Picornaviral polymerase fingers domain controls RNA binding and translocation, The

(1)

Targeted Mutations

Figure 5: Targeted mutations to the fingers. Targeted mutations within poliovirus 3Dpol _fingers

domain. Side view of 3Dpol_{down the RNA exit channel showing the three regions targeted for}

mutation within the fingers domain. Individual residues targeted are shown as spheres; kink residues shown in green, gateway residues in red, and sensor residues in yellow.

D47

P48

R49

P40

The Picornaviral Polymerase Fingers Domain

Controls RNA Binding and Translocation

Colleen L. Watkins and Olve B. Peersen

Department of Biochemistry & Molecular Biology

Colorado State University, Fort Collins, CO 80523-1870

Abstract: The picornavirus family of viruses includes poliovirus, the causative agent of paralytic polio and coxsackievirus, which is

responsible for viral-heart-disease. Picornaviruses contain a single-stranded positive-sense RNA genome replicated by 3D

pol

_{, an}

RNA-dependent RNA polymerase (RdRP). Crystal structures of 3D

pol

_{from multiple picornaviruses have shown a conserved polymerase fold}

analogous to a “right hand” composed of fingers, palm and thumb domains. These crystal structures also identified unique regions in the

fingers domain whose function in 3D

pol

_{were unknown. Through biochemical kinetic analysis we have now determined the purpose of these}

regions, and their effects on the catalytic cycle of 3D

pol

_.

Picornavirus Polymerase Structure

Poliovirus Lifecycle

The Sensor Region

This work was funded by NIH Grant R01-AI059130 to OBP.

The Gateway Region

The Kink Region

S115

T114

3Dpol RNA Initiation (min) EC Stability (min) Single Nucleotide kpol(nt/sec) Single Nucleotide KM(µM) Processive Elongation kpol(nt/sec) Processive Elongation KM (µM) Discrimination Factor WT 4 ± 2 130 ± 20 25 ± 1 20 ± 2 20 ± 1 49 ± 4 120 ± 10 Gateway Mutants T114A 10 ± 2 57 ± 10 25 ± 2 31 ± 5 14 ± 1 63 ± 6 100 ± 10 T114L 33 ± 7 5 ± 1 26 ± 3 90 ± 20 10 ± 1 100 ± 10 -T114S 4 ± 1 82 ± 9 26 ± 1 23 ± 1 20 ± 1 52 ± 4 -T114V 18 ± 4 9 ± 2 27 ± 6 150 ± 40 5 ± 1 80 ± 40 130 ± 100 S115A 5 ± 1 90 ± 20 28 ± 2 60 ± 7 11 ± 1 95 ± 6 -S115L 17 ± 3 15 ± 1 19 ± 4 110 ± 30 5 ± 1 30 ± 10 150 ± 20 S115T 5 ± 1 13 ± 1 23 ± 2 61 ± 7 8 ± 1 120 ± 10 130 ± 20 S115V 18 ± 3 25 ± 2 29 ± 1 38 ± 3 16 ± 1 74 ± 4 -3Dpol RNA Initiation (min) EC Stability (min) Single Nucleotide kpol(nt/sec) Single Nucleotide KM(µM) Processive Elongation kpol(nt/sec) Processive Elongation KM (µM) Discrimination Factor WT 4 ± 2 130 ± 20 25 ± 1 20 ± 2 20 ± 1 49 ± 4 120 ± 10 Kink Mutants P40G 13 ± 1 29 ± 2 22 ± 1 37 ± 3 13 ± 1 100 ± 10 170 ± 40 D47N 15 ± 1 19 ± 2 20 ± 1 46 ± 6 15 ± 1 55 ± 7 150 ± 30 P48G 6 ± 1 100 ± 20 27 ± 1 23 ± 3 20 ± 1 52 ± 4 -R49K 19 ± 2 29 ± 4 17 ± 2 40 ± 10 11 ± 1 90 ± 20 140 ± 20

3Dpol _InitiationRNA

(min) EC Stability (min) Single Nucleotide kpol(nt/sec) Single Nucleotide KM(µM) Processive Elongation kpol(nt/sec) Processive Elongation KM(µM) Discrimination Factor WT 2 ± 1 92 ± 16 25 ± 1 34 ± 2 23 ± 1 65 ± 4 90 ± 30 Sensor Mutants E161Q 10 ± 3 84 ± 15 22 ± 1 9 ± 1 17 ± 1 19 ± 2 38 ± 6 E161D 5 ± 1 65 ± 15 24 ± 1 19 ± 2 10 ± 1 32 ± 2 100 ± 10 R174K 3 ± 1 62 ± 12 2 ± 1 69 ± 7 1 ± 1 18 ± 1 12 ± 2 E161Q + R174K 8 ± 1 82 ± 18 5 ± 1 16 ± 2 7 ± 1 53 ± 9 90 ± 10 10 30 50 70 90 110 130 5 15 25 Discrim in atio n Fa ct o r

Maximal Elongation Rate (nt/s)

E161Q R174K _{PV WT} R174K E161D E161Q Primer Metal A Asp 328 Metal B Asp 233

Catalytic Cycle

General Mechanism of a Polymerase

30 50 70 90 110 130 150 170 5 15 25 Discrim in atio n Fa ct o r

Maximal Elongation Rate (nt/s) P40G D47N R49K PV WT 30 50 70 90 110 130 150 170 5 15 25 Discrim in atio n Fa ct o r

Maximal Elongation Rate (nt/s) S115L

T114V

S115T

PV WT

T114A

Figure 3: Six step catalytic cycle of 3Dpol _{(Gong & Peersen, 2010, PNAS, 107, 22505–10). Templating RNA base in the +1}

position unpaired with an NTP, 1. Incoming NTP positioning by base pairing, 2. Active site closure, 3. Catalysis, 4. Active site opening, 5. Translocation, 6.

Figure 2: The poliovirus polymerase is an RNA-dependent RNA-polymerase, with a structure analogous to a

“right hand” including a fingers, palm and thumb domain. Elongation complex shown with RNA passing

through the polymerase. The template strand is shown in cyan, and the product strand is shown in green.

Kinetic Assays

UCGGGCGCCA-3’ GGCCCGCGGUCACUACAGCACUA-5’ A A T* G RNA Template UCGGGCGCCAG-3’ GGCCCGCGGUCACUACAGCACUA-5’ A A T* G Elongation Complex UCGGGCGCCAGUGAUGU-3’ GGCCCGCGGUCACUACAGCACUA-5’ A A T* G UCGGGCGCCAG-3’ GGCCCGCGGUCACUACAGCACUA-5’ A A T* G or 0 0.2 0.4 0.6 0.8 1 5 15 25 % RNA Initiated % RNA Initia ted Time (minutes) 0.1 0.3 0.5 0.7 0.9 25 75 125 175 225 % Elongated % R N A el on ga ted Time (minutes) 0 Time (min) 10 15 30 45 75 120 5 60 90 180 240 0 0.5 1 1.5 2 5 10 15 Starting RNA +1 +7 Initiation Stability +3 ₊₂ +1 -1 -0.9 -0.5 0.0 0.33 1 1.7 ∆ 2 -Ap Flu oresce nce Time (sec) UCGGGCGACCAAAG-3’ GGCCCGCUGGUUUCGA_PCUUAGCU-5’ A A U _G +2 UCGGGCGACCAAAGC-3’ GGCCCGCUGGUUUCGA_PCUUAGCU-5’ A A U _G +1 CTP UCGGGCGACCAAAGC_d-3’ GGCCCGCUGGUUUCGA_PCUUAGCU-5’ A A U _G +1 dCTP or CTP Incorporation 5 14 23 30 80 130 180 [CTP] µM V ( n t/s) V_Max= 25 nt/s K_M= 34 µM 1 2 3 250 800 1350 [dCTP] µM V ( n t/s) dCTP Incorporation V_Max= 3.7 nt/s K_M= 460 µM 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝐹𝐹𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 = 𝑉𝑉𝐾𝐾𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝐶𝐶𝐶𝐶𝐶𝐶 𝑉𝑉_{𝑀𝑀𝑀𝑀𝑀𝑀} 𝐾𝐾_𝑀𝑀 𝑑𝑑𝐶𝐶𝐶𝐶𝐶𝐶 CCGGCCGGCCA-3’ GGCCGGCCGGUACUACAUGUCACUCUACUAACGCGC-

F

-5’ A A U _G +1

NTPs

CCGGCCGGCCAUGAUGUACAGUGAGAUGAUUGCGCG-3’ GGCCGGCCGGUACUACAUGUCACUCUACUAACGCGC-

F

-5’ A A U _G -0.30 0.20 0.70 1.20 10 30 50 Fluo re scein Sign al Time (sec) 2 6 10 14 18 100 300 500 700 Elo ng a tio n Ra te [NTP] V_Max = 24 nt/s K_M = 60 µM

Figure 6: Kinetic Assays. (A) Initiation and Stability. Polymerases were mixed with

RNA templates and NTPs, and incubated at room temperature before EDTA quench at multiple time points. Curve fitting was done to a single exponential equation for fraction elongated product at each time point,

representative graph shown. (B) Single

NTP rate and fidelity determination.

3Dpol_{-RNA complex was mixed with varying}

concentrations of CTP or dCTP and rate determined by 2-aminopurine fluorescence change with time to generate a Michaelis-Menten curve and calculate discrimination factor. (C) Processive elongation rate

determination. 3Dpol_{-RNA complex was}

mixed with varying NTP concentrations and rate determined by fluorescence change. Lag phase length was used to determine processive elongation rate (first ~20 NTPs added) at each NTP concentration.

A

B

C

Figure 1: The poliovirus lifecycle

during a viral infection. Virus

uptake leads to RNA genome

release and polyprotein

translation by host cell

ribosomes. Polyprotein

proteolytic processing occurs via

viral proteases. RNA genome is

copied by the viral polymerase

and packaged into new virus

particles capable of infecting

additional cells.

Figure 4: All polymerases have a conserved active site geometry and mechanism for nucleotide incorporation (Steitz, T.

A., 1998 Nature 391, 231–2). A general acid, general base, and two metal ions are required for chemistry to occur.