VPg Unlinkase, the Phosphodiesterase That Hydrolyzes the Bond between VPg and Picornavirus RNA: a Minimal Nucleic Moiety of the Substrate

A. Yu. Gulevich, R. A. Yusupova, and Yu. F. Drygin^*

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-3181; E-mail: drygin@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received November 28, 2001; Revision received January 4, 2002

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VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5´-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg-RNA complex were used: [¹²⁵I]Kp-pUpUpGp, [¹²⁵I]Kp-pUp, and [¹²⁵I]Kp-pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg-RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp-pUpUpGp and [¹²⁵I]Kp-RNA with similar velocity, but [¹²⁵I]Kp-pUp was split much slower. Under the same conditions [¹²⁵I]Kp-pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg-pU, [¹²⁵I]VPg-pUp, and [¹²⁵I]VPg-pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg-RNA and [¹²⁵I]VPg-pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.

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KEY WORDS: phosphodiesterase, picornavirus, VPg-RNA complex, radioactive iodination, substrate specificity

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