Related Experiment Videos
Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated
1Department of Pediatrics, San Francisco General Hospital, University of California 94110, USA.
Abstract:
The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.
Insights
The double-stranded RNA-dependent protein kinase (PKR) is crucial for interferon (IFN) gene induction and antiviral responses. Loss of PKR function impairs viral defense mechanisms and IFN-alpha induction.
Area of Science:
- Immunology
- Molecular Biology
- Virology
Background:
- Interferon (IFN) gene induction by viral infection or double-stranded RNA (dsRNA) signaling pathways are not fully elucidated.
- The dsRNA-dependent protein kinase (PKR) is an interferon-induced enzyme with known roles in protein synthesis regulation, mediating IFN actions, and tumor suppression.
- PKR activation by dsRNA binding suggests its potential role as a signal transducer in IFN expression regulation.
Purpose of the Study:
- To investigate the role of PKR in the regulation of IFN gene induction and antiviral activity.
- To understand how loss of PKR function affects cellular responses to viral infection and various inducers.
Main Methods:
- Generated PKR-deficient U-937 cells using antisense PKR transcript overexpression and dominant-negative PKR mutant gene expression.
- Assessed viral replication permissiveness in PKR-deficient cells compared to controls.
- Evaluated IFN-alpha and IFN-beta gene induction in response to encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate.
- Measured antiviral activity elicited by IFN-alpha and IFN-gamma in PKR-deficient cells.
Main Results:
- PKR-deficient cells exhibited increased permissiveness to viral replication.
- Impaired induction of IFN-alpha and IFN-beta genes was observed in PKR-deficient cells upon stimulation with various inducers.
- While IFN-alpha induction by dsRNA was impaired, IFN-beta induction remained intact in PKR-deficient cells.
- Loss of PKR function led to diminished antiviral activity mediated by IFN-alpha and IFN-gamma.
Conclusions:
- PKR plays a significant role in regulating IFN-alpha and IFN-beta gene induction.
- PKR is implicated in mediating antiviral activities induced by interferons.
- PKR is a key regulator in the cellular defense against viral infections and dsRNA stimuli.