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. 2001 Mar 1;20(5):1153-63.
doi: 10.1093/emboj/20.5.1153.

Oligomeric structures of poliovirus polymerase are important for function

S D Hobson  1 , E S Rosenblum, O C Richards, K Richmond, K Kirkegaard, S C Schultz
Affiliations

Oligomeric structures of poliovirus polymerase are important for function

S D Hobson et al. EMBO J. .

Abstract

Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

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Figures

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Fig. 1. Poliovirus polymerase–polymerase interactions as observed in the structure. (A) Ribbon representation of the structure of poliovirus polymerase. The thumb, fingers and palm subdomains are labeled. The conserved A and C sequence and structural motifs are colored red and yellow, respectively. The polypeptide regions involved in interactions at Interface I are blue and the N-terminal regions involved in interactions at Interface II are light gray. (B) Four polymerase molecules as packed within the crystals. Interfaces I and II are labeled. (C) Stereo view of the polymerase–polymerase interactions at Interface I. Side chain carbon atoms of residues in the thumb are shown in blue and those from the back of the palm are shown in yellow, oxygen atoms are colored red and nitrogen atoms blue. (D) Polymerase–polymerase interactions at Interface II. The N-terminal polypeptide regions are in light gray and the thumb subdomain is in dark gray. The three hydrophobic residues of the N-terminal strand that wedge into the hydrophobic core of the thumb subdomain are labeled. The Ca2+ site is shown in yellow and labeled.
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Fig. 2. Binding and extension analysis of wild-type and Interface I mutant polymerases. (A) Sequences of the self-complementary 0.20, 5.20 and 0.20.5 RNAs shown as duplexes. (B) Binding curves for wild-type polymerase with the annealed and single-stranded 0.20, 5.20 and 0.20.5 RNAs. (C) Binding curves for wild-type and Interface I mutant polymerases with annealed duplex 0.20 RNA. (D) Binding curves for wild-type and Interface I mutant polymerases with annealed duplex 5.20 RNA. (E) Substrate utilization by wild-type and Interface I mutant polymerases as measured by [α-32P]UMP incorporation into oligo(dT16)-primed poly(A) template. The values are normalized versus wild-type incorporation. (F) Native polyacrylamide gel analysis of extension reactions for wild-type and Interface I mutant polymerases using the 5.20 RNA substrate. The lower band is unelongated RNA, the middle band results from RNA that has been elongated on only one end and the upper band contains RNA that has been elongated on both ends, as indicated by the schemes to the right of the gel.
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Fig. 3. (A) Plot of specific activity versus concentration of wild-type and Interface I mutant polymerases. (B) Plot of specific activity versus concentration of wild-type polymerase in the presence and absence of 60 µM Zn2+.
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Fig. 4. Plaque assays from transfected cDNAs of wild type, L342A, D349R and mock transfection into KJT7 cells at 32.5, 37 and 39.5°C. Transfection of cDNAs that contained the R455D, R456D, L446A, L446A:R455D and V33A:F34A mutations did not give rise to viable virus.
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Fig. 5. Model of a poliovirus polymerase–dsRNA complex based on the structure of HIV-1 RT complexed to dsDNA (Huang et al., 1998).
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Fig. 6. (A) Interactions between the N-terminal strand (light gray) and the thumb subdomain of poliovirus polymerase. The positions of mutations introduced at Interface II are labeled and shown in yellow, modeled as the mutated amino acid. (B) Binding curves for wild-type, D6 and D65 mutant polymerases with the 5.20 RNA substrate. (C) Incorporation of [α-32P]UMP into oligo(dT16)-primed poly(A) template by wild-type and Interface II mutant polymerases. The values are normalized versus wild-type incorporation. (D) Intra- and intermolecular distances between residues 35 and 69 in crystals of poliovirus polymerase. (E) Non-reducing SDS–PAGE of the disulfide cross-linking reactions with wild-type and cysteine mutant polymerases. Individual lanes are as labeled. The reactions in lanes 13 and 14 were carried out for only 6 h because of extensive formation of larger cross-linked species for the A29C:I441C double mutant.
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Fig. 7. Structural representations of poliovirus polymerase oligomers. (A) Stereo image of two Interface I fibers interacting via Interface II. Each Interface I fiber contains four polymerase molecules colored orange and light gray in one strand and brown and dark gray in the second strand. The Interface I interaction surfaces are shown in blue. The N-terminal strands interacting via Interface II are shown in white. Duplex RNA, shown in red, is modeled into each fiber. (B) Stereo image of two Interface II fibers interacting via Interface I. Each Interface II fiber contains five polymerase molecules colored as in (A). The Interface I interaction surfaces are shown in blue and, therefore, illustrate additional sites of potential interactions via Interface I. The thumb subdomain of each polymerase molecule is indicated in stereo with a T. Duplex RNA, shown in red, has been modeled into the bottom two molecules of each Interface II fiber.
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References

    1. Ago H., Adachi,T., Yoshida,A., Yamamoto,M., Habuka,N., Yatsunami,K. and Miyano,M. (1999) Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Struct. Fold. Des., 7, 1417–1426. - PubMed
    1. Arnold J.J. and Cameron,C.E. (1999) Poliovirus RNA-dependent RNA polymerase (3Dpol) is sufficient for template switching in vitro. J. Biol. Chem., 274, 2706–2716. - PubMed
    1. Arnold J.J. and Cameron,C.E. (2000) Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub). J. Biol. Chem., 275, 5329–5336. - PubMed
    1. Baltimore D., Eggers,H.J., Franklin,R.M., and Tamm,I. (1963) Poliovirus induced RNA polymerase and the effects of virus-specific inhibitors on its production. Proc. Natl Acad. Sci. USA, 49, 843–849. - PMC - PubMed
    1. Beckman M.T.L. and Kirkegaard,K. (1998) Site size of cooperative RNA binding by poliovirus RNA-dependent RNA polymerase. J. Biol. Chem., 273, 6724–6730. - PubMed

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