Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis

Xu, Haixia; Si, Weiduo; Dobos, Peter

doi:10.1016/j.virol.2004.01.024

Cited by 26 publications

(33 citation statements)

References 39 publications

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“…Although the protein primer and the polymerase are usually two separate molecular moieties, the protein priming function in birnaviruses is carried out by the RNA-dependent RNA polymerase VP1 itself. It has been shown that both virion-associated and recombinant birnavirus VP1 have the self-guanylylation activity (12,17,22,26). VP1 selfguanylylation, which does not require viral RNA template, produces two products, 17,22,26).…”

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confidence: 99%

“…It has been shown that both virion-associated and recombinant birnavirus VP1 have the self-guanylylation activity (12,17,22,26). VP1 selfguanylylation, which does not require viral RNA template, produces two products, 17,22,26). It has been proposed that the pGpG moiety in VP1-pGpG binds to the conserved pCpC sequence at the terminal end of the viral RNA template during the initiation of nucleotide polymerization (17, 27).…”

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confidence: 99%

“…Intact birnavirus particles are transcription-competent, and they are capable of producing viral messengers in a semiconservative fashion (16). In vitro, isolated VP1 proteins have been shown to possess a replicase activity using viral (ϩ)RNA templates (17). Three-dimensional structures of birnaviruses show that the ordered capsid shell is made of VP2, whereas the viral genome, polymerase VP1, and VP3, another major capsid protein, are internally disordered (18)(19)(20).…”

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confidence: 99%

“…VP1 selfguanylylation, which does not require viral RNA template, produces two products, VP1-pG and VP1-pGpG (12,17,22,26). It has been proposed that the pGpG moiety in VP1-pGpG binds to the conserved pCpC sequence at the terminal end of the viral RNA template during the initiation of nucleotide polymerization (17,27). Consequently, the 5Ј ends of both genomic and messenger RNAs of birnaviruses are covalently linked to a VP1 molecule.…”

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confidence: 99%

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The structure of a birnavirus polymerase reveals a distinct active site topology

Pan

Vakharia

Tao

2007

Proc. Natl. Acad. Sci. U.S.A.

108

127

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Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 Å structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN-to -GDD-results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN-mutation to control their growth rate.evolution ͉ virus ͉ RdRp S ingle-subunit polymerases, including RNA/DNA-dependent RNA/DNA polymerases, are universally encoded in cellular organisms and viruses. Sequence analysis shows that RNAdependent RNA polymerases (RdRps) are a cluster of closely related enzymes that are mostly found in viruses, in which they assume critical functional roles by replicating and transcribing viral genome. The recently reported crystal structures of several RdRps and their functional complexes have provided important insights into the biological functions and catalytic mechanisms of these enzymes (1-10). These structures contain a well conserved core polymerase domain, the shape of which resembles a right hand with fingers, palm, and thumb subdomains. The palm, which is the most conserved region, contains a central, nonvariant, four-stranded ␤-sheet with five recurring catalytic sequence motifs (from A to E).In contrast to these conventional features, birnavirus polymerase VP1 exhibits several unusual characteristics (11,12). Most noticeably, the essential -X(G)DD-sequence, which is often referred to as the polymerase motif C (13, 14), is missing from VP1 (12). This raises the question as to whether VP1 promotes catalysis by the two-metal mechanism like in conventional polymerases, or whether VP1 employs a different mechanism for nucleotidyl transfer. Recent results indicate that birnavirus VP1, as well as the polymerases from some tetraviruses, may belong to a special group of unconventional polymerases with five essential RNA polymerase motifs arranged in the permuted order of C-A-B-D-E (15). In addition, the motif C in birnaviruses may have the sequence -ADN-, resulting in only two aspartate residues in the active site (15).Like polymerases from other dsRNA viruses, birnavirus VP1 catalyzes both replication and transc...

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confidence: 99%

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confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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The structure of a birnavirus polymerase reveals a distinct active site topology

Pan

Vakharia

Tao

2007

Proc. Natl. Acad. Sci. U.S.A.

108

127

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“…VP1, the viral RNA-dependent RNA polymerase (RdRp) encoded by segment B, is present in the virion in two forms, as a free protein and linked to the 5Ј ends of the two genomic segments by covalent binding, the so-called VPg (5,10,36). VP1 has been shown to be guanylated in vitro, and this VP1pGpG complex in turn serves as a primer for RNA synthesis (50).…”

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confidence: 99%

VP3, a Structural Protein of Infectious Pancreatic Necrosis Virus, Interacts with RNA-Dependent RNA Polymerase VP1 and with Double-Stranded RNA

Pedersen¹,

Skjesol²,

Jørgensen³

2007

J Virol

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Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of theBirnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.

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Significance of the genetic relationships deduced from partial nucleotide sequencing of infectious bursal disease virus genome segments A or B

Nouën¹,

Rivallan²,

Toquin³

et al. 2004

Arch Virol

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The rapid genomic characterization of infectious bursal disease virus (IBDV) requires determining which partial nucleotide (nt) sequences derived from IBDV segments A or B would produce phylogenetic information as significant as sequencing the whole corresponding segments. Long nt coding sequences of 27 IBDV segments A (aa 20-991) and 21 segments B (aa 7-stop codon) were retrieved from databanks and used to compute reference phylogenetic trees using Neighbor Joining (NJ) and Parsimony (P): clusters appearing in the NJ and P reference trees with a bootstrap value greater than 80% were considered as significant (Whole Segment Clusters, WSC). The sequences were then cut into overlapping regions. These were used to compute phylogenetic trees which were compared with reference ones. Of the partial sequences, the VP2 gene best represented IBDV segment A (10 out of 13 WSC were conserved), and the 5' two thirds of segment B best represented segment B (5 to 6 conserved WSC out of 6). Implementation of the Plato programme finally demonstrated that the region encoding VP2 variable domain (vVP2, segment A) is the only region of IBDV genome with a significantly different evolution rate, which result is consistent with vVP2 being subjected to a high selection pressure.

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Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis

Cited by 26 publications

References 39 publications

The structure of a birnavirus polymerase reveals a distinct active site topology

The structure of a birnavirus polymerase reveals a distinct active site topology

VP3, a Structural Protein of Infectious Pancreatic Necrosis Virus, Interacts with RNA-Dependent RNA Polymerase VP1 and with Double-Stranded RNA

Significance of the genetic relationships deduced from partial nucleotide sequencing of infectious bursal disease virus genome segments A or B

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