The Pennsylvania State University

The Graduate School

Department of Chemistry

INVESTIGATION OF THE NUCLEOTIDE SELECTION MECHANISM OF

RNA-DEPENDENT RNA POLYMERASE

A Thesis in

Chemistry

by

Jingjing Shi

© 2018 Jingjing Shi

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science,

December 2018

The thesis of Jingjing Shi was reviewed and approved* by the following:

David Boehr Associate Professor Thesis Advisor

Edward O’Brien Assistant Professor Tapas Mal Assistant Research Professor NMR Director Philip Bevilacqua Distinguished Professor of Chemistry and Biochemistry and Molecular Biology Head of the Department of Department or Graduate Program

*Signatures are on file in the Graduate School

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ABSTRACT

RNA viruses cause a number of acute and chronic diseases including the

common cold, severe acute respiratory syndrome (SARS), and a more recent

outbreak of Middle East respiratory syndrome (MERS). Vaccines developed

against viruses have saved many lives. A traditional vaccine is a preparation of

killed microorganisms, live attenuated organism, or living fully virulent organisms

that is administered to produce or artificially increase immunity to a disease.

However, safety concerns and efficacy are potential problems for the further

development of new vaccines. One promising strategy to develop vaccines is by

targeting the virally encoded RNA-dependent RNA polymerase (RdRp). The RdRp

is conserved in most RNA viruses and these enzymes share a conserved structure

and catalytical residues. It has been determined that RdRp error rate relates to

viral attenuation. A too accurate RdRp loses adaptability to the host environment;

RdRp with higher error rate are also detrimental because the potential of lethal

mutagenesis. It is crucial to understand the nucleotide selection mechanism to

further investigate the development of live, attenuated vaccines.

Poliovirus (PV) RdRp can be used as a model to study the nucleotide

selection mechanism. There have been great efforts in investigating the roles of

different conserved structural motifs in poliovirus RdRp regarding RdRp error rate.

This thesis will mainly focus on the motif D of RdRp and uses a combination of

kinetic and NMR experiments. Motif D undergoes an “open” to “closed”

conformational change during catalysis. Site-directed mutagenesis experiments

suggest that the importance of motif D is not just the conformational change during

catalysis but the rearrangement of the active site lysine residue. Other amino acid

changes in motif D may contribute to the repositioning of the active site lysine

residue and may be used to change the RdRp error rate.

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Besides motif D, long-range interactions also potentially contribute to the

change of PV RdRp fidelity by the repositioning of the catalytic lysine residue. NMR

is a great tool to study structural dynamic changes during catalysis. The 1H,13C-

methyl resonance of Met354 in motif D has been used as a probe to indicate the

structural change in motif D. Single amino acid substitutions (K228A and N370A)

in motif D change the open-closed conformational equilibrium and by perturbing

the motif D conformational state, the enzyme fidelity is altered. Experiments with

other protein variants (K359H, K359H/I331F) involving the motif D lysine suggest

a relationship between fidelity and replication speed.

.

Norovirus is also a single-stranded, positive-sense RNA virus and it also

uses RdRp as the replicative enzyme. However, unlike poliovirus, there is no FDA

approved norovirus vaccine. It is worthwhile to apply the knowledge from poliovirus

to study antiviral strategies for norovirus. This thesis will introduce preliminary trials

of applying tools developed from the poliovirus system to the study of norovirus

RdRp.

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TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................. vii

LIST OF TABLES .................................................................................................... x

ACKNOWLEDGEMENTS ....................................................................................... xi

Chapter 1 Introduction ............................................................................................. 1

1.1 Viruses ....................................................................................................... 1 1.2 Polioviruses ................................................................................................ 2 1.3 Noroviruses ................................................................................................ 2 1.4 Traditional vaccine development................................................................. 3 1.5 New Antiviral Strategies .............................................................................. 4 1.6 Altered RNA-dependent RNA Polymeraser (RdRp) Fidelity ........................ 4 1.7 Motif D conformational change is a critical consideration in RdRp fidelity ... 5 1.8 Summary .................................................................................................... 7 References ....................................................................................................... 9

Chapter 2 Engineering the Fidelity of the Viral RNA-dependent RNA polymerase by Modifying the Structural Dynamics of the Motif D Active Site Loop ................... 11

2.1 Introduction ................................................................................................. 11 2.2 Methods ...................................................................................................... 15

2.2.1 Materials ........................................................................................... 15 2.2.2 Plasmid Construction ........................................................................ 15

2.2.3 Overexpression of RdRp ................................................................... 16 2.2.4 Purificaiton of RdRp .......................................................................... 17 2.2.5 Purification, 5’-32P end labeling and annealing of sym/sub .............. 18 2.2.6 PV RdRp assay ................................................................................ 19

2.3 Results and Discussions ............................................................................. 20 2.3.1 The K228A and N370A substitution alter RdRp fidelity by perturbing motif

D conformational equilibrium. ............................................................. 20 2.4 Conclusions ................................................................................................ 26 References ....................................................................................................... 26

Chapter 3 Investigation of the Relationship Between Nucleotide Selection and Replicative Speed Using NMR Spectroscopy ................................................... 28

3.1 Introduction ................................................................................................. 28 3.2 Methods ...................................................................................................... 30

3.2.1 Materials ........................................................................................... 30

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3.2.2 Plasmid construction ......................................................................... 31 3.2.3 Overexpression and purification of RdRp .......................................... 31 3.2.4 NMR spectroscopy sample preparation ............................................ 31 3.2.5 1H-13C HSQC NMR experiment ......................................................... 32

3.3 Results ....................................................................................................... 32 3.3.1 Spectrum comparison of WT, K359H and K359H/I331F RdRp ......... 32 3.3.2 Optimal experimental condition for the formation of RdRp-RNA-NTP complex ..................................................................................................... 34 3.2.3 Nucleotide Selection Mechanism Investigation Using NMR .............. 36

3.4 Discussion .................................................................................................. 37 References ....................................................................................................... 38

Chapter 4 Investigation of Norovirus Polymerase Function in Well-Established Poliovirus Polymerase System .......................................................................................... 41

4.1 Introduction ................................................................................................. 41 4.2 Methods ...................................................................................................... 42

4.2.1 Materials ........................................................................................... 42 4.2.2 Plasmid construction ......................................................................... 42 4.2.3 Overexpression and purification of ProPol ........................................ 43 4.2.4 Active site titration assays ................................................................. 43

4.3 Results ....................................................................................................... 43 4.4 Discussion .................................................................................................. 45 References ....................................................................................................... 46

Chapter 5 Conclusion and Future Directions ........................................................... 48

5.1 Conclusion .................................................................................................. 48 5.2 Future Directions ........................................................................................ 50

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LIST OF FIGURES

Figure 1-1 Proposed interaction that govern the open and closed states of the motif-D loop that helps position the general acid Lys359. Based on molecular dynamics simulations, we proposed a model for the motif D conformation equilibrium, where Glu364 interacts with Lys228 in the “open” state, but this interaction breaks and a new interaction forms between Glu364 and Asn370 in the “closed” state. By making single amino acid substitution, these interactions could be modified. ......................................................................... 6

Figure 2-1: Extending two-metal-ion mechanism of nucleotidyl transfer to include general acid catalysis. Nucleoside triphosphate (green) enters the active site with a divalent cation (Mg2+, metal B). This metal ion is coordinated by the phosphates of the nucleotide, an aspartate residue located in structural motif A of all polymerases, and probably water molecules. Metal B orients the triphosphate in the active site and may contribute to charge neutralization during catalysis. A second divalent cation binds (Mg2+, metal A) that is coordinated by the 3’-hydroxyl of the primer terminus (cyan), the nucleotide a-phosphate and aspartate residues of structural motifs A and C. Metal A lowers the pKa of the 3’-hydroxyl, facilitating deprotonation and subsequent nucleophilic attack at physiological pH. As the transition state of nucleotidyl transfer is approached (indicated by dashed red lines), the primer 3’-hydroxyl proton, Ha, is transferred to an unidentified base... ........................................... 12

Figure 2-2: 1H-13C HSQC NMR experiment on PV RdRp WT, K228A and N370A. A. Proposed interaction that govern the open and closed states of the motif-D loop that helps position the general acid Lys359. Based on molecular dynamics simulations, we proposed a model for the motif D conformation equilibrium, where Glu364 interacts with Lys228 in the “open” state, but this interaction breaks and a new interaction forms between Glu364 and Asn370 in the “closed” state. By making single amino acid substitution, these interactions could be modified. B. The M354 resonances when protein bound with RNA and UTP (correct NTP). When correct NTP was bound to the protein, the WT and K228A proteins were in the closed state which allowed the subsequent reaction to occur. However, the N370A enzyme was in an open state which suggests that N370A could be a too accurate variant that even with the correct NTP bound, it would not form a catalytic active state. C. The M354 resonances when protein bound with RNA and 2’- UTP (incorrect NTP). When incorrect NTP was bound to the protein, most of the proteins were in the open state in which the proteins were not catalytic active. However, a part of the K228A variant was in the closed state even with the incorrect NTP bound which suggested that K228A variant lack in the recognition of correct and in correct NTP and leading to a lower fidelity variant. ..................... 14

Figure 2-3: PV RdRp fidelity can be manipulated by altering the conformational equilibrium of motif D. A, B. X-ray crystal structure of PV RdRp (PDB 2IM2). Colored motifs include motif A (red), B (green), C (yellow), D(blue), E (purple), F(brown). Lys228, Asn370, Lys359, Glu364, and probe Met354 are

viii

shown in blue spheres. We use Met354 as a sensor of the motif D loop conformation. C. The pre-steady-state kinetic mechanism of single nucleotide incorporation by PV RdRp in the presence of Mg2+. Kinetic scheme including binding, pre-chemistry conformational change, chemical reaction, post-chemistry conformational change, and the release of pyrophosphate group. For correct nucleotide insertion, conformational change and chemistry (in red) are both rate limiting. ........................................ 21

Figure 2-4: Formation and stability of RdRp-RNA complexes are not affected by the K228A and N370A substitutions. Reaction was conducted in buffer containing 50 mM HEPES pH7.5, 10mM BME, 5 mM MgCl2 and 60 μM ZnCl2. A. Sequence of sym/subU RNA. B, C. In the assembly assay, ATP and RNA were first incubated for 5 min followed by the addition of RdRp to initiate the reaction. Reaction was quenched by adding 25 mM EDTA. Final concentrations of RdRp, RNA, and ATP were 1, 1, and 500 μM respectively. The data for formed WT RdRp-RNA complexes (black circle), K228A RdRp-RNA complexes (cyan circle) and N370A RdRp-RNA complexes (red circle) was plotted as a function of time. D, E. In the dissociation assay, RdRp and RNA were incubated for 90 s to allow the formation of RdRp-RNA binary complexes, the addition of trap (100 μM ATP) were added to the complex. At each time point, ATP was allowed to react for 30 s and the reaction was quenched by the addition of 25 mM EDTA. Final concentrations of RdRp, RNA and ATP were 1, 0.1, and 500 μM respectively. Percentage of the remaining binary complex was plotted as a function of time for WT (black circle), K228A (cyan circle) and N370A (red circle). The solid lines represent the fit of data into single exponential function. The kdis of WT, K228A and N370A for sym/subU are 3.04±0.19×10-4s-1, 2.82±0.24×10-4s-1, and 3.19±0.20×10-4s-1, respectively. ..................................................................................................... 22

Figure 2-5: The K228A substitution lowers the fidelity while the N370A substitution shows a higher fidelity. A. Sym/sub U RNA structure. B. Reaction scheme for the stopped flow experiment. Reactions was carried out in 50 mM HEPES pH7.5, 10mM BME, 5 mM MgCl2 and 60 μM ZnCl2. RdRp and RNA were incubated for 2 min to allow the formation of RdRP-RNA binary complexes at room temperature. Reaction was then initiated by the addition of an equal amount of ATP at various concentrations. Changes in fluorescence were recalculated as product formed in time. Final concentration of RdRp and RNA were 0.5 and 0.5 μM respectively. C. Comparison of the correct ATP incorporation rates among WT (black), K228A (cyan) and N370A(red). The two variants were as efficient as WT in the incorporation of correct ATP incorporation. D. Reaction scheme of the benchtop assay. E. Comparison of the incorrect GTP incorporation rates among WT (black), K228A(cyan) and N370A(red). The K228A was more efficient at incorporating incorrect GTP while N370A was less efficient compared to WT enzyme. Solid lines represent the fit of the data into hyperbola function. The result kpol and KD,app for each nucleotide incorporation are shown in table 2-1... .............................................................. 24

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Figure 3-1: This figure was made based on the data from Ref. 54. The kpol for the correct nucleotide measures the speed of polymerization in vitro. The kpol,

corr/kpol, incorr is an in vitro surrogate for fidelity, as it measures the relative rates of incorporation for the correct and incorrect nucleotides. A higher ratio indicates higher fidelity. A. The measured speed of polymerization in vitro for WT, K359H and K359H/I331F. B. The measure relative fidelity for WT, K359H and K359H/I331F. ............................................................................................ 29

Figure 3-2: 1H-13C HSQC NMR spectrum of apo WT (black), K359H (red) and K359H/I331F (blue) RdRp. The NMR spectra were taken in D2O based buffer (25 mM tris phosphate buffer, pH 8.0, 150 mM NaCl, 1 mM DTT and 0.02% NaN3). Note: the left and right parts are the same spectrum with different contour level... .................................................................................... 32

Figure 3-3: 1H-13C HSQC NMR spectrum of PV K359H apo RdRp and PV K359H bound with different concentration of RNA. The black spectrum is of PV K359H apo protein, the red one is PV K359H bound with 1mM s/su RNA and the blue one is PV K359H bound with 2 mM s/su RNA. Note: the left and right parts are the same spectrum with different contour level. ......................... 35

Figure 3-4: 1H-13C HSQC NMR spectrum of PV K359H bound with different nucleotides. The black spectrum is of PV apo-K359H, the red spectrum is of PV K359H bound with 8mM UTP, the blue one is PV K359H bound with 8 mM 2’UTP and the purple one is PV K359H bound with 8mM CTP. Note: the left and right parts are the same spectrum with different contour level.................... 37

Figure 4-1: NV ProPol is self-cleaving in E-coli B834 cell culture. A.Map of the cleavage site for ORF 1 of the MD145-12 NV. Figure is adapted from reference1. B. Crystal structure of NV ProPol (PDB 2IPH). Three catalytic residues Glu 54, His 30 and Cys 139 are shown in blue spheres. C A closer view of the catalytic triad. .. .............................................................................. 44

Figure 4-2: A. Purification gel of inactivated NV 3CD. B. The NV H30A/C139A ProPol was not active in RNA replication. The reaction was repeated twice, the empty dot and black dot represented the first and second reaction respectively. ..................................................................................................... 45

x

LIST OF TABLES

Table 2-1: Comparison of nucleotide incorporation for WT, K228A and N370A RdRps reveal that K228A lowers the fidelity while N370A shows a higher fidelity... ............................................................................................................ 25

ACKNOWLEDGEMENTS

Firstly, I would like to express my sincere gratitude to my advisor Dr. David

Boehr for the continuous guidance of my research work and my study at Penn

State. His support helped in my research and writing of this thesis.

Besides my advisor, I would also like to thank the rest of my committee: Dr.

Edward O’Brien and Dr. Tapas Mal, for their insightful suggestions.

Especially, I would like to thank Dr. Craig Cameron who offered the

opportunity for me to conduct all the kinetic assays and his questions toward my

experiments often lead me to think further and broad my knowledge.

I would also thank my fellow labmates for the help in my experiments. I

would like to especially thank Dr. Xinran Liu for teaching me how to study the

enzyme function using kinetic methodologies and answering my questions

patiently.

The work in this thesis was funded by NIH grant AI104878.

Chapter 1

Introduction

1.1 Viruses

Viruses are infectious agents that can only replicate within the cells of living

hosts2.Bacterial viruses were used in studies of specific biochemical or genetic

events, and contributed tremendously to the development of molecular cell

biology3.Disease outbreaks caused by viruses often become significant risks to

public health. In the 18th century in Europe, 400,000 people died annually of

smallpox, and one third of the survivors went blind4. More recently, the Ebola virus

disease (EVD) outbreak in the north-eastern provinces of the Democratic Republic

of the Congo, which borders Uganda, Rwanda and South Sudan, resulted in a total

of 142 EVD cases including 97 deaths reported as of Sept. 18th, 2018. According

to World Health Organization (WHO) risk assessment, potential risk factors for

transmission of EVD at the national and regional levels were assessed to be high5.

Viral infection cycles are classified as lytic or lysogenic6. The lytic cycle of

viral replication, which is utilized in most viruses like poliovirus and norovirus,

includes the injection of the free virion into host cells (adsorption), the expression

of viral proteins and genetic materials, the assembly of new virus particles and the

release of new virus particles to infect other healthy cells. The production of a new

round of viral particles means death of host cells. In the lysogenic process, as

happens in some animal viruses, the viruses integrate into the host chromosome

and are replicate as parts of the host cell DNA 3.

Viruses are classified based on size and shape, identity of the nucleic acid

(RNA or DNA), configurations (double or single stranded), structure of the genome

2

(single or double stranded) and mode of synthesis (DNA polymerase, RNA

polymerase and reverse transcript)7. According to Baltimore classification system8,

there are six different classes; I: double-stranded (ds) DNA viruses (e.g.

Adenoviruses and Herpsesviruses); II: single-stranded (ss) DNA viruses (e.g.

Parvoviruses); III: dsRNA viruses (e.g. Reoviruses); IV: positive ssRNA viruses

(e.g. Picornaviruses); V: negative ss RNA viruses (e.g. Vesicular stomatitis virus);

VI: ssRNA reverse-transcript viruses ( e.g. the human immunodeficiency virus). In

this thesis, the two viruses, poliovirus and norovirus, that are mainly focused

belong to group IV, positive single-strand RNA viruses.

1.2 Poliovirus

Poliovirus is the cause of poliomyelitis. Poliomyelitis was very common in

the U.S. and caused severe illness in thousands of people each year before the

polio vaccine was introduced in 19559. Even though the global incidence of polio

cases has decreased by 99% since the beginning of the Global Polio Eradication

Initiative, there are still outbreaks of vaccine-derived poliovirus including a recent

outbreak in Sokoto State, Nigeria10.

Poliovirus belongs to the family of enteroviruses in Picornaviridae.

Picornaviruses are named for their small size, and they include a diverse array of

viruses11. The Picornaviridae family causes most illnesses among all virus families.

Infection with various picornaviruses may cause clinical syndromes such as

aseptic meningitis (the most common acute viral disease of the central nervous

system12, the common cold13, hand-foot-and-mouth disease14, and hepatitis14.

1.3 Norovirus

Norovirus is a highly contagious virus that causes severe vomiting and

diarrhea15. Infections can be acquired through food or water that is contaminated

3

during preparation or contaminated surfaces, or through contact with infected

people16. In children and elderly adults with compromised immune systems,

norovirus infections sometimes lead to severe complications including

dehydration, malnutrition and even death.

Norovirus is also a Group IV single-stranded positive-sense RNA, which

belongs to the family Caliciviridae17. Diseases associated with this family include

feline calicivirus (respiratory disease)18, rabbit hemorrhagic disease virus19 and

Norwalk group of viruses (gastroenteritis).

Norovirus was not well studied because of a lack of suitable cell culture and

animal model20. Since norovirus and poliovirus share the same type of RdRp in

viral replication, tools developed for study poliovirus RdRp can be applied towards

study norovirus RdRp.

1.4 Traditional vaccine development

Vaccines developed against viruses have saved many lives21. Successful

vaccine strategies have been utilized for smallpox, poliovirus and measles22. Most

human vaccines were developed based on empirical approaches designed to

mimic, by vaccination, the natural infection induced immunity by iterating over

successful treatment of viral infection23. A traditional vaccine is a preparation of

killed microorganisms, live attenuated organism, or living fully virulent organisms

that is administered to produce or artificially increase immunity to a particular

disease24. During the production of a traditional vaccine, the virus is weakened in

a laboratory to the point where it’s alive and able to reproduce, but not able to

cause severe disease. Live attenuated vaccines against human viral diseases

have been very successful25. For example, the MMR (measles, mumps and

rubella) vaccine that combines the attenuated virus measles, mumps and rubella

has been very effective at protecting people against these viruses26.

4

However, the chances of finding effective vaccines empirically are low22.

One rational vaccine strategy can be derived from current vaccines. Polio is one

of the classical examples of live, attenuated vaccine27. Due to the success of the

polio vaccine and the similarities between the RNA viruses, a more efficient

strategy may be to design new antiviral drugs and RNA vaccines based on the

knowledge obtained from poliovirus.

1.5 New antiviral strategies

RNA viruses have the highest replicative error rates in nature, with

approximately one mistake made per 1,000-100,000 nucleotides in one genome

replication cycle28. Because of the high error rates, there are population diversities

among different RNA viruses. This complex population is called quasispecies29.

Under selective pressure, quasispecies are important for survival since a few

viruses that contain beneficial mutations would survive and act as founders for the

next generation. However, most errors that are made during replication are

deleterious, resulting in debilitation of a high percentage of the population.

Therefore, RNA viruses live on the edge of “error catastrophe” , in other words,

RNA viruses are in a dynamic balance between the cost of deleterious mutations

with the benefit of adaptive mutations. A new idea of developing RNA vaccines

takes advantage of the cost of error catastrophe. Ribavirin is a nucleotide analog

mutagen29, which is incorporated in to the viral-genome during replication and

increases error frequency by promiscuous base-pairing. Ribavirin debilitates the

viral population over several rounds of replication by causing accumulation of

deleterious mutations and error catastrophe.

1.6 Altered RNA-dependent RNA polymerase (RdRp) fidelity

5

The enzyme responsible for RNA genome replication of RNA-based viruses

is the virally encoded RNA-dependent RNA polymerase (RdRp). Polymerase

fidelity is biochemically defined as kpol/KD of single nucleotide incorporation30. The

maximum rate of polymerization constant is defined as kpol and KD is the apparent

equilibrium dissociation constant. Enhanced replication fidelity results in reduced

genetic variation, and it leads to a reduce in virus fitness. The enhanced fidelity

also reduces the frequency of genetic reversion back to wild-type. There is now

evidence in several systems that RdRp fidelity is a determinant of viral

pathogenesis and virulence31.

In poliovirus RdRp, the active site lysine residue (i.e. Lys359 in poliovirus),

which is conserved in all nucleic acid polymerases serves as a general acid that

catalyzes nucleotide incorporation. The conserved motif D Lys has already been

identified as an important residue to modify to attenuate the virus. Weeks et al

have shown that substituting the lysine residue in poliovirus RdRp for arginine

produces a RdRp that replicates slower with higher fidelity32. This virus variant is

genetically stable and replicates well in cell culture, and in vivo, this variant is

attenuated, and elicits a protective immune response. It provides the same level

immunoprotective effects as Sabin I33.Since this lysine residue is conserved in all

viral polymerases, similar studies can be done in a wide range of viruses,

suggesting that a universal, mechanism-based strategy may exist for viral

attenuation and vaccine development.

1.7 Motif D Conformational change is a critical consideration in RdRp fidelity

The Boehr and Cameron lab groups have previously determined that the

structural motif D active-site loop is important for nucleotide selection34. When

correct nucleotide binds, the active-site loop forms a ‘closed’ conformation that

helps to bring in the highly conserved motif D Lys (Lys359 in PV RdRp), which acts

6

as a general acid to protonate the pyrophosphate leaving group (Figure 1-1).

Binding of incorrect nucleotide does not result in the same conformational change,

and instead, the enzyme remains in an ‘open’ conformation. They have suggested

that manipulating the conformational dynamics of the active-site loop will lead to

changes in RdRp fidelity. The RdRp of the Sabin I vaccine strain has Thr362

changed to Ile. Such a drastic change so close to Lys359 might alter RdRp function

and contribute in some way to the attenuated phenotype of Sabin type I. Yang et

al. used solution-state nuclear magnetic resonance (NMR) to study PV RdRp 33,

and discovered that the T362I induces structural rearrangements within motif D

and showed that the Sabin-derived T362I substitution in PV RdRp shows

decreased fidelity by affecting the conformational equilibrium between the open

and closed states. PV strains encoding the T362I substitution have reduced

virulence in mice. Thus, the general concept of modifying the conformational

dynamic of motif D to impact RdRp fidelity and virus biology may be a guiding

principle in rational vaccine design.

Figure 1-1 Proposed interactions that govern the open and closed states of the motif-D loop that

helps position the general acid Lys-359. Based on molecular dynamics simulations, we proposed

a model for the motif D conformation equilibrium, where Glu364 interacts with Lys228 in the “open”

state, but this interaction breaks and a new interaction forms between Glu364 and Asn370 in the

“closed” state. By making single amino acid substitution, these interactions could be modified.

7

1.8 Summary

Altogether, there have been great efforts in investigating the nucleotide

selection mechanism of the RdRp, however more details still require further

investigation. Previous experiments showed the importance of motifs A, C, D and

F in poliovirus RdRp, and motif D has been shown to undergo an “open” to “closed”

transition during catalysis based on MD simulations and NMR results. The Sabin

vaccine strain encodes one motif D substitution T362I, and T362I shows a

significant change in RdRp fidelity. Other substitutions in motif D may also

contribute to the repositioning of the active site lysine residue and may serve as a

framework to rationally design other vaccine candidates.

Besides motif D, there are still long-range interactions that can contribute to

the change of PV RdRp fidelity. The investigation of other motifs could lead to a

more complete molecular understanding of RdRp function, and could provide

valuable information in the rational development of vaccines.

In this thesis, I studied two protein variants (K228A and N370A) that are

predicted to change the conformational equilibrium of the motif D loop. Consistent

with the model (Figure 1-1), K228A substitution lowers the fidelity while the N370A

substitution shows a higher fidelity. Motif D undergoes an “open” and “closed”

conformational state by kinetic study and this leads to a rational design of site-

directed mutagenesis in motif D by combination of MD simulation and NMR.

Chapter 3 describes the long-range interactions that could reposition the active

site Lys 359 from other motifs and this may help a better understanding of the roles

of other motifs. Chapter 4 discusses that the potential to use PV kinetic study as a

model system to learn norovirus RdRp.

8

References 1. Belliot, G.; Sosnovtsev, S. V.; Chang, K.-O.; Babu, V.; Uche, U.; Arnold, J. J.; Cameron, C. E.; Green, K. Y. J. J. o. v., Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. 2005, 79 (4), 2393-2403. 2. Wessner, D. R. J. N. E., The origins of viruses. 2010, 3 (9), 37. 3. Lodish, H.; Berk, A.; Zipursky, S. L.; Matsudaira, P.; Baltimore, D.; Darnell, J., Viruses: Structure, Function, and Uses. 2000. 4. Belongia, E. A.; Naleway, A. L., Smallpox Vaccine: The Good, the Bad, and the Ugly. Clinical Medicine and Research 2003, 1 (2), 87-92. 5. report, W. Ebola virus disease – Democratic Republic of the Congo; 2018. 6. Evans, C.; Brussaard, C. P. D., Regional Variation in Lytic and Lysogenic Viral Infection in the Southern Ocean and Its Contribution to Biogeochemical Cycling. Applied and Environmental Microbiology 2012, 78 (18), 6741-6748. 7. Gelderblom, H. R., Structure and classification of viruses. 1996. 8. Baltimore, D., Expression of animal virus genomes. Bacteriological Reviews 1971, 35 (3), 235-241. 9. 10 facts on polio eradication. 10. Circulating vaccine-derived poliovirus type 2 – Nigeria. 11. Massilamany, C.; Koenig, A.; Reddy, J.; Huber, S.; Buskiewicz, I. J. C. o. i. v., Autoimmunity in picornavirus infections. 2016, 16, 8-14. 12. Irani, D. N., Aseptic Meningitis and Viral Myelitis. Neurologic clinics 2008, 26 (3), 635-viii. 13. Worrall, G., Common cold. 2011, 57 (11), 1289-1290. 14. Logan, G.; Freimanis, G. L.; King, D. J.; Valdazo-González, B.; Bachanek-Bankowska, K.; Sanderson, N. D.; Knowles, N. J.; King, D. P.; Cottam, E. M., A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq. BMC Genomics 2014, 15 (1), 1-10. 15. Robilotti, E.; Deresinski, S.; Pinsky, B. A., Norovirus. 2015, 28 (1), 134-164. 16. Green, K. J. C. M.; Infection, Norovirus infection in immunocompromised hosts. 2014, 20 (8), 717-723. 17. Portal, T. M.; Siqueira, J. A. M.; Costa, L. C. P. d. N.; Lima, I. C. G. d.; Lucena, M. S. S. d.; Bandeira, R. d. S.; Linhares, A. d. C.; Luz, C. R. N. E. d.; Gabbay, Y. B.; Resque, H. R. J. b. j. o. m., Caliciviruses in hospitalized children, São Luís, Maranhão, 1997-1999: detection of norovirus GII. 12. 2016, 47 (3), 724-730. 18. Schwartz-Porsche, D.; Kaiser, E. J. P. i. v. m., Feline epilepsy. 1989, 1 (4), 628-649. 19. Abrantes, J.; van der Loo, W.; Le Pendu, J.; Esteves, P. J., Rabbit haemorrhagic disease (RHD) and rabbit haemorrhagic disease virus (RHDV): a review. Veterinary Research 2012, 43 (1), 12.

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20. Subba-Reddy, C. V.; Yunus, M. A.; Goodfellow, I. G.; Kao, C. C., Norovirus RNA synthesis is modulated by an interaction between the viral RNA-dependent RNA polymerase and the major capsid protein, VP1. Journal of virology 2012, 86 (18), 10138-10149. 21. Huang, D. B.; Wu, J. J.; Tyring, S. K., A review of licensed viral vaccines, some of their safety concerns, and the advances in the development of investigational viral vaccines. Journal of Infection 2004, 49 (3), 179-209. 22. Rueckert, C.; Guzman, C. A., Vaccines: from empirical development to rational design. PLoS Pathog 2012, 8 (11), e1003001. 23. Doolan, D. L.; Apte, S. H.; Proietti, C., Genome-based vaccine design: the promise for malaria and other infectious diseases. International Journal for Parasitology 2014, 44 (12), 901-913. 24. Minor, P. D., Live attenuated vaccines: Historical successes and current challenges. Virology 2015, 479–480, 379-392. 25. De Gregorio, E.; Rappuoli, R., From empiricism to rational design: a personal perspective of the evolution of vaccine development. Nat Rev Immunol 2014, 14 (7), 505-514. 26. Cardemil, C. V.; Dahl, R. M.; James, L.; Wannemuehler, K.; Gary, H. E.; Shah, M.; Marin, M.; Riley, J.; Feikin, D. R.; Patel, M. J. N. E. J. o. M., Effectiveness of a third dose of MMR vaccine for mumps outbreak control. 2017, 377 (10), 947-956. 27. Theiler, M.; Smith, H. H., THE USE OF YELLOW FEVER VIRUS MODIFIED BY IN VITRO CULTIVATION FOR HUMAN IMMUNIZATION. The Journal of Experimental Medicine 1937, 65 (6), 787-800. 28. Pfeiffer, J. K.; Kirkegaard, K., Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice. PLoS Pathogens 2005, 1 (2), e11. 29. Crotty, S.; Cameron, C. E.; Andino, R., RNA virus error catastrophe: Direct molecular test by using ribavirin. Proceedings of the National Academy of Sciences of the United States of America 2001, 98 (12), 6895-6900. 30. Freistadt, M. S.; Vaccaro, J. A.; Eberle, K. E., Biochemical characterization of the fidelity of poliovirus RNA-dependent RNA polymerase. Virology Journal 2007, 4 (1), 44. 31. Korboukh, V. K.; Lee, C. A.; Acevedo, A.; Vignuzzi, M.; Xiao, Y.; Arnold, J. J.; Hemperly, S.; Graci, J. D.; August, A.; Andino, R.; Cameron, C. E., RNA Virus Population Diversity, an Optimum for Maximal Fitness and Virulence. The Journal of Biological Chemistry 2014, 289 (43), 29531-29544. 32. Weeks, S. A.; Lee, C. A.; Zhao, Y.; Smidansky, E. D.; August, A.; Arnold, J. J.; Cameron, C. E., A Polymerase Mechanism-based Strategy for Viral Attenuation and Vaccine Development. The Journal of Biological Chemistry 2012, 287 (38), 31618-31622. 33. Liu, X.; Yang, X.; Lee, C. A.; Moustafa, I. M.; Smidansky, E. D.; Lum, D.; Arnold, J. J.; Cameron, C. E.; Boehr, D. D., Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity. J Biol Chem 2013, 288 (45), 32753-65.

10

34. Yang, X.; Smidansky, E. D.; Maksimchuk, K. R.; Lum, D.; Welch, J. L.; Arnold, J. J.; Cameron, C. E.; Boehr, D. D., Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure (London, England : 1993) 2012, 20 (9), 1519-1527.

Chapter 2

Engineering the Fidelity of the Viral RNA-dependent RNA Polymerase by

Modifying the Structural Dynamics of the Motif D Active Site Loop

In this chapter, I was responsible for protein overexpression, purification

and all the kinetic studies except the GTP mis-incorporation assay, which was

conducted by Jacob M. Perryman from the Cameron lab.

2.1 Introduction

Single-strand positive-sense RNA viruses cause a number of acute and

chronic diseases, including the common cold35, severe acute respiratory syndrome

(SARS)36, and the recent outbreak of Ebola in the Republic of Congo5. The enzyme

responsible for genome replication of RNA-based viruses is the virally encoded

RNA-dependent RNA polymerase (RdRp). RdRps have a conserved cupped ‘right

hand’ structure, which have three major subdomains: palm, fingers and thumb37.

The RdRps belongs to a polymerase superfamily including DNA-dependent

DNA polymerase (DdDp), DNA-dependent RNA polymerases (DdRp）, and RNA-

dependent DNA polymerases (reverse transcriptase, RT)38. Most of these

polymerases share a two-metal-ion mechanism for phosphodiester bond

formation39 and share common structural motifs. Nucleoside triphosphate enters

the active site with a divalent cation (Mg2+, metal B). This metal ion is coordinated

by the phosphates of the nucleotide, an aspartate residue located in structural

motif A, and water molecules (Figure 2-1). Metal B orients the triphosphate in the

active site and likely contributes to charge neutralization during catalysis. A second

divalent cation binds (Mg2+, metal A) and is coordinated by the 3’-hydroxyl of the

12

primer terminus the nucleotide α-phosphate and aspartate residues of structural

motifs A and C. Metal A lowers the pKa of the 3’-hydroxyl, facilitating deprotonation

and subsequent nucleophilic attack at physiological pH. As the transition state of

nucleotidyl transfer is approached (indicated by dashed red lines in (Figure 2-2),

the primer 3’-hydroxyl proton, Ha, is transferred to an unidentified base (B), and it

has been proposed that then pyrophosphate leaving group is protonated (Hb) by

a basic amino acid on the enzyme.

Figure 2-1. Extending two-metal-ion mechanism of nucleotidyl transfer to include general acid

catalysis. Nucleoside triphosphate (green) enters the active site with a divalent cation (Mg2+, metal

B). This metal ion is coordinated by the phosphates of the nucleotide, an aspartate residue located

in structural motif A of all polymerases, and probably water molecules. Metal B orients the

triphosphate in the active site and may contribute to charge neutralization during catalysis. A

second divalent cation binds (Mg2+, metal A) that is coordinated by the 3’-hydroxyl of the primer

terminus (cyan), the nucleotide a-phosphate and aspartate residues of structural motifs A and C.

Metal A lowers the pKa of the 3’-hydroxyl, facilitating deprotonation and subsequent nucleophilic

attack at physiological pH. As the transition state of nucleotidyl transfer is approached (indicated

by dashed red lines), the primer 3¢-hydroxyl proton, Ha, is transferred to an unidentified base

Polymerase fidelity is related to viral pathogenesis and virulence31. Previous

studies have suggested that a too accurate RdRp also attenuates the virus38, 40, 41.

Mg2+

O

O

O

O

O

OH

Mg2+O

O

O

O

OH

P

O

PO

O

O

O

O

PO

O

B?

Ha

O

Primer

Base

(O)HO

OH

(O)H

Base

Motif C

Motif A

a

g

3'

B

b

Hb A

PV RdRp Lys359(Motif D)

13

Virus with lower fidelity are more error-prone and may lead to fatal mutations. From

these studies, it has been suggested that viruses encoded with RdRps having

altered fidelity may serve as potential vaccine strains42, 43.

The Boehr and Cameron lab groups have previously determined that the

structural motif D active-site loop is important for nucleotide selection34. When

correct nucleotide binds, the active-site loop forms a ‘closed’ conformation that

helps to bring in the highly conserved motif D Lys (Lys359 in PV RdRp), which acts

as a general acid to protonate the pyrophosphate leaving group. Binding of

incorrect nucleotide does not result in the same conformational change, and

instead, the enzyme remains in an ‘open’ conformation. We have suggested that

manipulating the conformational dynamics of the active-site loop will lead to

changes in RdRp fidelity. Recent studies in our group showed that the Sabin-

derived T362I substitution in PV RdRp decreases fidelity by affecting the

conformational equilibrium between the open and closed states33. PV strains

encoding the T362I substitution have reduced virulence in mice. Thus, the general

concept of modifying the conformational dynamic of motif D to impact RdRp fidelity

and virus biology may be a guiding principle in rational vaccine design.

Based on structures modeled from MD simulations, we have established a

model (Figure 2-2 A) in motif D where Glu364 interacts with Lys228 in the “open”

state, but in the “closed” state, Glu364 interacts with Asn370 instead. By making

single amino acid substitutions at these two sites, further knowledge of this model

will be obtained. The N370A variant should be in the open state since the

interaction between Glu364 and Asn370 in the closed state will be affected by the

amino acid substitution. Similarly, the K228A variant will prefer to be in the closed

state while the interaction in the open state between Glu364 and Lys228 is

perturbed. Previously, Dr. Xiaorong Yang in our group the NMR spectra showing

that N370A stays in the open state even the RdRp was bound to correct NTP and

part of the K228A was in the “closed” state when the RdRp was bound the incorrect

14

NTP (Figure 2-2 B, C). The NMR experiment is consistent with the proposed model

and confirmed the existence of two conformational states. However, kinetic data is

needed to determine any effects of the substitutions on the RdRp fidelity.

Figure 2-2. 1H-13C HSQC NMR experiment on PV RdRp WT, K228A and N370A. A. Proposed

interactions that govern the open and closed states of the motif-D loop that helps position the

general acid Lys359. Based on molecular dynamics simulations, we proposed a model for the motif

D conformation equilibrium, where Glu364 interacts with Lys228 in the “open” state, but this

interaction breaks and a new interaction forms between Glu364 and Asn370 in the “closed” state.

By making single amino acid substitution, these interactions could be modified. B. The Met354

resonances when protein bound with RNA and UTP (correct NTP). When correct NTP was bound

to the protein, the WT and K228A proteins were in the closed state. However, the N370A enzyme

was in an open state which suggests that N370A could be a too accurate variant that even with the

correct NTP bound, it would not form a catalytic active state. C. The Met354 resonances when

protein bound with RNA and 2’- UTP(incorrect NTP). When incorrect NTP was bound to the protein,

most of the proteins were in the open state in which the proteins were not catalytically active.

However, a part of the K228A variant was in the closed state even with the incorrect NTP bound

15

which suggested that K228A variant lack in the recognition of correct and in correct NTP, and

leading to a lower fidelity variant.

In this Chapter, we showed that the K228A substitution lowers the fidelity

for more efficient incorrect nucleotide incorporation while the N370A substitution

shows a higher fidelity. Altogether, our data support the notion that RdRp fidelity

can be altered through rational modification of the motif D loop and surrounding

residues. These studies give us a better understanding of RNA polymerase

fidelity by using PV as a model system.

2.2 Methods

2.2.1 Materials

[γ-32P] ATP and [α-32P] UTP (>7000Ci/mmol) were from VWR-MP

Biomedical. Nucleoside 5’-triphosphates and 2’-deoxynucleoside 5’-triphosphates

(all nucleotides were ultrapure solutions) were from GE Healthcare. 3’-

Deoxyadenosine 5’-triphosphate (cordycepin) was from Trilink Biotechnologies. All

RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO). T4

polynulceotide kinase was from New England Biolabs, Inc. [Methyl-13C] Methionine

was from Cambridge Isotope laboratories. HisPur Ni-NTA resin was from Thermo

Scientific. Q-Sepharose fast flow was from Amersham Pharmacia Biothce, Inc.

The QuickChange site-directed mutagenesis kit was from Stratagene. The plasmid

DNA isolation Miniprep kit was from Qiagen. All other reagents were of the highest

grade available from Sigma or Fisher.

2.2.2 Plasmid construction

16

The K228A and N370A variants were generated using the QuickChange

method and appropriate primers. The mutation was confirmed by DNA sequencing

(Nucleic Acid Facility, The Pennsylvania State University). There are two interface

amino acid substitutions (L446D and R455D) to prevent polymerase dimerization

in wild-type (WT), K228A and N370A RdRps used in the kinetic and NMR studies.

2.2.3 Overexpression of RdRp

The autoinducing system was introduced by Studier44. The pET plasmid

was transformed into Met-auxotroph Escherichia coli B834(DE3) pRARE cells in

which the pRARE is for rare codon adaptation. At first the cells were in a M9

minimal media with glucose to prevent pre-mature induction and then were used

to inoculate a large scale autoinducing M9 media with lactose to start auto-

induction.

Overexpression of PV RdRps was performed as described previously41.

The pSUMO-RdRp plasmid was transformed into B834(DE3) pRARE cells. The

cells with the plasmid were used to inoculate 100 mL M9 minimal media with

glucose and shaken at 250 rpm at 37 ℃ until the optical density at 600 nanometer

(OD600) reached 1.0. The cells were then inoculated into a 500 mL M9 minimal

media with lactose with a starting OD600 at 0.05 at 250 rpm at 37 ℃ until the OD600

reached 0.5, after which the temperature was reduced to 25 ℃ to continue the

overexpression of PV RdRp for 18 hours. The cells were harvested by

centrifugation at 3900 rpm for 30 min at 4 ℃, washed with 10 mM Tris (pH 8.0) and

1mM EDTA buffer (T10E1), and harvested again by centrifugation in the same

condition. The cell pellets were stored at -80℃ until further use.

Protein samples used in NMR experiments were labeled by [Methyl-

13C]Met, while RdRps involved in kinetic studies did not incorporate this isotope.

17

Previous experiments demonstrated there were no kinetic effects with this isotope

incorporation.

2.2.4 Purification of RdRp

Protein purification of PV wild-type and N370A variant were performed as

described previously with some minor adjustments41. Frozen cells were thawed on

ice and suspended in lysis buffer (20% glycerol, 100mM potassium phosphate pH

8.0, 1μg/mL pepstatin, 1μg/ml leupeptin, 500 μM EDTA, 60 μM ZnCl2 and 5mM

β-mercaptoethanol (BME) with a ratio of 1g pellet in 5mL lysis buffer. Cells were

lysed by passing through a French pressure cell at 10.000 psi for three times.

Phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM

and a third of total PMSF was added after each French press. The nonidet P-

40(NP-40) was added after lysis to final concentration 0.1% v/v. Polyethylenimine

(PEI) was added over 30 min to precipitate the nucleic acid to a final concentration

of 0.25 (v/v). The lysate was stirred for an additional 15 min at 4℃ after complete

addition of PEI. The lysate was centrifuged for 30 min at 16,500 rpm at 4℃. The

PEI supernatant was transferred to another container, and ammonium sulfate was

slowly added to a final concentration of 60% saturation to precipitate protein. The

supernatant was stirred for an additional 15 min at 4℃ and lysate was centrifuged

for 30 min at 16,500 rpm at 4℃. The supernatant was decanted and the pellet was

suspended in buffer B (20% glycerol, 100mM potassium phosphate pH 8.0,

1μg/mL pepstatin, 1μg/ml leupeptin, 500 μM EDTA, 60 μM ZnCl2, 5mM β-

mercaptoethanol(BME),500 mM NaCl,1mM PMSF ,0.1% NP40 and 5mM

imidazole.) The protein solution was diluted to 5.4 mg/mL before the Ni-NTA

column. The diluted protein solution is then loaded onto a pre-equilibrated Ni-NTA

column with buffer B at a flow rate of 1mL/min. The column was washed the first

time at a low rate of 1mL/min with 10 column volumes of buffer C ( 100mM

phosphate pH 8.0, 5mM BME, 20% glycerol, 60uM ZnCl2) containing 500mM

NaCl, 1mM PMSF, 0.1% NP40 and 5mM imidazole. The column was washed a

18

second time to the baseline (monitored by absorbance at 280nm) with 10 column

volumes of buffer C containing 500mM NaCl, 1mM PMSF and 5mM imidazole

without NP-40, leave out NP-40 in all subsequent buffers. The column was washed

a third time with 5 column volumes of buffer C containing 500mM NaCl, 1mM

PMSF and 50mM imidazole. The protein was eluted with 10 column volumes of

buffer C containing 500mM NaCl, 1mM PMSF and 500mM imidazole. The eluted

protein with Ulp1 enzyme (1ug/1mg SUMO fusion) to cleave the SUMO fusion was

dialyzed (1214,000 Da MWCO membrane) overnight against 1L buffer D (20%

glycerol, 500mM NaCl, 50mM Tris, 60uM ZnCl2 and 5mM BME). The dialyzed

protein was dialyzed a second time for 4h against 1L buffer E (20% glycerol,

100mM potassium phosphate, 60uM ZnCl2 and 5mM BME). The cleaved protein

was then passed through a Ni-NTA column equilibrated with 3 column volumes of

buffer E at a flow rate of 1mL/min. The pass-through protein was then dialyzed

against 1L buffer F (20% glycerol, 50mM Tris pH 8.0 and 1mM DTT) containing

25mM NaCl. The dialyzed protein was then loaded onto the Q-sepharose column

pre-equilibrated with 3 column volumes of buffer F containing 25mM NaCl at a flow

rate of 1mL/min. The column was washed with 10 column volumes of buffer F

containing 25mM NaCl and eluted with buffer F containing 500mM NaCl. The

purified protein was aliquoted and stored at -80 ℃.

For the purification of K228A RdRp protein, a few changes were made to

the original procedures. After the cells were lysed using the French press, cell

lysates were centrifuged for 30 min at 5,000 rpm at 4℃. The supernatant was then

loaded onto a pre-equilibrated Ni-NTA column with buffer B at a flow rate of

1mL/min. This Ni-NTA column with protein was incubated for 30 min at 4℃ while

shaking. All the following purification procedures were the same as previously

mentioned.

2.2.5 Purification, 5’-32P end labeling and annealing of sym/sub

19

For kinetic studies, the RNA oligonucleotide sym/sub was gel purified as

described previously41. The nucleotide sequence of the sym/sub RNA is 5’-

GCAUGGGCCC.2’-ACE (bis(2-acetoxyethoxy) methyl) protected sym/sub RNA

was purified by 23% denaturing PAGE gel containing 1.7% bisacrylamide, 7M urea

and TBE buffer (89 mM Tris base, 89 mM boric acid, and 2 mM EDTA). The

sym/sub RNA was visualized via short length UV light. The gel containing RNA

was cut and chopped into pieces. The RNA was electroeluted from the gel in TBE

buffer in Elutrap followed by a desalting column. The sym/sub RNA was dried by

vacuum evaporation and was stored at -20℃. The purified sym/sub RNA was

deprotected and desalted immediately prior to use.

The procedure for 5’-32P end-labeling of sym/sub at the 5’-end was as

described previously. 32P r-labelled ATP and T4 polynucleotide kinase were added

into the sym/sub RNA in T4 reaction buffer at 37 ℃ for 1h to a final concentration

of 1 uM and 0.4 unit/uL, respectively. The reaction was quenched by incubation at

65 ℃for 5 min.

For annealing of sym/sub RNA, the RNA was diluted to a 10mM Tris pH 8.0

and 0.1 mM EDTA buffer (T10E0.1). The annealing process was conducted in a

thermocycler using a program with denaturing at 95 ℃for 1 min and deceasing to

10 ℃with a rate of 5℃/min.

2.2.6 PV RdRp assay

The reactions were conducted in buffer containing 50 mM HEPES pH7.5,

10mM BME, 5 mM MgCl2 and 60 μM ZnCl2. Reaction buffer was first incubated for

5 min followed by the addition of either RdRp or ATP to initiate the reaction. RdRp

enzyme was diluted with dilution buffer (20% glycerol, 50 mM HEPES pH7.5,

10mM BME, and 60 μM ZnCl2) immediately prior to use. Reaction was quenched

20

by adding 25 mM EDTA at each time point. Final concentrations of RdRp, RNA,

and ATP were indicated specifically in the appropriate figure legend.

2.3 Results and Discussions

2.3.1 The K228A and N370A substitutions alter RdRp fidelity by perturbing

the motif D conformational equilibrium.

The K228 and N370 residues are located in the

palm” subdomain of PV RdRp (Figure 2-3). Based on molecular dynamics

simulations, we proposed a model for the motif D conformational equilibrium

(Figure 2-2), where E364 interacts with K228 in the “open” state, but in the “closed”

state, E364 interacts with N370 instead. To test this model, we generated the

K228A and N370A variants. The single amino acid substitution could change

enzyme fidelity by perturbing the motif D conformational equilibrium including a

repositioning of the general acid Lys359. Before testing this prediction, it was

necessary to know whether these substitutions affect RNA binding affinity and

stability of the RdRp-RNA binary complexes. Assembly and dissociation assays

were performed to investigate the formation and the stability of binary complexes,

respectively for these two variants and wild-type (WT) enzyme. In kinetic studies,

the RNA template used was sym/subU, which contains six complimentary base

pairs with four nucleotide overhangs (Figure2-4). For the sym/subU RNA, ATP is

the next correct nucleotide. In the assembly assay (Figure 2-4C), the two variants

formed a similar level of catalytically competent enzyme complexes compared to

WT. The N370A variant was slightly better in the formation of enzyme complexes,

which suggested a more accessible active site.

In the dissociation assay, the N370A RdRp complexes were less stable

compared to K228A variant and WT enzyme complexes. The differences between

the three RdRps were not significant (Figure 2-4). The half-lives of the RdRp-RNA

21

complexes were about 30mins, which was sufficiently long for the subsequent

transient kinetic studies. Altogether, the N370A and K228A substitutions did not

affect the formation and stability of the enzyme complexes.

Figure 2-3 PV RdRp fidelity can be manipulated by altering the conformational

equilibrium of motif D. A, B. X-ray crystal structure of PV RdRp (PDB 2IM2). Colored motifs

include motif A (red), B (green), C (yellow), D(blue), E (purple), F(brown). Lys228, Asn370,

Lys359, Glu364, and probe Met354 are shown in blue spheres. We use Met354 as a

sensor of the motif D loop conformation. C. The pre-steady-state kinetic mechanism of

single nucleotide incorporation by PV RdRp in the presence of Mg2+. Kinetic scheme

including binding, pre-chemistry conformational change, chemical reaction, post-

chemistry conformational change, and the release of pyrophosphate group. For correct

nucleotide insertion, conformational change and chemistry (in red) are both rate limiting.

22

Figure 2-4 Formation and stability of RdRp-RNA complexes are not affected by the

K228A and N370A substitutions. Reaction was conducted in buffer containing 50 mM

HEPES pH7.5, 10mM BME, 5 mM MgCl2 and 60 μM ZnCl2. A. Sequence of sym/subU

RNA. B, C. In the assembly assay, ATP and RNA were first incubated for 5 min followed

by the addition of RdRp to initiate the reaction. Reaction was quenched by adding 25 mM

EDTA. Final concentrations of RdRp, RNA, and ATP were 1, 1, and 500 μM respectively.

The data for formed WT RdRp-RNA complexes (black circle), K228A RdRp-RNA

complexes (cyan circle) and N370A RdRp-RNA complexes (red circle) was plotted as a

function of time. D, E. In the dissociation assay, RdRp and RNA were incubated for 90 s

to allow the formation of RdRp-RNA binary complexes, the addition of trap (100 μM ATP)

were added to the complex. At each time point, ATP was allowed to react for 30 s and the

reaction was quenched by the addition of 25 mM EDTA. Final concentrations of RdRp,

RNA and ATP were 1, 0.1, and 500 μM respectively. Percentage of the remaining binary

complex was plotted as a function of time for WT (black circle), K228A (cyan circle) and

N370A (red circle). The solid lines represent the fit of data into single exponential function.

The kdis of WT, K228A and N370A for sym/subU are 3.04±0.19×10-4s-1,

2.82±0.24×10-4s-1, and 3.19±0.20×10-4s-1, respectively.

Single nucleotide incorporation assays were then performed to study the

change of enzyme fidelity of the two variants. In this kinetic study, fidelity is defined

23

as the ratio of the catalytic efficiency of correct nucleotide over that of incorrect

nucleotide. The catalytic efficiency in these assays equals the ratio of the maximal

rate constant kpol over the apparent dissociation constant Kd,app. The two variants

showed a comparative catalytic efficiency in correct ATP incorporation while The

K228A was more efficient at incorporating incorrect GTP while N370A was less

efficient compared to WT enzyme (Figure 2.4, Table 2-1). These results suggested

that the K228A substitution lowers the fidelity while the N370A substitution shows

a higher fidelity.

24

Figure 2-5. The K228A substitution lowers the fidelity while the N370A substitution shows a higher

fidelity. A. Sym/sub U RNA structure. B. Reaction scheme for the stopped flow experiment.

Reactions was carried out in 50 mM HEPES pH7.5, 10mM BME, 5 mM MgCl2 and 60 μM ZnCl2.

RdRp and RNA were incubated for 2 min to allow the formation of RdRP-RNA binary complexes at

room temperature. Reaction was then initiated by the addition of an equal amount of ATP at various

concentrations. Changes in fluorescence were recalculated as product formed in time. Final

concentration of RdRp and RNA were 0.5 and 0.5 μM respectively. C. Comparison of the correct

ATP incorporation rates among WT (black), K228A (cyan) and N370A(red). The two variants were

as efficient as WT in the incorporation of correct ATP incorporation. D. Reaction scheme of the

benchtop assay. E. Comparison of the incorrect GTP incorporation rates among WT (black),

K228A(cyan) and N370A(red). The K228A was more efficient at incorporating incorrect GTP while

N370A was less efficient compared to WT enzyme. Solid lines represent the fit of the data into

hyperbola function. The result kpol and KD,app for each nucleotide incorporation are shown in table

2-1.

25

Table 2-1 Comparison of nucleotide incorporation for WT, K228A and N370A RdRps

reveal that K228A lowers the fidelity while N370A shows a higher fidelity.

26

2.4 Conclusions

The fidelity change caused by the amino acid substitution is very subtle.

And there is no virulence study done to these two substitutions. It is hard to

conclude that by making this single amino acid substitution, it leads to a huge

change in the virus. But the importance of this study is that we point out the motif

D conformational equilibrium and by changing residues that affect the equilibrium

rather than the active site residue, we can change the RdRp fidelity by 2 folds. The

altered RdRp fidelity is directly related to virulence, so the study of motif D

equilibrium can lead to a new way to rational design RNA virus vaccines.

References 1. Hayden, F. G., Update on influenza and rhinovirus infections. In Antiviral Chemotherapy 5, Springer: 1999; pp 55-67. 2. Li, L.; Wo, J.; Shao, J.; Zhu, H.; Wu, N.; Li, M.; Yao, H.; Hu, M.; Dennin, R. H., SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients. Journal of Clinical Virology 2003, 28 (3), 239-244. 3. report, W. Ebola virus disease – Democratic Republic of the Congo; 2018. 4. Ng, K. K.-S.; Arnold, J. J.; Cameron, C. E., Structure-function relationships among RNA-dependent RNA polymerases. In RNA Interference, Springer: 2008; pp 137-156. 5. Castro, C.; Smidansky, E. D.; Arnold, J. J.; Maksimchuk, K. R.; Moustafa, I.; Uchida, A.; Gotte, M.; Konigsberg, W.; Cameron, C. E., Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat Struct Mol Biol 2009, 16 (2), 212-218. 6. Carvalho, A. T. P.; Fernandes, P. A.; Ramos, M. J., The Catalytic Mechanism of RNA Polymerase II. Journal of Chemical Theory and Computation 2011, 7 (4), 1177-1188. 7. Korboukh, V. K.; Lee, C. A.; Acevedo, A.; Vignuzzi, M.; Xiao, Y.; Arnold, J. J.; Hemperly, S.; Graci, J. D.; August, A.; Andino, R.; Cameron, C. E., RNA Virus Population Diversity, an Optimum for Maximal Fitness and Virulence. The Journal of Biological Chemistry 2014, 289 (43), 29531-29544. 8. Arnold, J. J.; Vignuzzi, M.; Stone, J. K.; Andino, R.; Cameron, C. E., REMOTE-SITE CONTROL OF AN ACTIVE-SITE FIDELITY CHECKPOINT IN A VIRAL RNA-DEPENDENT RNA POLYMERASE. The Journal of biological chemistry 2005, 280 (27), 25706-25716.

27

9. Yang, X.; Welch, J. L.; Arnold, J. J.; Boehr, D. D. J. B., Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance. 2010, 49 (43), 9361-9371. 10. Vignuzzi, M.; Stone, J. K.; Arnold, J. J.; Cameron, C. E.; Andino, R., Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 2006, 439 (7074), 344-348. 11. Vignuzzi, M.; Wendt, E.; Andino, R., Engineering attenuated virus vaccines by controlling replication fidelity. Nature medicine 2008, 14 (2), 154-161. 12. Yang, X.; Smidansky, E. D.; Maksimchuk, K. R.; Lum, D.; Welch, J. L.; Arnold, J. J.; Cameron, C. E.; Boehr, D. D., Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure (London, England : 1993) 2012, 20 (9), 1519-1527. 13. Liu, X.; Yang, X.; Lee, C. A.; Moustafa, I. M.; Smidansky, E. D.; Lum, D.; Arnold, J. J.; Cameron, C. E.; Boehr, D. D., Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity. J Biol Chem 2013, 288 (45), 32753-65. 14. Studier, F. W. J. P. e.; purification, Protein production by auto-induction in high-density shaking cultures. 2005, 41 (1), 207-234.

Chapter 3

Investigation of the Relationship Between Nucleotide Selection and Replicative Speed Using NMR Spectroscopy

3.1 Introduction

Mutation rate is the probability of new mutations in a single gene or the

frequency of a mutation occurring in the lifetime of an organism over time in

genetics45. Mutations are the ultimate source of intraspecific variability in many

viruses and natural selection acts as the driving force. There has been discussion

on the mutation rate in viruses over a long time29. Viruses with high mutation rate

are more error-prone and may lead to fatal mutations. However, viruses with lower

mutation rate result in reduced genetic variation, and it leads to a reduce in virus

fitness. Previous work suggests that the selective pressure for higher mutation

rates is due to the increased supply of potential beneficial mutations. However, it

is hard to determine the actual reason of a higher mutation rate separating from its

consequences.

RNA viruses are the ideal model to study the mutation rate driving force.

RNA viruses have the highest replicative error rates in nature, with approximately

one mistake made per 1,000-100,000 nucleotides in one genome replication

cycle28. Because of the high error rates, there are population diversities among

different RNA viruses. This complex populations is called quasispecies29. Under

selective pressure, quasispecies are important for survival since a few viruses that

contain beneficial mutations would survive and act as founders for the next

generation. It has been discussed that RNA viruses require high error rates, and

RNA viruses with high fidelity would be less able to adapt to complex

environment46. The observed attenuation of antimutator RNA viruses further

indicated the adaptive benefit of high mutation rates29.

29

One simple possible explanation of the adaptive value of high mutation rate

is that viruses evolve under a trade-off between replication speed and fidelity47. In

other word, faster replication is preferred even along with a decreased fidelity as

the cost48. RNA viruses are of the extreme example of r-selection where the

optimal evolutionary solution is the high replication rate49.

The G64S PV RdRp is a well-studied antimutator variant of poliovirus28 and

a recent study determined the selective force of mutation rate50. The higher fidelity

G64S leads to a significant fitness cost, and this fitness cost was directly linked to

viral replication kinetics. Further experiments confirmed that direct selection for

increased replication speed would result in an indirect selection of higher mutation

rates RdRp. The polymerases of other RNA viruses are likely to have the similar

speed-fidelity trade-off systems because of the structural and functional

similarities. By investigating another PV antimutator, K359H, they found out that

K359H has a slower polymerization kinetics and a higher fidelity compared to WT.

While they added another amino acid substitution I331F combined with K359H,

similar characteristics were found (Figure3-1).

Figure 3-1. This figure was made based on the data from Ref. 54. The kpol for the correct nucleotide

measures the speed of polymerization in vitro. The kpol, corr/kpol, incorr is an in vitro surrogate for fidelity,

as it measures the relative rates of incorporation for the correct and incorrect nucleotides. A higher

ratio indicates higher fidelity. A. The measured speed of polymerization in vitro for WT, K359H and

K359H/I331F. B. The measure relative fidelity for WT, K359H and K359H/I331F.

30

Nuclear magnetic resonance (NMR) has been used widely in modern

molecular biology to generate structural and dynamic information51. NMR is one of

the major techniques capable to determine the structural of bio-marcromolecules

at atomic level. Heteronuclear single-quantum correlation (HSQC) experiment is

used to correlate the chemical shift of a nitrogen or carbon to an attached proton52.

Particularly, in [Methyl-13C] methionine labeled protein sample, each methionine

in the protein will be represented by a single peak in the NMR spectrum, which

forms an atypical bond. To use HSQC experiment to interpret binding data, the

binding interactions are interpreted by monitoring peaks that have perturbed

chemical shifts as a result of ligand binding.

In this chapter, we will use NMR to investigate the difference in structural

and dynamic change in the RdRp while incorporating different nucleotides. Single

amino acid substitution of the active site lysine was introduced and conformational

state change upon different nucleotides incorporation was studied using NMR. The

NMR results will be a complementary to the kinetic study and will help us better

understand the relationship between polymerase fidelity and nucleotide

incorporation rate.

3.2 Methods

3.2.1 Materials

Nucleoside 5’-triphosphates and 2’-deoxynucleoside 5’-triphosphates (all

nucleotides were ultrapure solutions) were from GE Healthcare. 3’-

Deoxyadenosine 5’-triphosphate (cordycepin) was from Trilink Biotechnologies. All

RNA oligonucleotides were from Intergrated DNA Technologis. [Methyl-13C]

Methionine was from Cambridge Isotope laboratories. HisPur Ni-NTA resin was

from Thermo Scientific. Q-Sepharose fast flow was from Amersham Pharmacia

Biothce, Inc. The QuickChange site-directed mutagenesis kit was from Stratagene.

31

The plasmid DNA isolation Miniprep kit was from Qiagen. All other reagents were

of the highest grade available from Sigma or Fisher.

3.2.2 Plasmid construction

Plasmid K359H RdRp, K359H/I331F were from the Cameron lab, and the

sequences were confirmed by DNA sequencing at the Nucleic Acid Facility at the

Pennsylvania State University.

3.2.3 Overexpression and purification of RdRp

Overexpression and protein purification were the same as previously

described (see Methods in Chapter 2).

3.2.4 NMR spectroscopy sample preparation

Purified protein with [Methyl-13C] methionine labeling was concentrated to

greater than 500 uM, desalted using spin columns (Thermo scientific). RNA was

diluted to 50 mM HEPES pH7.6 in D2O and reannealed just prior to use.

Reannealing procedures were the same as previously described (see Methods in

Chapter 2).

The reannealed RNA and 3’-dATP were added to NMR buffer containing 10

mM HEPES pH 8.0, 200 mM NaCl, 5 mM MgCl2, 20 uM ZnCl2 and 0.02% NaN3

in D2O to a final concentration of 1 mM and 4 mM, respectively. The RNA and 3’-

dATP was incubated in NMR buffer at room temperature for 5 min. This binary

mixture was added to a desalting column in a falcon tube containing the purified

RdRp. The sample that has been buffer exchanged was collected and then the

RdRp-RNA-3’dATP mixture was transferred into a new microcentrifuge tube. This

tertiary mixture was incubated at room temperature for 4 hours. The desired NTP

32

was added to this mixture to a final concentration of 4 mM. A centrifuge step was

needed to remove any precipitate if necessary. Another buffer exchange is

required to remove any excess NTP.

3.2.5 1H-13C HSQC NMR experiment

NMR experiments were performed on a Bruker Avance III 600 MHz

spectrometer equipped with a 5mm inverse detection triple resonance

(1H/13C/15N) single axis gradient TCI cryoprobe. 1H-13C heteronuclear single

quantum coherence (HSQC) spectra were generally acquired at 64 (t1) *512 (t2)

complex matrix, with 64-128 scans per increment and 1.0 s recovery delay.

3.3 Results

3.3.1 Spectrum comparison of WT, K359H and K359H/I331F RdRp

Figure 3-2 1H-13C HSQC NMR spectrum of apo WT (black), K359H (red) and K359H/I331F (blue)

RdRp. The NMR spectra were taken in D2O based buffer (25 mM tris phosphate buffer, pH 8.0,

150 mM NaCl, 1 mM DTT and 0.02% NaN3). Note: the left and right parts are the same spectrum

with different contour level.

33

In 1H-13C HSQC NMR, each peak stands for a methionine group in the

protein. It is very useful in large protein since large protein spectrum is very

complex and contains many peaks that may overlap with each other. By only

investigating methionine groups in the protein, the spectrum is simplified, and it is

easier to find the peak of interest.

This methionine strategy has some disadvantages too. One major problem

is that many methyl groups have similar resonances and it will form the bulk

methionine group area that it is hard to distinguish between peaks. The methionine

groups are of very different chemistry environments all over the protein, it is difficult

to observe all the peaks at one contour level. The spectrum (Figure 4-1) was shown

in two different contour level, the left part was of high contour level in order to show

individual peak in the bulk methionine arear, and the right part was of low contour

level to show chemical shift of interesting methionine probes.

Different methionine probes are used to indicate chemical environment

change near this specific methyl group. For example, Met354 is the probe in motif

D, the resonance shift of Met354 indicates a change in motif D, probably

conformational change. The Lys359 residue is important both structurally and

catalytically. It can be inferred that by making single amino acid substitution

K359H, there will be a conformational change in motif D, and results in a chemical

shift of Met354 resonance. Interestingly, the spectrum of apo K359H RdRp not

only shows the predicted change in Met354, there are other changes that remote

from the mutation site. For example, the Met225 and Met323 resonances are also

shifted. This suggest a potential long-range interaction between the Lys359 and

34

other motifs in the palm domain. One other interesting change is that by change

lysine to histidine, the Met187 shows up on the spectrum and this suggests that

the K359H amino acid substitution also cause chemistry environment change in

the buried helix methionine groups.

With the introduction of the second amino acid substitution I331F, there will

be chemical changes in Met225 and Met323 from the crystal structure since they

are close to each other. The spectrum shows that the Met225 resonances shift

more compared to K359H and the Met323 spilt into two peaks which indicates a

possible change into a slow conformational change of Met323.

There are other changes on the methionine map of RdRp. For example,

Met394, a methionine probe that is important for nucleotide incorporation also

changes. It can be inferred from the resonance change of Met394 that the two

amino acid substitutions have effect on the nucleotide incorporation.

3.3.2 Optimal experimental conditions for the formation of RdRp-RNA-NTP

complex

35

Figure 3-3 1H-13C HSQC NMR spectrum of PV K359H apo RdRp and PV K359H bound with

different concentration of RNA. The black spectrum is of PV K359H apo protein, the red one is PV

K359H bound with 1mM s/su RNA and the blue one is PV K359H bound with 2 mM s/su RNA.

Note: the left and right parts are the same spectrum with different contour level.

As for spectrum quality, the apo protein (black) spectrum are the best. The

protein bound with RNA, either 1mM (red) or 2mM (blue), shows a higher level of

noise. This might result in an impurity in the RNA.

In order to test the difference in the nucleotide incorporation of these two

variants, I used K359H as the example to optimize the RNA incorporation condition

first. After the incorporation of RNA, it can be predicted there should be further

chemical shifts of Met394 which is the methionine probe for nucleotide

incorporation and Met 354 which is the methionine probe for motif D.

At first, 1mM RNA was added to the apo protein. Compare the apo spectrum

and protein bound with 1mM RNA spectrum, there are some differences in

predicted methionine groups. There are also resonance changes of Met225 and

36

Met323 which suggest that there are conformational changes in other motifs also.

However, the chemical shift change with 1mM RNA was very subtle, in other

words, the protein is not saturated with RNA.

The RNA concentration was doubled to investigate the conformational

change. With 2 mM RNA, the resonance shifts of Met354 and Met394 were more

obvious. The chemical shift changes in Met6 and Met74 indicates a long-range

interaction in the thumb domain. The Met187 that shows up on the apo-K359H

spectrum disappeared, and this may due to the conformational change in the

buried methionine group by the incorporation of RNA. In conclusion, 2 mM of

reannealed RNA is sufficient to investigate conformational change of nucleotide

incorporation.

3.3.3 Nucleotide Selection Mechanism Investigation Using NMR

With the success in formation of RdRp-RNA complexes, the investigation of

nucleotide selection mechanism in RdRp is now available using NMR as a tool.

All kinds of nucleotides can be used to study the differences in the conformational

state inside RdRp upon binding.

37

Figure 3-4 1H-13C HSQC NMR spectrum of PV K359H bound with different nucleotides. The black

spectrum is of PV apo-K359H, the red spectrum is of PV K359H bound with 8mM UTP, the blue

one is PV K359H bound with 8 mM 2’UTP and the purple one is PV K359H bound with 8mM CTP.

Note: the left and right parts are the same spectrum with different contour level.

There are resonances changes to different methionine probes with different

nucleotides incorporation. However, the differences between those nucleotides

are hard to tell. More experiments on other variants (I331F) and further study on

nucleotide analogs bound with RdRp will provide a more complete map of the

nucleotide selection mechanism. These experiments altogether suggest that the

standard protocol of RdRp incorporation RNA and nucleotides are established.

3.4 Discussion

38

It is important to finally optimized the RdRp-RNA-NTP complex formation buffer

condition. Since NMR is a powerful tool to study dynamic change during chemistry

reaction, NMR can be used to investigate motif D equilibrium change upon binding

different nucleotide. And this will help us understand the nucleotide selection

mechanism better and will provide a complementary study of the kinetic

experiment of speed-fidelity trade-off system.

There are other nucleotide analogs like valopicitabine, a prodrug of the nucleoside

analog 2’-C’-Methylctindine with anti-hepatitis C virus, which can be used in NMR

experiment. The standard protocol for regular nucleotide incorporation fits well in

these analogs and by using NMR, it is easy and clear to investigate specific

conformational change inside RdRp. And hopefully by a series of study of different

nucleotide analogs, there will be a better understanding of the mechanism of

nucleotide selection in RdRp.

References

1. Crow, J. F., The high spontaneous mutation rate: is it a health risk? Proceedings of the National Academy of Sciences of the United States of America 1997, 94 (16), 8380-8386. 2. Crotty, S.; Cameron, C. E.; Andino, R., RNA virus error catastrophe: Direct molecular test by using ribavirin. Proceedings of the National Academy of Sciences of the United States of America 2001, 98 (12), 6895-6900. 3. Pfeiffer, J. K.; Kirkegaard, K., Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice. PLoS Pathogens 2005, 1 (2), e11. 4. Pfeiffer, J.; Kirkegaard, K., A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. PNAS 2003, 100. 5. Regoes, R. R.; Hamblin, S.; Tanaka, M. M. J. P. R. S. B., Viral mutation rates: modelling the roles of within-host viral dynamics and the trade-off between replication fidelity and speed. 2013, 280 (1750), 20122047.

39

6. Elena, S. F.; Sanjuán, R., Adaptive Value of High Mutation Rates of RNA Viruses: Separating Causes from Consequences. 2005, 79 (18), 11555-11558. 7. Borderıa, A. V.; Elena, S. F. J. I., Genetics; Evolution, r-and K-selection in experimental populations of vesicular stomatitis virus. 2002, 2 (2), 137-143. 8. Fitzsimmons, W.; Woods, R. J.; McCrone, J. T.; Woodman, A.; Arnold, J. J.; Yennawar, M.; Evans, R.; Cameron, C. E.; Lauring, A. S. J. b., A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus. 2018, 309880. 9. Sugiki, T.; Kobayashi, N.; Fujiwara, T., Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists. Computational and structural biotechnology journal 2017, 15, 328-339. 10. Sørensen, O.; Eich, G.; Levitt, M. H.; Bodenhausen, G.; Ernst, R. J. P. i. n. m. r. s., Product operator formalism for the description of NMR pulse experiments. 1984, 16, 163-192.

Chapter 4

Investigation of Norovirus Polymerase Function in Well-Established Poliovirus Polymerase System

4.1 Introduction

Norovirus (NV) is a positive stand RNA, non-enveloped virus belonging to

Caliciviridae family(caliciviruses)15. NV is considered to be a major public health

problem in developed countries since NV is a major causal factor of

gastroenteritis53. Norwalk virus, a norovirus, is thought to be responsible for

roughly 90% of epidemic, non-bacterial outbreaks of gastroenteritis in humans

around the work54. However, effective treatments for gastroenteritis caused by NV

are not currently available. The lack of suitable cell culture system for human

calicivirus has prevented the study of effective treatments over previous

decades55.

With the development of gene analysis and experimental determination of

3D structural techniques56, the structural and nonstructural proteins from a range

of caliciviruses were studied further, and these structural studies provide a

molecular framework for understanding many aspects of their replication

strategies. Structural studies of the recombinant viral proteinase and polymerase

in complex with substrates and inhibitors provide a basis for understanding

substrate recognition and enzymatic mechanisms, thus setting the stage for the

design of new antiviral compounds.

Even though the norovirus and poliovirus are both single-strand RNA

viruses, the enzyme responsible for RNA replication is slightly different. In

41

poliovirus, the polymerase (3Dpol) is the only active enzyme for RNA replication,

the 3CD in poliovirus is a fully functional protease without polymerase activity57;

however, previous studies suggest that norovirus proteinase-polymerase(pro-pol)

and polymerase(pol) are both active forms of RNA-dependent RNA polymerase1.

The ProPol protein of Feline calicivirus (FCV) is the predominant for of the RdRp

observed in FCV-infected cells58. The 3D-like (3DL) polymerase of Rabbit

hemorrhagic disease virus (RHDV), a member of the genus Lagovirs in

Caliciviridae, was also enzymatically active as an RdRp when produced in

bacteria59. Both the FCV ProPol and RHDV Pol were able to synthesize RNA from

a heteropolymeric template in the absence of added primer, FCV ProPol was more

active than RHDV Pol, which indicates the ProPol part was more efficient than Pol

alone1. Previous studies in our group suggests a rational design of poliovirus

vaccine by manipulating the polymerase activity, this success could be applied to

NV since they have a similar RNA replication mechanism.

Ribavirin is the only FDA approved broad-spectrum viral inhibitor aimed at

positive strand RNA viruses and is used to treat severe patients infected with NV60.

However, the error-prone nature of RNA viruses facilitates viruses to evade

ribavirin treatment. Recently, Dr. Goodfellow and colleagues tried to treat NV

infections of two young patients with ribavirin. While the treatment appeared to be

initially successful, the viral load eventually increased again. Dr. Goodfellow and

colleagues were able to isolate clinical samples of the NV before and after ribavirin

treatment, and nucleic acid sequencing indicated that there were changes in the

coding sequence of the RdRp following ribavirin treatment. Possibly, some

mutations were involved into 3Dpol were involved to reduce sensitivity to ribavirin.

Several interesting mutations found in the coding region of 3Dpol validate this

hypothesis, when the genome encoding 3Dpol of the resistant NV strain (post2)

from the patients after ribavirin treatment failed was aligned with that of the NV

strain from the patients before (pre1) and after (post1) successful ribavirin

treatment in Dr. Goodfellow’s group. This observation suggests that these

42

mutations were used to enhance the ability of NV RdRp to filter out ribavirin from

correct nucleotide. Such fidelity mutant will also be helpful for the fidelity

mechanism studies of NV 3Dpol.

In this chapter, some preliminary experiments were conducted to test the

possibility of applying kinetic assays developed for PV RdRp to norovirus proteins.

Under these poliovirus kinetic reaction conditions, the norovirus pro-pol showed

no activity at all, which suggests a different RNA primer may be required and

further protein engineering might be needed to fully understand the norovirus RNA

replication mechanism.

4.2 Methods

4.2.1 Materials

[γ-32P] ATP and [α-32P] UTP (>7000Ci/mmol) were from VWR-MP

Biomedical. Nucleoside 5’-triphosphates and 2’-deoxynucleoside 5’-triphosphates

(all nucleotides were ultrapure solutions) were from GE Healthcare. 3’-

Deoxyadenosine 5’-triphosphate (cordycepin) was from Trilink Biotechnologies. All

RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO). T4

polynulceotide kinase was from New England Biolabs, Inc. [Methyl-13C] Methionine

was from Cambridge Isotope laboratories. HisPur Ni-NTA resin was from Thermo

Scientific. Q-Sepharose fast flow was from Amersham Pharmacia Biothce, Inc.

The QuickChange site-directed mutagenesis kit was from Stratagene. The plasmid

DNA isolation Miniprep kit was from Qiagen. All other reagents were of the highest

grade available from Sigma or Fisher.

4.2.2 Plasmid construction

43

Pro-Pol mutants used in this chapter include H30A, C149A, and

H30A/C149A were made based on the pre1 (NV plasmid from patient 1 pre-

ribavirin treatment), post1(NV plasmid from patient 1 post-ribavirin treatment) and

post 2((NV plasmid from patient 2 post-ribavirin treatment) plasmid from Dr.

Goodfellow’s lab with the design of forward and reverse primers following the

instructions of the QuichChange site-directed mutagenesis kit. The mutagenesis

products were confirmed by DNA sequencing at the Nucleic Acid Facility at the

Pennsylvania State University.

4.2.3 Overexpression and purification of ProPol

Overexpression and protein purification were the same as previously

described (see Methods in Chapter 2).

4.2.4 Active site titration assays

Active site titration assays were the same as previously described (see

Methods in Chapter 2). Briefly, the reaction was carried out in 50-ul mixture

containing 50mM HEPES buffer (pH 7.5), 10 uM BME, 5 mM MgCl2, 60 uM ZnCl2,

10 uM S/Su*, 500 uM ATP and 1uM NV Pre1. Reactions were incubated at 30 ℃

and quenched by addition of EDTA to a final concentration of 50 mM.

4.3 Results

The previous studies suggest that both ProPol and Pol are active forms of

norovirus RNA-dependent RNA polymerase. At first, the norovirus ProPol was

purified with no modification to the plasmid, which was a ProPol with an active

protease domain. The purification of the active norovirus ProPol showed that the

ProPol would cleave itself and modifications were required to get an intact

norovirus ProPol.

44

Two forms of recombinant proteinase-polymerase (Pro-Pol) were used

previously to study the mechanism of NV replication strategy1, one is Pro-Pol with

an inactivated proteinase domain to prevent autocleavage, and the other one is

mutant His-E1189A-ProPol protein with active proteinase but with the natural Pro-

Pol cleavage site blocked. This suggested that the inactivation of the active site in

proteinase domain would help us to get NV ProPol sample without self-cleavage.

Examination of the Chiba virus 3C-like protease (CVP) structure confirms

that His30 and Cys139 are catalytic residues and shows that Glu54 is in the

position of the third active site residue (Figure 3-1). The Chiba virus was isolated

form a patient with gastroenteritis acquired in Chiba, Japan in 198761. The active

site is located at the center of a deep cleft between the N- and C-terminal domains,

as are the active sites of other chymotrypsin-like proteases. His30, Glu54, and

Cys139 are conserved in all NV 3C-like proteases.

Figure 4-1 NV ProPol is self-cleaving in E-coli B834 cell culture. A.Map of

the cleavage site for ORF 1 of the MD145-12 NV. Figure is adapted from

reference1. B. Crystal structure of NV ProPol (PDB 2IPH). Three catalytic residues

Glu 54, His 30 and Cys 139 are shown in blue spheres. C A closer view of the

catalytic triad.

45

After the examination of NV ProPol, I used plasmid pre 1 as an example to

block the active site of NV ProPol. With site-directed mutagenesis, the active site

histidine and cysteine were changed to alanine. The plasmid with two mutations

(H30A/C149A) was used in further overexpression, purification and kinetic studies.

I successfully obtained intact ProPol protein without self-cleavage. In order to apply

our PV model system to study NV, the first step was to test the polymerase activity

using the same reaction condition. However, the H30A/C149A ProPol showed no

activity in the PV kinetic assay condition (Figure 3-2).

Figure 4-2. A. Purification gel of inactivated NV 3CD. B. The NV H30A/C139A ProPol was

not active in RNA replication. The reaction was repeated twice, the empty dot and black

dot represented the first and second reaction respectively.

4.4 Discussion

The NV activity assays with sym/sub-U RNA showed nearly no activity. This

suggests that the sym/sub-U primer/template system is not suitable for studying

46

nucleotide incorporation in NV. It will be easier to investigate if there is any

conformational change upon sym/sub U RNA binding to the NV ProPol using NMR.

Other possible reasons for the loss of polymerase activity include that the

ProPol extracted from the patients did not have the authentic N terminus of NV

ProPol. Studies in the family of caliciviridae tend to believe that unlike

picornaviridae, ProPol is not necessary to be cleaved to have polymerase function,

ProPol showed the best activity in the polyr(U) activity assay, and 3D(pol) along

shows a weaker activity than ProPol. It is possible that some mutations from the

patients’ genome would prohibit 3Dpol function and shut down the ProPol as the

main polymerase.

Another interesting fact from previous NV polymerase studies was that by

changing the active site histidine other residues, the NV ProPol protease is of no

activity. While by changing the active site cysteine to alanine, the NV ProPol

protease retains about 30% of the protease function61. Two forms of recombinant

Pro-Pol were used in previous NV polymerase studies, one is Pro-Pol with an

inactivated proteinase domain to prevent autocleavage, and the other one is

mutant His-E1189A-ProPol protein with active proteinase but with the natural

ProPol cleavage site blocked. Another recombinant protein PRO-, which does not

have any protease activity show no RdRp activity. This suggests a potential

communication between the NV protease and polymerase domain. However, the

protein in our kinetic study is with two active site mutations which will cause the

protease to lose activity. Thus, a NV ProPol with lowered protease function might

be used to study the polymerase function.

References

1. Robilotti, E.; Deresinski, S.; Pinsky, B. A., Norovirus. 2015, 28 (1), 134-164. 2. Elliott, E. J., Acute gastroenteritis in children. BMJ (Clinical research ed.) 2007, 334 (7583), 35-40.

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3. Glass, R. I.; Parashar, U. D.; Estes, M. K., Norovirus gastroenteritis. The New England journal of medicine 2009, 361 (18), 1776-1785. 4. Vashist, S.; Bailey, D.; Putics, A.; Goodfellow, I., Model systems for the study of human norovirus Biology. Future virology 2009, 4 (4), 353-367. 5. Clarke, I. N.; Lambden, P. R., Organization and Expression of Calicivirus Genes. The Journal of Infectious Diseases 2000, 181 (Supplement_2), S309-S316. 6. Marcotte, L. L.; Wass, A. B.; Gohara, D. W.; Pathak, H. B.; Arnold, J. J.; Filman, D. J.; Cameron, C. E.; Hogle, J. M., Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase. Journal of virology 2007, 81 (7), 3583-3596. 7. Belliot, G.; Sosnovtsev, S. V.; Chang, K.-O.; Babu, V.; Uche, U.; Arnold, J. J.; Cameron, C. E.; Green, K. Y. J. J. o. v., Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. 2005, 79 (4), 2393-2403. 8. Wei, L.; Huhn, J. S.; Mory, A.; Pathak, H. B.; Sosnovtsev, S. V.; Green, K. Y.; Cameron, C. E., Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase. Journal of virology 2001, 75 (3), 1211-1219. 9. Urakova, N.; Strive, T.; Frese, M., RNA-Dependent RNA Polymerases of Both Virulent and Benign Rabbit Caliciviruses Induce Striking Rearrangement of Golgi Membranes. PloS one 2017, 12 (1), e0169913-e0169913. 10. Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y.; Hong, Z.; Andino, R.; Cameron, C. E., The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat Med 2000, 6. 11. Nakamura, K.; Someya, Y.; Kumasaka, T.; Ueno, G.; Yamamoto, M.; Sato, T.; Takeda, N.; Miyamura, T.; Tanaka, N., A norovirus protease structure provides insights into active and substrate binding site integrity. Journal of virology 2005, 79 (21), 13685-13693.

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Chapter 5

Conclusion and Future Directions

5.1 Conclusions

Outbreaks of RNA viruses cause a number of acute and chronic diseases,

and the emergence of new viruses lead to severe issues in human health, food

security and even social stability. Take polio for example, in the early 20th century,

polio was one of the most threatened diseases among the world, and polio caused

hundreds of thousands of children paralyzing every year. After the investigation of

effective vaccines in 1960s and the beginning of the Global Polio Eradication

Initiative in 1988, polio was under control and the polio was eradicated 99% in

2013 according to WHO report. However, there are still vaccine-derived

polioviruses (VDPVs) that may cause the similar polio outbreak and wild endemic

polioviruses are found in Afghanistan, Nigeria and Pakistan. Polioviruses are one

of the most well-studied viruses and the Sabin I vaccine is also one of the most

successful RNA vaccines. However, there are still severe issues in completely

eradicating poliovirus. As for other RNA viruses, the unknown identity of RNA

viruses, endemic transmission among people and animals, limited availability of

antiviral strategies and the high mutation rates of RNA viruses will result in

infectious disease.

There have been great efforts in the development of viral vaccines and

antiviral drugs. Ribavirin has proven to be a useful treatment of a variety of viral

infection for decades5. It has now been proposed that ribavirin treatment leads to

“lethal mutagenesis”6, owing to the ability of ribavirin to become incorporated into

the viral RNA and base pair with either uracil or cytosine. The polymerase of RNA

viruses can also be used as a potential drug target. RdRps have a conserved

structure of a cupped ‘right hand’ structure, and have three major subdomains:

palm, finger and thumb. It is now established that the accuracy or ‘fidelity’ of RNA

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synthesis is closely tied to virus biology. Previous studies have also suggested that

viruses encoded with RdRps having altered fidelity may serve as potential vaccine

strains.

In this thesis, I studied the relationship between the motif D conformational

equilibrium and enzyme fidelity, the effects of enzyme fidelity on the virus

replication rates and began preliminary kinetic study on norovirus polymerase.

In Chapter 2, it was shown that N370A and K228A substitutions on motif D

in PV RdRp altered the RdRp fidelity by perturbing the motif D conformational

state. It was concluded that motif D undergoes an “open” to “closed”

conformational change and this investigation leads to a rational design of site-

directed mutagenesis in motif D by combination of MD simulation and NMR.

In Chapter 3, the reposition of active site lysine residue was caused either

by direct amino acid substitution of lysine residue or by long-range interactions

from other motifs. The standard protocol of RdRp-RNA-NTP complexes formation

in NMR study was established and this can help the study of the effects of RNA

analogs as potential antiviral drugs in NMR.

In Chapter 4, the potential use of PV kinetic study as a model system was

discussed. Norovirus polymerase was successfully purified, and first stage kinetic

study showed that further investigation of norovirus polymerases using PV system

required modifications.

Altogether, these studies suggest that motif D and other motifs determine

enzyme fidelity by repositioning of the active site lysine and by a rearrangement of

the motif D conformational state. These studies provide a better understanding in

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nucleotide selection mechanism and this will facilitate the study of antiviral

strategies against RNA viruses.

5.2 Future Directions

The preliminary study on NV suggests a possible communication between

the protease motif (3C) and the polymerase motif (3D) in viruses. Since there are

both structural and functional similarities in NV and PV, it is possible to conclude

that these allosteric pathways are also important in poliovirus 3CD. Our group has

been working on optimizing NMR experiment conditions for poliovirus 3CD, and

there is significant progress recently. The further investigation of the activity of NV

3CD can be studied in NMR first. NMR is a great tool to show the conformational

change upon ligand binding. Using the standard protocol establish for 3D-RNA-

NTP complexes formation, the NV 3CD-RNA-NTP complexes could be formed.

The activity of NV 3CD can be determined by looking for the conformational

change.