Abstract
The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of uncapped RNA significantly reduced L375 decapping activity, suggesting that L375 may recognize its substrate through interaction with the RNA body.
Highlights
The giant Mimivirus, which infects Acanthamoeba species, possesses a 1.2 Mb genome encoding over 900 proteins [1,2,3]
To evaluate if recombinant Mimivirus L375 could decap mRNA, a maltose binding protein (MBP)-L375 fusion protein terminated with C-terminal 10X histidine epitope tag (MBP-L375-HIS10) was synthesized in Escherichia coli and purified through successive amylose and nickel-nitrilotriacetic acid columns
The 32Pcap-labeled RNA substrate was combined with MBP-L375-HIS10 and the products of the reactions were separated on polyethyleneimine (PEI)-cellulose thin layer chromatography (TLC) plates
Summary
The giant Mimivirus, which infects Acanthamoeba species, possesses a 1.2 Mb genome encoding over 900 proteins [1,2,3]. Mimivirus rivals some small bacterial species with respect to physical and genome size, and was the first virus identified to encode some of its own translational components [1,2,3]. Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group that currently includes seven viral families: Poxviridae, Asfarviridae, Iridoviridae, Phycodnaviridae, Mimiviridae, Ascoviridae, and Marseilleviridae [4,5,6,7,8]. The Nudix hydrolase motif is a conserved amino acid sequence found in a diverse group of enzymes that typically cleave nucleoside diphosphates linked to another moiety X [10]. Two NCLDV families, Poxviridae and Asfarviridae, encode Nudix enymes that possess
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