Shiftless (SHFL) is a host antiviral factor that inhibits the viral -1 programmed ribosomal frameshifting (-1PRF). How the antiviral activity of SHFL is regulated is not known. Here, we report that the phosphorylation of SHFL by the casein kinase 1 delta (CK1δ) and epsilon (CK1ε) regulates its antiviral activity. The casein kinases were identified as SHFL-interacting proteins. Downregulation of the expression of the endogenous casein kinases or pharmacological inhibition of kinase activity significantly impaired the activity of SHFL to inhibit HIV-1 -1PRF and viral production. The residues T250 and T253 were identified as critical phosphorylation sites; substitution of either of the residues with alanine, resulting in the mutants SHFL-T250A or SHFL-T253A, abolished the antiviral activity of SHFL against HIV-1. The T250A mutation did not affect SHFL interaction with target RNA or the ribosomal proteins uL5 and eS31 but significantly reduced its interaction with IGF2BP proteins. Furthermore, SHFL-T250A displayed reduced association with the actively translating polysomes. These results are consistent with the notion that SHFL interaction with ribosomes is critical for its activity to inhibit -1PRF. Taken together, our results indicate that phosphorylation of SHFL by CK1δ/ε is required for its antiviral activity.IMPORTANCEInnate immune responses and antiviral defense mechanisms are regulated through a variety of mechanisms, among which post-translational modifications, especially phosphorylation, play important roles. Deciphering the regulatory mechanisms helps understand the innate immune responses more comprehensively. SHFL is an interferon-stimulated host antiviral factor that inhibits the replication of multiple viruses. The present study revealed that phosphorylation of SHFL by casein kinase 1 δ/ε is required for its antiviral activity against HIV-1. These results provide an additional example for how immune responses are regulated. Furthermore, given that SHFL inhibits -1PRF, the present studies provide tools for further exploring the mechanisms of -1PRF.
Keywords: -1 programmed ribosomal frameshifting; Shiftless; antiviral factor; casein kinase 1; phosphorylation.