IFN Signal Transduction Research Articles

Human Cytomegalovirus Inhibits IFN-α-Stimulated Antiviral and Immunoregulatory Responses by Blocking Multiple Levels of IFN-α Signal Transduction

AbstractThe type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-α confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-α in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-α-stimulated responses by disrupting multiple levels of the IFN-α signal transduction pathway. We demonstrate that HCMV inhibits IFN-α-stimulated MHC class I, IFN regulatory factor-1, MxA and 2′,5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-α signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence.

STAT3 complements defects in an interferon-resistant cell line: evidence for an essential role for STAT3 in interferon signaling and biological activities.

STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFNalpha/beta) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFNalpha/beta signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFNalpha/beta. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-kappaB DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-kappaB DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFNalpha/beta.

Interferon-resistant Human Melanoma Cells Are Deficient in ISGF3 Components, STAT1, STAT2, and p48-ISGF3γ

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3gamma, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.

Resistance of melanoma cell lines to interferons correlates with reduction of IFN-induced tyrosine phosphorylation. Induction of the anti-viral state by IFN is prevented by tyrosine kinase inhibitors.

Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN-induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN-resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN.

E1A repression of IL-6-induced gene activation by blocking the assembly of IL-6 response element binding complexes.

Some viral products interfere with host antiviral defense mechanisms. Adenovirus E1A represses IFN signal transduction pathways which induces gene activation and an antiviral state. Both IFN and IL-6 activate Jak/Tyk protein tyrosine kinases and the STAT (signal transducer and activator of transcription) family proteins. We showed that 12S E1A repressed IL-6 signals activating the junB promoter and the two IL-6 response elements (REs), JRE-IL6 and type II IL-6 RE (also called acute phase response element), required for IL-6-induced activation of the junB promoter and the type II acute phase reactant genes, respectively, in hepatocytes. Conserved region 1 of the 12S E1A was responsible for the repression. Target molecules of the repression by E1A appeared to be IL-6-inducible DNA-binding proteins acting on the IL-6 REs. In a rat 3Y1 cell line stably expressing E1A, the levels of IL-6-induced IL-6 RE binding proteins were severely reduced compared with those in a parental 3Y1 cell line. Moreover, we found that the levels of the STAT family proteins including Stat1-alpha (p91), Stat1-beta (p84), Stat2 (p113), and Stat3 were decreased by the stable expression of adenovirus E1A. The E1A-induced reduction in the amount of DNA-binding proteins seemed to be partly responsible for the decreased transcriptional activity of the IL-6 RE-driven gene expression in response to IL-6. This repression mechanism may be applicable to the E1A repression of IFN-gamma-induced gene activation.

Interferon-induced nuclear signalling by Jak protein tyrosine kinases

Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling.