Imiquimod suppresses propagation of herpes simplex virus 1 by upregulation of cystatin A via the adenosine receptor A1 pathway

Imiquimod is recognized as an agonist for Toll-like Receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent Antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible Antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine Protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of Cystatin A was suggested to be mediated by antagonizing Adenosine Receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with Adenosine Receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the Cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for Adenosine Receptor A(1) and induced a host Antiviral protein, Cystatin A. The process occurred independently of TLR7 and type I IFNs.