NMR structure of the viral peptide linked to the genome (VPg) of poliovirus

fWe report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

Section: Discussionmentioning

confidence: 74%

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confidence: 99%

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

Leen

Kwok

Birtley

et al. 2013

“…Two of the models, based on mutation and computation studies, assume that VPg and the VPg precursor 3AB bind to the same surface on 3D pol and conclude that VPg enters the polymerase active site from the back of the polymerase molecule (45,46). In contrast, structural evidence from the FMDV 3D pol -VPg complex suggests an alternative arrangement, since the last ordered residues at the carboxyl end of VPg are seen to bind to the front face of 3D pol .…”

Section: Vol 81 2007 Crystal Structure Of Poliovirus 3cd Protein 3591mentioning

confidence: 99%

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

Marcotte

Wass

Gohara

et al. 2007

109

123

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C pro and the viral polymerase 3D pol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3D pol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D pol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D pol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.Poliovirus (PV), a member of the Picornaviridae family of RNA viruses, must simultaneously perform many tasks inside a host cell for efficient and successful viral replication, and only a small number of gene products are responsible for these processes. The virus produces a single polyprotein that is cleaved by virally encoded proteases. Many of the viral proteins, including precursor proteins, play multiple roles in viral replication.The viral protein 3CD is a multifunctional precursor to the poliovirus protease 3Cpro and the RNA-dependent RNA polymerase 3Dpol . The 3CD molecule retains its ability to function as a protease but lacks polymerase activity (20). Its proteolytic functions include cleavages, resulting in the production of structural proteins VP0, VP1, and VP3 and nonstructural proteins 3AB, 3CD, 3C pro , and 3D pol . In many of these functions, 3CD serves as a better protease than 3C pro , suggesting that the 3D region of 3CD contributes to that activity (34).3CD is also a crucial component of the viral replication complex. The 5Ј-terminal region of the poliovirus genome adopts a cloverleaf structure to which 3CD can bind in the presence of the viral protein 3AB or the cellular protein PCBP2 (2, 35, 49). Both forms of this ribonucleoprotein complex appear to play important roles in inducing RNA synthesis (49). The initiation of RNA synthesis is primed by the addition of two uridines to the 22-amino-acid protein primer VPg (3B) at the side-chain hydroxyl of Tyr3. Positive-strand synthesis is thought to use an RNA hairpin structure as its template within the PV genome (37,42). One model suggests that this cisacting replication signal in the 2C-noncoding region of the PV genome, cre(2C), binds to 3CD and forms a mu...

“…The nuclear magnetic resonance structure of PV VPg in solution has shown that it is intrinsically flexible in low-salt buffer (58). A major conformational change was observed in a stabilizing solution, perhaps mimicking induced folding upon binding to 3D pol (58). Concerning the VPg-binding site on 3D pol , two different sites seem to coexist.…”

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confidence: 99%

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

Gruez

Selisko

Roberts

et al. 2008

119

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D pol at this site excludes its uridylation by the carrier 3D pol . We suggest that VPg at this position is either uridylated by another 3D pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.