Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Structure, Function, and Mechanism

Craig Ε. Cameron; David W. Gohara; Jamie J. Arnold

doi:10.1128/9781555817916.ch21

Chapter 21 : Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Structure, Function, and Mechanism

Authors: Craig Ε. Cameron¹, David W. Gohara¹, Jamie J. Arnold¹

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Affiliations: 1: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802

Content Type: Monograph

Publication Year: 2002

Category: Viruses and Viral Pathogenesis

Book DOI: 10.1128/9781555817916

Chapter DOI: 10.1128/9781555817916.ch21

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Abstract:

Replication of the poliovirus genome has been studied for many decades by using a variety of molecular, genetic, biochemical, and structural approaches. These studies have uncovered most, if not all, of the virus encoded proteins and RNA sequences/structures required for genome replication. Proteins encoded by the P3 region of the genome, however, are thought to participate more directly in the genome replication process. The fourth and final protein domain of the P3 region of the viral polyprotein is the RNA-dependent RNA polymerase (RdRP) 3Dpol, the core component of the replication machinery. One of the most important contributions to one’s understanding of 3Dpol function was the solution of the crystal structure of 3Dpol by researchers in 1997. This structure provided the first glimpse into the architecture of an RdRP. The preceding discussion highlights the similarity of 3Dpol with other classes of nucleic acid polymerase. While features unique to 3Dpol may exist, for example, the so-called fingertips, this subdomain likely exists in all RdRPs based on the two RdRP structures available to date. In contrast, two potential interaction/oligomerization domains were also observed in the crystal structure of 3Dpol. These interaction surfaces have no structural homologues in any other polymerases for which structural information is available, including the RdRP from hepatitis C virus (HCV). It is clear that the availability of structural information for 3Dpol has shed light on one’s understanding of 3Dpol function.

Citation: Cameron C, Gohara D, Arnold J. 2002. Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Structure, Function, and Mechanism, p 255-267. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi: 10.1128/9781555817916.ch21

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Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Structure, Function, and Mechanism

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/content/10.1128/9781555817916.chap21.fig21-1

FIGURE 1

Substrates employed to study poliovirus polymerase in vitro. (A) Hairpin substrate ( 67 ). (B) Homopolymeric primer/template substrate (dT15/rA30) ( 6 ). (C) Heteropolymeric primer/template substrate ( 8 ). (D) sym/sub ( 7 ). (E) 3Dpol-catalyzed incorporation of AMP into sym/sub ( 7 ).

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10.1128/9781555817916/fig21-1.gif

FIGURE 1

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Tables

Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Structure, Function, and Mechanism

/content/10.1128/9781555817916.chap21.tab21-1

TABLE 1

Conservation of residues among structural features of poliovirus 3Dpol

a Conservation was determined based on sequence alignment of the RNA-dependent RNA polymerases from foot-and-mouth disease virus, encephalomyocarditts virus, mengovirus, hepatitis A virus, poliovirus, echovirus, Coxsackie A virus, Coxsackie Β virus, and human rhinovirus (A. C. Palmenberg, http://www.bocklabs.wisc.edu/acp; A. C. Palmenberg, personal communication).

b Interactions were determined using the program “CONTACT” from the CCP4 suite of programs. Interatomic distance cutoffs were set between 2.8 and 3.3 Å.

c Residues in boldfaced type are from the same side of either interface I or II (i.e., blue molecule in Color Plates 20 and 22).

d Residues that are underlined have been mutated. The mutations and phenotypes are listed in Table 2 .

e Interactions are based on a comparison of the 3Dpol ternary complex model with HIV-1 RT.

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10.1128/9781555817916/tab21-1.gif

TABLE 1

Conservation of residues among structural features of poliovirus 3Dpol

b Interactions were determined using the program “CONTACT” from the CCP4 suite of programs. Interatomic distance cutoffs were set between 2.8 and 3.3 Å.

c Residues in boldfaced type are from the same side of either interface I or II (i.e., blue molecule in Color Plates 20 and 22).

d Residues that are underlined have been mutated. The mutations and phenotypes are listed in Table 2 .

e Interactions are based on a comparison of the 3Dpol ternary complex model with HIV-1 RT.

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TABLE 2

Mutations introduced in poliovirus 3Dpol

a Residues on the surface are in boldfaced type.

b See footnote a, Table 1 .

c Inserted residues are indicated as capitalized letters flanked by residues amino- and carboxy-terminal to the insertion.

d Gohara and Cameron, unpublished.

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10.1128/9781555817916/tab21-2.gif

TABLE 2

Mutations introduced in poliovirus 3Dpol

a Residues on the surface are in boldfaced type.

b See footnote a, Table 1 .

c Inserted residues are indicated as capitalized letters flanked by residues amino- and carboxy-terminal to the insertion.

d Gohara and Cameron, unpublished.

/content/10.1128/9781555817916.chap21.tab21-3

TABLE 3

Kinetic and thermodynamic constants for 3Dpol catalyzed nucleotide incorporation

a Values taken from reference 32 .

b Vafues taken from Arnold and Cameron, unpublished.

c Values taken from reference 21 .

d R, ribavirin, which is l-β-D-ribofuranosyl'l,2,4'triazole-3-carboxamide.

e Values taken from D. Maag and C. E. Cameron, unpublished results.

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10.1128/9781555817916/tab21-3.gif

TABLE 3

Kinetic and thermodynamic constants for 3Dpol catalyzed nucleotide incorporation

a Values taken from reference 32 .

b Vafues taken from Arnold and Cameron, unpublished.

c Values taken from reference 21 .

d R, ribavirin, which is l-β-D-ribofuranosyl'l,2,4'triazole-3-carboxamide.

e Values taken from D. Maag and C. E. Cameron, unpublished results.