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Protein-primed RNA synthesis by purified poliovirus RNA polymerase

Nature volume 393, pages 280–284 (1998)Cite this article

Abstract

A small protein, VPg, is covalently linked to the 5′ end of the plus-stranded poliovirus genomic RNA1,2,3. Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5′ end by the methylated structure that is common to most eukaryotic mRNAs. These discoveries presented two problems. First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5′ mRNA end, has solved the first conundrum4,5,6. Here we describe the resolution of the second problem. VPg is linked to the genomic RNA through the 5′-terminal uridylic acid of the RNA. We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol. Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U). Initiation of transcription of the poliovirus genome from the polyadenylated 3′ end therefore depends on VPg.

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Figure 1: Uridylylation of VPg and synthesis of VPg–poly(U) in vitro by poliovirus polymerase 3Dpol.
Figure 2: Uridylylation of VPg and synthesis of VPg–poly(U) as a function of UTP concentration.
Figure 3: Identification of the elongated product as VPg–poly(U).
Figure 4: Uridylylation of VPg and synthesis of VPg–poly(U) as a function of 3Dpol concentration.
Figure 5: Uridylylation of VPg and synthesis of VPg–poly(U) by wild-type (WT) and mutant M394T-3Dpol at 30 °C and 36 °C.
Figure 6: The use of wild-type and mutant VPg peptides as substrates for uridylylation by 3Dpol.
Figure 7: Model of the initiation of poliovirus RNA replication.

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Acknowledgements

We thank M. Salas for helpful discussion; J. Lyle and K. Kirkegaard for purified M394T-3Dpol; and A. Wimmer and M. Shepley for critical reading of the manuscript. This work was supported by the National Institute of Allergy and Infectious Diseases of the NIH.

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Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, School of Medicine, Health Science Center, State University of New York, Stony Brook, 117945222, New York, USA

    Aniko V. Paul, Dmitri Filippov & Eckard Wimmer

  2. Leiden University, Gorlaeus Laboratory, PO Box 9502, Leiden, 2300 RA, The Netherlands

    Jacques H. van Boom & Dmitri Filippov

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Correspondence to Aniko V. Paul.

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Paul, A., van Boom, J., Filippov, D. et al. Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393, 280–284 (1998). https://doi.org/10.1038/30529

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