RIG-I and MDA5 responsible for replication detection
Alphaviruses are the group of widely distributed human and animal pathogens. In cultured cells, their replication results in a very rapid inhibition of cellular transcription, which prevents type I IFN induction and activation of cellular antiviral mechanisms. These effects promote efficient spread of infection. However, it is a different matter in vivo. In animal models, alphaviruses do induce IFN, suggesting that cells are capable of detecting their replication. Our new data demonstrate that replication of alphaviruses is sensed by two intracellular receptors, RIG-I and MDA5. No type I IFN response is induced in their absence, and both RIG-I and MDA5 alone are capable of initiating IFN-b expression in response to alphavirus replication. RIG-I and MDA5 promote IFN induction with different kinetics and in concentration-dependent modes. The dependence of the induction of the antiviral response on intracellular RIG-I and MDA5 concentrations provides a plausible explanation for the discrepancy of the data generated from in vivo and in vitro studies, as most of the stable cell lines appear to express RIG-I and MDA5 at very low basal levels.
The previous studies in the lab found that two groups of alphaviruses; the Old World and the New World alphaviruses, use completely different mechanisms to rapidly and completely shut down cellular transcription, and thus, very efficiently prevent development of the cellular antiviral response. As a result, continuous cell lines infected with wild type viruses produce type I IFN very inefficiently if at all. However, replication of alphaviruses is readily sensed in vivo and in some primary cells. Infected animals do produce type I IFN, which plays a prominent inhibitory role in alphavirus infection. Therefore, we decided to investigate the source of this discrepancy between the in vitro and in vivo data and to identify cellular receptors that sense alphaviruses. To achieve this, we developed cell lines that either lack one or both RIG-I and MDA5 receptors, or express them at different levels. Using this approach, we demonstrated efficient IFN induction by wild type alphaviruses in cells expressing high basal levels of RIG-I or MDA5. The most important conclusion drawn from these experiments was that proper interpretation of the roles of pattern recognition receptors in the antiviral response, in different cells, must be supported by analysis of their expression levels.
The kinetics of IFN induction by wt alphaviruses and their mutants were different in RIG-I- and MDA5-expressing cells. The MDA5-expressing cells induced type I IFN earlier than those expressing RIG-I. This suggests that these proteins recognize different viral RNA species, which remain to be identified. The MDA5-expressing cells also produced significantly more IFN-ß than the parental or RIG-I-expressing cells in response to wild type viruses. Thus, development of the antiviral response in cells infected with alphaviruses depends on basal concentrations of RIG-I and MDA5, but MDA5 appears to be more efficient in IFN-ß induction.
Left panel: NIH 3T3 cells release very low amount of INF-b in response to infection with Venezuelan equine encephalitis virus (VEEV). Downregulation of both RIG-I and MDA5 (dKD cells) completely blocks INF-b production. High basal level of MDA5 (KI MDA5 cells) lead to significant increase INF-b production in infected cells, which strongly inhibits virus spread as demonstrated by the reduced plague size in the right panel.
About the author
Ivan Akhrymuk is a graduate student in the Department of Microbiology at the University of Alabama at Birmingham.