Inhibition of Chikungunya Virus Replication by Harringtonine, a Novel Antiviral That Suppresses Viral Protein Expression

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Reunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting >= 70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC50] = 0.24 mu M) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.