Biochemical and Genetic Studies of the VPg Uridylylation Reaction Catalyzed by the RNA Polymerase of Poliovirus

Paul, Aniko V.; Peters, J.; Mugavero, JoAnn; Jiang, Yin; Boom, Jacques H. van; Wimmer, Eckard

doi:10.1128/jvi.77.2.891-904.2003

Cited by 79 publications

(100 citation statements)

References 58 publications

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“…9). Most interestingly, the PV VPg in vivo and in vitro uridylylation function is also sensitive to the double mutation Lys-9 to Ala-9,Lys-10 to Ala-10 resulting in a nonviable virus phenotype (39). In addition, an NTP binding function was predicted by the same authors for the amino acids Tyr-3, Gly-5, Lys-9, Lys-10, and Arg-17 in PV VPg.…”

Section: Figmentioning

confidence: 89%

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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Section: Figmentioning

confidence: 89%

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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“…Mutational analysis of PV VPg has led to the identification of several key amino acid residues, including Tyr3, Gly5, Lys9, Lys10, and Arg17, which are essential for both uridylylation and viral growth (12,28,30). Exactly how these residues participate with the 3D pol in the uridylylation of VPg remains unknown.…”

Section: Discussionmentioning

confidence: 99%

“…pol -directed in vitro VPg uridylylation have been reported (12,(28)(29)(30)33), we sought to extend these studies by using chimeric PV/HRV16 in order to identify critical residues in VPg necessary for assessing combined uridylylation and in vivo replication potentials.…”

Section: Discussionmentioning

confidence: 99%

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Viability of Poliovirus/Rhinovirus VPg Chimeric Viruses and Identification of an Amino Acid Residue in the VPg Gene Critical for Viral RNA Replication

Cheney¹,

Naim²,

Shim³

et al. 2003

J Virol

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to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D pol in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.Human rhinoviruses (HRV) are members of an extensive genus of the Picornaviridae family that are most frequently associated with viral infections causing symptoms of the common cold. They are small, nonenveloped positive-stranded RNA viruses possessing genomes of approximately 7,200 nucleotides. Like all members of the Picornaviridae, the rhinovirus genome encodes a single polyprotein that is posttranslationally cleaved by viral proteases. In addition to these characteristics, rhinoviruses possess a long 5Ј untranslated region, a shorter 3Ј untranslated region, a poly(A) tail at their 3Ј termini, and a small covalently bound virus-encoded protein termed VPg (3B) at their 5Ј termini (6,11,22,42).Rhinovirus replication is believed to follow the same basic strategy outlined for the prototypic picornavirus poliovirus (PV). This replication strategy involves transcription of the viral genome into a cRNA (negative strand) followed by synthesis of new genomic RNA strands which template from the negative strand. Central to this process is the viral RNA-dependent RNA polymerase (RdRp) 3D pol which is responsible for the synthesis of both strands. VPg has been shown to be essential for protein-primed initiation of both the positive and negative RNA strand replication (42).Perhaps the most well studied rhinovirus serotype is HRV14, and many details of its structure (2, 7, 10, 36, 38) and function have been elucidated (24,25,29,35). However, HRV14 represents a less significant target molecule for antiviral drug discovery since it belongs to the rhinovirus subgroup A, which generally lacks pathogenicity and circulates infrequently in human populations (1, 21). Phylogenetic tree analysis of picornaviruses suggests that HRV14 is more divergent than many other rhinoviruses (1). HRV16, on the other hand, is included in the more pathogenic subgroup B and is more closely related to other circulating rhinoviruses (21). At the present time, no effective therapeutics are available for prevention or treatment of rhinovirus infections. Current development of rhinovirus antivirals has focused on the inhibition of viral capsid function or 3C protease activity (reviewed in reference 40). The RdRp 3D pol of rhinovirus represents another target for the development of antiviral compounds since viral RdRp is a key enzyme in viral RNA replication. The corresponding polymerase from PV has been th...

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“…In addition, RNA viruses, such as Poliovirus, characterized by the presence of a protein covalently linked to the 5Ј end of the viral genome, have been also shown to initiate at internal positions with a further recovery of the terminal sequence by a sliding-back mechanism (18)(19)(20). Bacteriophage Nf belongs to the group of phages that infect Bacillus (21).…”

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confidence: 99%

Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

Longás¹,

Villar²,

Lázaro³

et al. 2008

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

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Bacteriophages 29 and Nf from Bacillus subtilis start replication of their linear genome at both DNA ends by a protein-primed mechanism, by which the DNA polymerase, in a template-instructed reaction, adds 5 -dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP. Mutational analysis of the 3 terminal thymines of the Nf DNA end indicated that initiation of Nf DNA replication is directed by the third thymine on the template, the recovery of the 2 terminal nucleotides mainly occurring by a stepwise sliding-back mechanism. By using chimerical TPs, constructed by swapping the priming domain of the related 29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3 nucleotide used as template during initiation.chimerical terminal protein ͉ polymerase ͉ linear genomes ͉ protein-primed replication ͉ sliding-back M any organisms, such as bacteriophages; animal viruses, such as adenovirus, mitochondrial plasmids, linear chromosomes and plasmids of Streptomyces (1); and more recently virus infecting Archaea, such as halovirus (2, 3), possess replication origins constituted by inverted terminal repetitions (ITR) with a terminal protein (TP) linked to both 5Ј ends of their linear chromosomes (4). In these cases, the location of the 2 replication origins allows both strands to be replicated continuously, without requiring asymmetric complexes of DNA polymerase with other accessory proteins to control the different mechanics of continuous and discontinuous DNA synthesis (5). Additionally, the TP provides the OH Ϫ group of a specific serine, threonine or tyrosine to prime initiation of DNA replication from the very ends of the linear chromosome, the TP remaining covalently linked to the 5Ј-DNA ends (parental TP) (1,4,6).The development of an in vitro replication system with highly purified proteins and DNA from bacteriophage 29 of B. subtilis has allowed to lay the foundations of the so-called proteinprimed mechanism of DNA replication (4, 6). 29 has a linear dsDNA, 19,285 bp long, containing a TP of 31 kDa covalently linked to each 5Ј end [TP-DNA (7)] that, together with a 6-bp inverted terminal repeat (3Ј-TTTCAT-5Ј) (8, 9) form part of a minimal replication origin. Once the replication origins are specifically recognized by a heterodimer formed by the DNA polymerase and a free TP molecule (10, 11), the DNA polymerase catalyzes the formation of a covalent bond between dAMP and the hydroxyl group of Ser 232 of the TP, a reaction directed by the second T at the 3Ј end (12). Then, the TP-dAMP initiation product translocates backwards 1 position to recover the template information corresponding to the first T, the so-called sliding-back mechanism, which requires a terminal repetition of 2 bp (12) and provides a way to prevent mutations at the 29 DNA ends during initiation, since the 3Ј-5Ј exonuclease activity of 29 DNA polymerase cannot proofread the TP-linked nucleotide (13).The sliding-back mechanism, occurring also in the 29-related phage GA-1 (14); S...

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Biochemical and Genetic Studies of the VPg Uridylylation Reaction Catalyzed by the RNA Polymerase of Poliovirus

Cited by 79 publications

References 58 publications

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Viability of Poliovirus/Rhinovirus VPg Chimeric Viruses and Identification of an Amino Acid Residue in the VPg Gene Critical for Viral RNA Replication

Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

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