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Protein-primed RNA synthesis by purified poliovirus RNA polymerase
A V Paul1, J H van Boom, D Filippov
1Department of Molecular Genetics and Microbiology, School of Medicine, Health Science Center, State University of New York, Stony Brook 11794-5222, USA. apaul@asterix.bio.sunysb.edu
Abstract:
A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus genomic RNA. 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 conundrum. 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.
Insights
Poliovirus RNA uses a protein called VPg, not a cap, for translation initiation. This VPg protein is essential for initiating poliovirus RNA transcription, linking RNA polymerase activity to viral genome replication.
Area of Science:
- Virology
- Molecular Biology
- RNA Replication
Background:
- Poliovirus genomic RNA is linked to a protein, VPg, at its 5' end.
- Unlike most eukaryotic mRNAs, poliovirus mRNA lacks a 5' cap structure.
- The function of VPg and the mechanism of translation for uncapped viral RNA were previously unclear.
Purpose of the Study:
- To elucidate the function of the VPg protein in poliovirus replication.
- To understand the role of VPg in viral RNA transcription and translation.
Main Methods:
- Investigated the linkage of VPg to the 5'-terminal uridylic acid of poliovirus RNA.
- Demonstrated VPg uridylylation by the poliovirus RNA-dependent RNA polymerase (3Dpol).
- Showed that uridylylated VPg primes polyadenylate RNA synthesis by 3Dpol.
Main Results:
- VPg is covalently attached to the 5'-terminal uridylic acid of poliovirus genomic RNA.
- The poliovirus RNA polymerase (3Dpol) can uridylylate VPg.
- Uridylylated VPg serves as a primer for the synthesis of poly(U) RNA by 3Dpol.
Conclusions:
- VPg is essential for the initiation of poliovirus RNA transcription.
- The VPg protein plays a critical role in viral genome replication by priming RNA synthesis.
- This mechanism explains how poliovirus initiates transcription from its polyadenylated 3' end.