The Mimivirus L375 protein possesses intrinsic mRNA decapping activity

The giant Mimivirus is a member of a group of viruses termed the nucleocytoplasmic large DNA viruses, which also includes poxviruses, asfarviruses, iridoviruses, and phycodnaviruses. Both poxviruses and asfarviruses produce mRNA decapping enzymes during infection that are thought to accelerate mRNA turnover and promote the shutoff of host protein synthesis. These viral mRNA decapping enzymes contain a Nudix hydrolase motif, a signature sequence characteristic of enzymes that cleave nu cleoside di phosphates linked to another moiety X . Mimivirus encodes two putative Nudix enzymes within its genome, one of which is termed L375. To determine if L375 can mediate mRNA decapping, the L375 protein was expressed in Escherichia coli and purified by affinity chromatography. Incubation of recombinant L375 with a 32 P‐cap‐labeled RNA substrate resulted in cleavage of the mRNA cap, liberating m 7 GDP as a product. Mutations introduced into the L375 Nudix motif abolished mRNA decapping activity, confirming that the L375 protein was responsible for cleavage of the mRNA cap. The decapping activity of L375 was inhibited by certain methylated cap analogs and uncapped RNA, suggesting that L375 recognizes its substrate through interaction with both cap and RNA moieties. Funding for this research was provided by NIH and McDaniel College.