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Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site.

ABSTRACT: Protein priming is an important mechanism that many viruses use to initiate genomic DNA or RNA synthesis. Birnaviruses are the only double-stranded (ds) RNA viruses that use protein priming. The viral-encoded VP1 of birnavirus functions as both a polymerase and a protein primer and is able to undergo self-guanylylation to acquire a covalently linked rGMP. By employing biochemical assays using recombinant proteins, we have shown that VP1 self-guanylylation does not require an RNA template but is dependent on divalent metal ions. VP1 reacts with all four types of rNTPs but strongly prefers rGTP. Unexpectedly, two fatal polymerase mutants D402A and E421Y, each having an essential catalytic residue mutated and unable to catalyze RNA synthesis, remain active in self-guanylylation. The guanylylation site was further mapped to the VP1 N-terminal domain. Our results support a mechanism in which VP1 self-guanylylation is catalyzed by a novel active site different from the polymerase active site.

SUBMITTER: Pan J

PROVIDER: S-EPMC2783171 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site.

Pan Junhua J Lin Li L Tao Yizhi Jane YJ

Virology 20091004 1

Protein priming is an important mechanism that many viruses use to initiate genomic DNA or RNA synthesis. Birnaviruses are the only double-stranded (ds) RNA viruses that use protein priming. The viral-encoded VP1 of birnavirus functions as both a polymerase and a protein primer and is able to undergo self-guanylylation to acquire a covalently linked rGMP. By employing biochemical assays using recombinant proteins, we have shown that VP1 self-guanylylation does not require an RNA template but is ...[more]

PMID: 19801157

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Project description:Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We report that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV-infected target organs and different cells but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C terminus. The K751 ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C terminus of VP1 enhanced its polymerase activity. The K751-to-R mutation of VP1 protein did not block the rescue of IBDV but decreased the replication ability of IBDV. Our data demonstrate that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.IMPORTANCE Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression, and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C terminus of VP1 is the target site for ubiquitin, and its ubiquitination is independent of VP1's interaction with VP3 and eukaryotic initiation factor 4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We conclude that VP1 is the ubiquitin-modified protein and reveal the mechanism by which VP1 promotes avibirnavirus replication.

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Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site.

Publications

Self-guanylylation of birnavirus VP1 does not require an intact polymerase activity site.

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