) virus. Using single-cycle assays of virus replication our results indicate that PGAs only inhibit when present in the culture medium after the cells are infected, and that they are most effective during incubation periods including from 3 to 5 h post-infection. Furthermore, viral RNA synthesis is blocked in infected cells treated with PGA and, as a result, viral antigens are greatly reduced in the cytoplasm of the cells 5 h post-infection. Since the antiviral effect of PGAs is unperturbed by actinomycin D, when cellular RNA synthesis is greatly reduced, it appears unlikely that induction of new cellular proteins is the reason for the antiviral activity of PGAs. In separate experiments we were unable to demonstrate directly the induction of interferon, or of the two dsRNA-dependent enzymes, 2,5-oligoadenylate synthetase and protein kinase, which are greatly increased in interferon-treated cells. Thus, we conclude that the antiviral activity of PGAs is unrelated to the antiviral action of interferons and involves a unique mechanism independent of cellular protein synthesis.

Inhibition of replication of herpes simplex virus in mouse macrophages by interferons.

The replication of herpes simplex virus (HSV) type 1 in macrophages grown from spleen cells of mouse strains susceptible to HSV infection in vivo was very sensitive to interferon (IFN). Different types of mouse IFN (alpha, beta, gamma) exhibited similar antiviral activities. However, treatment of cells with IFN-gamma in combination with IFN-alpha or IFN-beta resulted in a synergistic inhibition of virus growth. As shown by assaying HSV DNA polymerase, IFN inhibited expression of the beta-genes. Inhibition of enzyme induction correlated well with the reduction of viral yield. Induction of HSV DNA polymerase was delayed by IFN in a dose-dependent manner. These results show that IFN inhibits HSV replication at an early step prior to or during the synthesis of beta-proteins.

Different effects of IFNγ and IFNα/β on “immediate early” gene expression of HSV-1

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.

SELECTION AND CHARACTERIZATION OF INTERFERON-SENSITIVE CELLS DERIVED FROM AN INTERFERON-RESISTANT NIH 3T3 LINE

We have developed a selection protocol to isolate interferon (IFN)-sensitive subclones directly from an IFN-resistant cell population. The protocol uses encephalomyocarditis virus (EMCV) as a selection agent in combination with pretreatment with low doses of IFN and subsequent growth in the presence of virus-neutralizing antiserum. We have applied this protocol to the partially IFN-resistant NIH 3T3 clone 1 line and have obtained a number of IFN-sensitive subclones. Sensitivity to IFN was restricted to protection against EMCV. Replication of vesicular stomatitis virus as well as cell growth were resistant to IFN treatment as in the original clone 1 line. We have compared levels of 2,5-oligoadenylate (2-5A) synthetase, dsRNA-activated protein kinase and 2-5A-dependent RNase in some IFN-sensitive subclones and found no difference from the resistant clone 1 cells. Markedly decreased levels of 2-5A-dependent RNase and thus a defective 2-5A pathway have been implicated as a possible cause for the partial resistance of clone 1 cells to IFN. Since the selected IFN-sensitive subclones are of the same phenotype in this respect as the clone 1 line we suggest that inhibition of EMCV in these lines occurs through a mechanism independent of the 2-5A system.