The Role of RNA Secondary Structure in Replication of Nodamura Virus RNA2 John Howard Upton University of Texas at El Paso, [email protected]

The Role of RNA Secondary Structure in Replication of Nodamura Virus RNA2 John Howard Upton University of Texas at El Paso, Jhupton@Miners.Utep.Edu

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2009-01-01 The Role of RNA Secondary Structure in Replication of Nodamura Virus RNA2 John Howard Upton University of Texas at El Paso, [email protected]

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THE ROLE OF RNA SECONDARY STRUCTURE IN REPLICATION OF NODAMURA VIRUS RNA2

JOHN H. UPTON, III

Department of Biological Sciences

APPROVED:

______Kyle L. Johnson, Ph.D., Chair

______Germán Rosas-Acosta, Ph.D.

______Ming-Ying Leung, Ph.D.

______Patricia D. Witherspoon, Ph.D. Dean of the Graduate School

DEDICATION

This thesis is dedicated to my family and friends who have encouraged me through the years. Without their patience, understanding, support, and most of all love, the completion of this work would not have been possible.

THE ROLE OF RNA SECONDARY STRUCTURE IN REPLICATION OF NODAMURA VIRUS RNA2

JOHN H. UPTON III, B.S.

THESIS

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

in Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Biological Sciences

THE UNIVERSITY OF TEXAS AT EL PASO

August 2009 ACKNOWLEDGEMENTS

I express my gratitude and love to my wife, Janeen, and our three children for their continued support and encouragement. She never stopped believing in me, always reminding me that I could achieve anything I set my mind to. You gave me immense support, desire to be a role model to our children, and the drive to complete my tasks without pressure. Thank you.

To the Command and the members of the 7220th Medical Support Unit, thank you for the support and understanding while I worked on this project. My military mentors gave me more than they realize. The person I am today is a result of all the great individuals that I have encountered throughout my tours: Leaders, Peers, and subordinates alike.

I would like to convey my appreciation to Dr. Kathy Hanley and her laboratory staff, particularly Tammy Romero, for guiding me through the nuclease mapping procedures. Without you, I would not be where I am today. I am certain that without your support this project would not be done. Thank you for your confidence in me and for helping me achieve what seemed impossible.

A special thanks to all of the UTEP Biology staff members who assisted me while using unfamiliar laboratory equipment: Lani Alcazar in the Biomolecule Analysis Core facility, Berenice Arriaga in the HHMI Student Molecular Research Lab Core facility, and especially Omar Hernandez in the DNA Analysis Core facility whose patience and assistance with the optimization of this project’s sequencing and mapping gels was greatly appreciated.

v I would also like to acknowledge some of the outstanding faculty members at

UTEP whose dedication and influence furthered my love of the sciences: Thank you, Dr.

Stephen Aley for your recommendation letters and guidance throughout my time at

UTEP. Thank you, Dr. Donna Ekal for your recommendations and your vital role in helping me achieve all my goals. Thank you, Dr. Tina Garza for your recommendation letters and for the wonderful experience working as your T.A. in Molecular Cellular

Biology. Finally, thank you, Dr. Mahesh Narayan for your recommendations, advice, and great sense of humor. You each were an integral part of my success and have made a lasting impression.

My sincere gratitude to all the members of the Johnson Lab, especially John

Rosskopf, my right hand man, technical advisor and good friend for putting up with my onslaught of endless questions, volleying my ideas and concerns as they came up, and helping to keep my spirits high throughout this project.

I would like to thank the members of my committee; Dr. Ming-Ying Leung for her guidance and assistance with computer modeling and predictions; Dr. German Rosas-

Acosta for his advice and wisdom, which were greatly appreciated; and finally I would like to express my deep appreciation to my committee chair and mentor, Dr. Kyle L.

Johnson. Your instruction on this project and beyond has helped me grow in ways I am just beginning to comprehend. Thank you for showing me that this was not an impossible task. You came through for me while I was under pressure, without hesitation. You are more of a role model to me than you know.

vi ABSTRACT

Our laboratory studies genome replication of positive-strand RNA viruses, using a reverse-genetic system in yeast cells. Nodamura virus (NoV) provides an excellent model system for the study of RNA replication due to its genetic simplicity, its robust yield of replication products, and its ability to replicate in a wide variety of host cells.

NoV contains a bipartite positive strand RNA genome: RNA1 encodes the viral RNA replicase, while RNA2 encodes the capsid protein. The role of RNA secondary structure in the genome replication of other RNA viruses has been well established. For NoV, sequences at the 3’ end of RNA2 are critical for RNA replication and we hypothesized that the secondary structure adopted by these RNA sequences is essential for RNA replication. Three different software programs were used to generate predicted secondary structures of the 3’-terminal end of NoV RNA2; all three consistently predicted the presence of a conserved stem-loop structure within this region. We tested whether the predicted structure has biological relevance in the viral life cycle and whether its formation could be verified experimentally. The structure was deleted from a cDNA clone of RNA2 using site directed mutagenesis. Yeast cells were transformed with wildtype or mutant RNA2 together with RNA1 as a source of RdRp and RNA replication products were detected by Northern blot hybridization. Our results showed that deletion of the predicted stem-loop structure resulted in a severe defect in RNA replication. We also used nuclease mapping to confirm that NoV RNA2 forms this structure in solution. These data suggest that the 3’-terminal region plays a significant role in RNA2 replication. Its exact role is, as yet, unknown.

vii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... v

ABSTRACT...... vii

TABLE OF CONTENTS...... viii

LIST OF FIGURES...... x

Chapter

1. INTRODUCTION...... 1

1.1. RNA Secondary Structure...... 2

1.2. Nodaviruses………………...... 5

1.3. Nodavirus RNA Replication...... 7

1.4. Prediction of NoV RNA2 Secondary Structure...... 10

1.5. Summary………………………………………………...... 13

2. MATERIALS AND METHODS ...... 14

2.1. Cells and Growth Conditions ...... 14

2.2. Plasmids Used...... 15

2.3. RNA Isolation and Analysis...... 18

2.4. Nuclease Mapping and Primer Extension Analysis...... 18

3. RESULTS ...... 20

3.1. Prediction of a Stem-Loop Structure in NoV RNA2……...... 20

3.2. Effect of Stem-Loop Deletion on RNA2 Replication ...... 21

3.3. The Predicted Stem-Loop Can Form Under Biological Conditions...... 27

viii 4. SUMMARY AND CONCLUSIONS ...... 31

4.1. Summary ...... 31

4.2. Conclusions ...... 32

LIST OF REFERENCES ...... 36

CURRICULUM VITA...... 40

ix LIST OF FIGURES

1. Three types of structures adopted by RNA………………………………………………..3

2. Nodavirus genome organization schematic……………………………………………….7

3. Schematic of nodavirus RNA replication strategy………………………………………..8

4. Initiation of NoV RNA replication in transformed yeast cells…………………………….9

5. RNA Secondary Structure Predictions for NoV2 nucleotides 1287-1336…………..11

6. RNA Secondary Structure Predictions for NoV2 nucleotides 1237-1336…………..11

7. Schematic of plasmids pN2GFP and pN2GFPΔSL1.………………………………...... 22

8. Schematic of plasmids TpG-NoV1 (pN1) and TpG-NoV1R59Q (pN1R59Q).………..23

9. ΔSL1 mutant doesn’t affect RNA1 replication or RNA3 synthesis………………...... 24

10. ΔSL1 mutant exhibits a defect in RNA2 replication. ……………………………...... 25

11. The predicted structure can form in solution …………………………………………..29

x CHAPTER 1

INTRODUCTION

Secondary structures of many viral RNA genomes and subgenomic sequences have been shown to play important roles in a wide variety of processes that comprise the viral life cycle. These structures exist for varied functions, such as protection of the viral sequence from enzymatic digestion by host cellular defenses, for initiation of viral sequence translation both internally and near capped or uncapped ends, and even for the initiation of RNA replication (3, 9, 10, 24, 29). Our laboratory studies the mechanism of RNA replication for members of the virus family Nodaviridae. These viruses, which contain bipartite positive-strand RNA genomes, have been used for studies of virus structure, assembly of progeny viruses, and RNA replication. Here we describe studies that investigate the role of RNA secondary structure in nodavirus RNA replication, using

Nodamura virus (NoV) in transformed yeast cells as a model system.

Our hypothesis is that a predicted RNA secondary structure at the 3’ end of

NoV RNA2 is required for its replication. This 3’ RNA secondary structure may have some specific affinity for host cellular proteins and/or viral proteins that participate in the replication complex including the binding of the RNA1 encoded viral RdRp and/or RNA3 association directing the RdRp to the template. In testing this hypothesis, we will gain valuable knowledge relating to RNA secondary structure along with the mechanisms of initiating NoV RNA replication. These studies have involved the use of site-directed mutagenesis to delete the NoV RNA2 sequence involved in the predicted secondary structure and to determine its function (if any) in the viral replicative cycle, as described in Chapter 3. We then go on to show that the predicted secondary structure at the 3’ end of NoV RNA2 can form under biological conditions, using nuclease mapping.

1.1. RNA Secondary Structure

RNA secondary structure is formed by intramolecular base pairing interactions in the sequence of a single-stranded RNA molecule. There are a few different types of secondary structures commonly adopted by RNA molecules. Most of these structures are either helical or varied types of loops which have unpaired nucleotides closed by helical structures. As shown in Figure 1, a stem-loop structure consists of a short unpaired region closed with a base paired helix and is one of the most common RNA structures. Stem loops are also the basis for larger RNA structural motifs like the cloverleaf, which creates a four part structure similar to that of tRNA (61). Other structures (Figure 1) include internal loops, short stretches of unpaired bases within a helical structure, and bulge loops, where one side of a strand in a helix has unpaired bases. Pseudoknots are yet another RNA structure that complicate matters. These occur when a stem loop folds over and the unpaired loop region base pairs with another unpaired region. The unpaired region with which it pairs designates what specific type of pseudoknot is formed. The simplest pseudoknot is referred to as the H-type. Several other types of pseudoknot have been described, including B-types, I-types, M-types, and H-H-types, but the reader is referred to an excellent recent review for details, since they are beyond the scope of the project (9).

2 Figure 1. Three types of structures adopted by RNA. Stem-loop structures consist of base-paired regions (double-stranded stems) connected by single-stranded loops at the end of a stem (hairpin loops), in the body of a stem by bulge (B) or interior (I) loops, or at the junction of multiple stems (multi-branched (M) loops). These secondary structures can combine to form higher-order structures (tertiary structures. One such tertiary structure is the pseudoknot, formed when nucleotides in a loop base pair with a single- stranded region elsewhere in the overall structure. The hairpin type (H-type) pseudoknot involves bases in the loop of a hairpin loop. An H-type pseudoknot contains two stems, S1 and S2, connected by single- stranded loops. In some cases, there may be no unpaired bases present between the two stems and the stems form a co-axial stack that appears helical. The figure was taken from Brierly et al. (9).

RNA secondary structures have been found to have a wide range of functions in viral processes. These structures are so important for the function and/or preservation of the RNA molecule that often the structures are preserved even when the sequences are not. Stem-loops and other secondary structures at the ends of RNA molecules may act to protect the sequence from being degraded by nucleases that would otherwise recognize and destroy the ends (29). Stem-loops found in the positive-strand genomic

RNA of the plant virus tomato bushy stunt virus (TBSV) have been shown to regulate minus-strand synthesis and subsequent rounds of RNA replication (15, 44, 45). In other viruses, similar structures aid in recognition of translational start sites or are required for initiation of RNA replication. For example, a bulged stem-loop structure found in the 3’

UTR of mouse hepatitis virus (a coronavirus) was determined to be essential for viral

3 RNA replication (24). Pseudoknots and other structures have been shown to be essential for circularization of the genomes of some viruses, have various catalytic activities such as formation of peptide bonds between amino acids during protein synthesis, or create internal ribosomal entry sites (IRES) that can initiate translation of mRNAs in the absence of a 5’ cap structure (9). A hairpin-type pseudoknot located in the 3' UTR of the bovine coronavirus has been suggested as a requirement for RNA replication (58). Finally, a cloverleaf structure found at the 5’ end of poliovirus RNA serves as a cis-acting element that directs initiation of minus-strand synthesis (35).

So, how can one find or predict where RNA secondary structures occur in a particular RNA genome? One of the most popular bioinformatic tools used for this today is the Mfold web server. This server is commonly used because of its user friendly interface which is able to quickly provide access to RNA and DNA folding predictions

(61). The Mfold or “Multiple fold” software was developed at the end of the 1980s for folding RNA using an algorithm which predicts the total minimum free energy along with minimum free energies for folding which involve any particular base pair (59). Since its development, Mfold has undergone many revisions to reach its current server-based form. This server is available to any and all users and the input and setup of the software is fairly intuitive and easy to use, as it allows users to simply click and paste.

The disadvantage is that, for reasons of efficiency and the use of minimum free energy as a basis for its structural predictions, the program does not allow for prediction of pseudoknots.

There are other programs available that incorporate pseudoknot predictions, including NuPack, Pseudoknots RE (PknotsRE), and Pseudoknots RG (PknotsRG).

These programs are also available on a web-based server at UTEP, called RNAVLab.

4 The NuPack program package, by Dirks and Pierce, enables thermodynamic analysis of secondary structures of nucleic acid for single strands with and without pseudoknots and for multiple interacting strands without pseudoknots (11, 12). PknotsRE, an algorithm by Rivas and Eddy, generates the optimal minimum energy for a structure.

This tool has been implemented for a single-stranded RNA sequence using standard thermodynamic parameters but is limited to predictions of 150 nucleotides at present

(47). PknotsRG was created by Jens Reeder and Robert Giegerich and is an improvement of the Rivas and Eddy algorithm and considers the class of simple recursive pseudoknots (46). Although the class of pseudoknots it predicts is more restricted than PknotsRE, the same pseudoknots can be found within a longer nucleotide sequence. If time is a factor, consider that this program, at present, can predict the folded structure of a sequence of 1000 nucleotides in approximately 12 hours (46). We have used NuPack, PknotsRE, and PknotsRG to predict RNA secondary structures adopted by the genomic RNAs of members of the virus family

Nodaviridae, as described in section 1.4, and in reference (55).

1.2. Nodaviruses

The virus family Nodaviridae is comprised of two genera: alphanodaviruses and betanodaviruses. The alphanodaviruses, which infect predominantly insects, include

Flock House virus (FHV), black beetle virus (BBV), Boolarra virus (BoV), Pariacoto virus

(PaV), and the type species of the genus, Nodamura virus (NoV). The betanodaviruses, including barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), greasy grouper nervous necrosis virus (GGNNV), redspotted grouper nervous necrosis virus (RGNNV), and the type species of the genus, striped jack

5 nervous necrosis virus (SJNNV), have been isolated only from fish (52). The namesake of the family, NoV, infects not only insects, but it has also been shown to lethally infect suckling mice and hamsters (19, 20). The ability of NoV to replicate its genome to tremendously high levels in a wide variety of cells, including those of the yeast

Saccharomyces cerevisiae, presents unique circumstance for studying its intricate processes (5, 41-43, 50, 51, 53). Therefore, our laboratory uses members of the nodavirus family to study the basic mechanism of viral RNA replication.

The members of the Nodaviridae contain bipartite single-stranded, positive-sense

RNA genomes with a total length of approximately 4.5 kb (6, 7). Both nodavirus genomic RNA segments possess a 5’ cap structure and lack poly(A) tails at the 3’ end

(25, 27). As shown schematically in Figure 2, the RNA1 segment contains an open reading frame that encodes the viral RNA-dependent RNA-polymerase (RdRp), protein

A, while the smaller RNA2 segment contains an open reading frame for the capsid protein precursor (25). These viruses also synthesize a subgenomic RNA3 that is co- terminal with the 3’ end of RNA1. RNA3 contains overlapping open reading frames that encode the nonstructural proteins B1 and B2 (25, 26). Although the function of B1 is not yet known and its presence does not appear to have an effect on the viral life cycle, the B2 protein functions as a suppressor of the host cellular defense mechanism called

RNA interference (RNAi) (26, 31, 54).

Figure 2. Nodavirus genome organization. All members of the virus family Nodaviridae have the same general genome organization. Nodamura virus (NoV) is used as an example.

1.3. Nodavirus RNA Replication

The single-stranded, positive-sense RNA genomes of the nodaviruses are translated immediately upon entry into a host cell, because they are recognized as messenger RNAs (mRNAs) by the host protein synthesis machinery. As described above, the bipartite viral genome is recognized as two separate mRNAs: translation of

RNA1 produces the RdRp and translation of RNA2 produces the capsid precursor protein. Newly translated RdRp and viral genomic RNA will associate to form replication complexes (RC’s), which are the site of RNA replication in the host cell. These RC’s are contained within “spherules” that form in the outer mitochondrial membrane and project into the space between the outer and inner mitochondrial membranes (36-38). As replication proceeds, the RdRp catalyzes synthesis of negative-strand intermediates of the genomic RNAs. These intermediates serve as templates for synthesis of more positive strand genome copies, each of which is identical to those initially introduced to

7 the host cell (17, 18). During RNA replication, a small subgenomic RNA, RNA3, is synthesized from the RNA1 template; RNA3 is co-3’-terminal with RNA1 and encodes the nonstructural proteins B1 and B2 (see above). RNA3 has also been shown to replicate via negative strand intermediates (13).

Figure 3. Schematic of nodavirus RNA replication strategy. The RNA-dependent RNA polymerase (RdRp) encoded by RNA1 catalyzes replication of both genomic RNA segments. Negative strand RNA replication intermediates serve as templates for further positive strand synthesis.

Several different mechanisms exist in different viruses to regulate viral gene expression and help coordinate the competing processes of translation, replication, and packaging of the virus. For the nodaviruses, controlling transcription through production of a subgenomic RNA is the mechanism of choice (1, 13, 14, 32, 39). FHV RNA2 replication appears dependent on the presence of RNA3 to begin and it is thought that the subgenomic RNA3 acts in trans to activate RNA2. Further it was observed for FHV that RNA2 contains a cis-acting replication sequence, which is dependent on the RNA3 to initiate minus strand synthesis (1, 13, 14, 32). This negative strand intermediate acts as the template for mass production of positive sense RNA2 molecules and as levels of

8 RNA2 build, RNA3 transcription declines in response (1, 13, 14, 60). As the coordinated effort continues and positive strand copies build up, they begin to associate with the translated viral structural proteins to form new virus particles. Each new virus particle packages one molecule each of RNA1 and RNA2, so this coordination may represent a means to keep the levels of progeny genomic RNAs at an equimolar level to facilitate assembly of progeny viruses.

Figure 4. Initiation of NoV RNA replication in transformed yeast cells. Yeast cells are transformed with plasmids that express NoV genomic RNA1 from an inducible yeast RNA polymerase II (polII) promoter (GAL1). Primary RNA1 transcripts are synthesized by cellular RNA polII and translated by the cellular machinery to yield the viral RNA-dependent RNA polymerase (RdRp). The RdRp then initiates RNA replication, producing negative strand replication intermediates that are used to make further positive strands. Subgenomic RNA3 is synthesized and replicated by the RdRp. In the presence of a similar GAL1-RNA 2 plasmid (not shown), RNA2 is similarly replicated via negative strand intermediates.

The study of nodavirus RNA replication mechanisms has been greatly facilitated by their broad host range. While NoV can infect and kill both insect and mammalian hosts (19, 56), introduction of the isolated RNA genomes of NoV or FHV into cultured cells of mammalian, insect, plant, or yeast origin results in exponential RNA replication

(4, 5, 25, 26, 33, 41, 43, 53, 56). In the yeast Saccharomyces cerevisiae, the entire replicative cycles of both FHV and NoV can be initiated from cDNA plasmids, resulting the production of infectious virus particles (41, 43). These plasmids express cDNA copies of NoV RNA1 and RNA2 under transcriptional control of an inducible yeast GAL1 promoter (41). On induction with galactose, the cells produce primary transcripts of

9 RNA1 that are translated to produce the viral RdRp, which initiates RNA1 replication and subgenomic RNA3 synthesis. If yeast cells are co-transformed with RNA1 and

RNA2, RNA2 is replicated by the RdRp in trans (41).

1.4 Prediction of NoV RNA Secondary Structure

Cis-acting replication signals RNA sequences that are required for an RNA to be used as a template for RNA replication. Other examples of cis-acting replication elements defined for a nodavirus genome segment are two cis-acting sequences located in FHV RNA1. These sequences, described by Lindenbach et al. (32), control the subgenomic RNA3 production from that same segment of RNA1. The authors showed that the two cis-acting elements, one near the RNA3 start site and another far upstream, participate in long-distance base pairing with one another (32).

Research on the related alphanodaviruses NoV and FHV has shown that major deletions of the capsid ORF while leaving the ends of RNA2 intact, has little or no effect on virus replication (14, 60). In fact, FHV deletion mutants that retain only 50 nucleotides at the 3’end of RNA2 are still able to replicate in cultured cells (1). With this in mind, we hypothesize that any RNA elements required for RNA2 replication reside in this 3’ terminal region. Many positive strand RNA viruses use secondary structure in such RNA elements to regulate and/or initiate RNA replication (15, 24, 35, 44, 45, 58).

Any secondary structure that exists within that element, particularly the remaining 50 bases at the 3’ end of nodavirus RNA2 could contribute to these viruses’ ability to replicate their genomic RNAs.

Therefore, we performed RNA secondary structure prediction analysis in collaboration with investigators in UTEP’s Bioinformatics Program and the Department

10 of Computer and Information Science at the University of Delaware. When the structure of the 3’-terminal 50 nt was predicted, programs PknotsRE (47), PknotsRG (46), and

NuPack (11, 12) all predicted a 24 nucleotide stem-loop structure that was positioned

14 bases upstream of the 3’ terminus of NoV2 (55), as shown in Figure 5.

Figure 5. RNA Secondary Structure Predictions for NoV2 nucleotides 1287-1336. A sequence file containing the 3’-terminal 50 nucleotides of NoV RNA2 was used for prediction by three different computer programs as described in the text. All three programs generated the identical prediction, shown here. Nucleotides marked in red indicate single-stranded regions, while blue indicates double-stranded regions. Predictions were performed by Taufer et al., 2008 (55).

Computer prediction analysis of the RNA2 segments from seven other members of the same virus family revealed the presence of a similar stem-loop structure near the

3’ end of each segment, suggesting that the structure is conserved within the family

Nodaviridae (55). When we predicted the structure of the 3’-terminal 100 nucleotides of the same RNA2 segments, PknotsRE (47), PknotsRG (46), and NuPack (11, 12) all predicted the same 24 nucleotide stem-loop structure in NoV RNA2, this time in the larger context of predicted pseudoknots that differed slightly from program to program

(55), as shown in Figure 6.

PKnotsRE

PKnotsRG

NuPack

Figure 6. RNA Secondary Structure Predictions for NoV2 nucleotides 1237-1336. A sequence file containing the 3’-terminal 100 nucleotides (nt) of NoV RNA2 was used for prediction by three different computer programs as described in the text. Top panel, PKnotsRE; middle panel, PKnotsRG; lower panel, NuPack. In each case, nucleotides marked in red indicate single-stranded regions, blue indicates double-stranded regions, and yellow indicates regions predicted to be involved in formation of a pseudoknot. Predictions were performed by Taufer et al., 2008 (55).

1.5 Summary

The studies described here were designed to test whether the structure predicted to form near the 3’ end of NoV RNA2 plays a functional role in the viral life cycle. We want to understand the importance of this 3’ end, the RNA secondary structure it is predicted to contain, and the role that structure plays in initiating RNA minus strand synthesis. This is of great interest in furthering our understanding of the mechanisms of

RNA replication for this virus and subsequently other, more complex replication systems. Greater understanding of this system and these processes could assist in the design of NoV-based vectors for transient gene expression and vaccine development.

Therefore, discovering exactly what at the 3’ end of these sequences is necessary to maintain the integrity of replication is of great value.

13 CHAPTER 2

MATERIALS AND METHODS

2.1. Cells and growth conditions

Plasmids were amplified in various Escherichia coli strains, as follows. Subclones of PCR products introduced into the TA cloning vector pGEM-T Easy® (Promega) were amplified in the JM109 strain (Promega), while all other plasmids were amplified in strains NEB5α and NEB10β (New England Biolabs). Transformed E. coli cells were grown in Luria-Bertani (LB) broth, or on LB agar plates, each supplemented with ampicillin. RNA replication studies were performed in the yeast Saccharomyces cerevisiae synthetic deletion strain BY4733 (MATa his3Δ200 leu2Δ0 met15Δ0 trp1Δ63 ura3Δ0) (8 .) Competent yeast cells were prepared and transformed using a Frozen-EZ

Yeast Transformation II™ kit (Zymo Research Corp., Orange, CA). Plasmids containing appropriate selectable markers were co-transformed with experimental plasmids to allow growth of yeast cells in media selective for LEU2 and TRP1. For selection of leu+ trp+ colonies, yeast cells were plated on glucose-containing solid minimal medium

(YNB) supplemented with histidine, methionine, and uracil and lacking leucine and tryptophan and incubated at 30oC. For induction of the GAL1 promoter, leu+ trp+ cells were inoculated into selective liquid medium containing 2% galactose and grown at

30oC for 24h prior to harvest.

14 2.2. Plasmids Used

Parental plasmids. Plasmids TpG-NoV1 (pN1) and LpG-NoV2 (pN2) contain cDNA clones of NoV genomic RNAs 1 and 2, respectively, flanked by an inducible yeast

RNA polymerase II (GAL1) promoter and a hepatitis delta virus (HDV) antigenomic ribozyme, as described previously; primary transcription of the NoV cDNAs can be induced by growth of yeast cells in galactose as a carbon source (41). Plasmids pN1 and pN2 also contain yeast TRP1 and LEU2 selectable markers, respectively, which allow us to select for yeast cells that have successfully taken up the plasmids. Plasmid pN2 also contains the yeast ADH1 termination and polyadenylation sites immediately downstream of the HDV ribozyme (41). Parental plasmids YEplac112 (21) and Yep351

(23) lack NoV sequences but contain the same TRP1 and LEU2 selectable markers, respectively.

Plasmid pNoV2(0,0), which contains the NoV RNA2 cDNA and HDV ribozyme sequences under transcriptional control of a bacteriophage T7 promoter, was described previously (25); This plasmid also contains an ampicillin resistance gene and an origin of replication required for selection and replication in E. coli, respectively.

Plasmid LpG-NoV2-GFP. We constructed a plasmid, LpG-NoV2-GFP (pN2-

GFP), which contains the coding region for mammalian-codon-optimized green fluorescent protein (GFP), flanked by sequences from the NoV RNA2 cDNA. Upstream of the GFP ORF are NoV2 nt 1-17, followed by a unique NcoI restriction site; downstream of GFP is a unique MluI site, followed by NoV2 nt 1099-1336. This plasmid differs from the N2G plasmid described previously (41) in that the GFP ORF has now been positioned at the AUG codon normally used to initiate translation of the NoV

15 capsid protein precursor, so that GFP is now expressed in the absence of amino- terminal capsid protein sequences.

Plasmid pN2GFPΔSL1. We deleted the stem-loop structure predicted to form between NoV2 nt 1299 and 1322, using PCR-based circular mutagenesis followed by

Dpn1 selection, as described (49). Plasmid pNoV2(0,0) was amplified using Pfu-Turbo

DNA polymerase (Stratagene) and the following mutagenic oligonucleotides, which are complementary to one another. The oligonucleotide sequences, respectively, are 5’-

CGTCCCCAAGCTCGTAGCACCTTCAACCTCTTGGTGGGTCG-3’ (identical to nt

1278-1298 and 1323-1336 of NoV2, then the first 6 nt of the HDV ribozyme) and 5’-

CGACCCACCAAGAGGTTGAAGGTGCTACGAGCTTGGGGACG-3’ (complementary to the first 6 nt of the HDV ribozyme, followed by nt 1336-1323 and 1298-1278 of NoV2).

The region of interest was confirmed by sequencing and a small DNA fragment containing the desired mutation was re-ligated into plasmid pN2-GFP using standard techniques (49). The resulting plasmid, LpG-N2GFPΔSL1, contains the entire sequence of pN2-GFP except nt 1299-1322 of the NoV2 cDNA, which form stem loop SL1.

Plasmid pT7-N2ΔMluI-N2. For use as a template for in vitro transcription in

RNase mapping studies, we constructed a plasmid containing a head-to-tail dimer of

NoV RNA2 flanked by opposing bacteriophage T7 and SP6 promoters positioned such that T7 transcripts were positive sense and the SP6 negative sense. This plasmid, pT7-

N2DMluI-N2, contains an upstream copy of NoV2 from which a 319 bp MluI fragment has been deleted, followed by a complete downstream copy of NoV2, within the plasmid backbone of pT7-N2 (25). The junction between the two cDNA sequences is 5’-

…CTTGGT/GTAAAC…-3’ where the 3’-terminus of the upstream copy of the NoV

RNA2 cDNA is juxtaposed with the 5’ terminus of the downstream copy. We generated

16 the dimer junction by overlapping PCR reactions, using the following oligonucleotide primers: Primer 1, 5’-GCAATTGCGATCCTTGTGACTACGCC-3’ (identical to NoV2 nt

881-906 Primer 2, 5’-GGTCTTCAACCTCTTGGTGTAAACAACCAATAACATCAT-

GGTATCCAAAGCAGCAC-G-3’ (complementary to NoV2 nt 39-1, followed by NoV2 nt

1336-1319); primer 3, 5’-GGTCTTCAACCTCTTGGTGTAAACAACCAATAACATCAT-

GGTATCCAAAGCAGCACG-3’ (identical to NoV2 nt 1319-1336, followed by NoV2 nt 1-

39); primer 4, 5’-GCTGTCTTCGAGTACGGCG-3’ (complementary to NoV2 nt 137-119).

Plasmid pT7-N2 was used as a template for independent PCR reactions using primers

1 and 2 or 3 and 4. The resulting two PCR products were annealed together, by virtue of internal primers 2 and 3 being complementary to one another, and used as a template for PCR using the flanking primers 1 and 4). The resulting junction PCR product was subcloned into the TA cloning vector pGEM-T Easy® (Promega) and confirmed by sequencing. To generate the full-length dimer construct, a small fragment

(MluI1099-SapI) that contains the dimer junction was ligated to SapI-BglI and BglI-MluI780 fragments from pT7-N2, to generate plasmid pT7-N2ΔMluI-N2.

Plasmid pG4-N2dimerPvuII. We also constructed an NoV2 dimer subclone in the pGEM®-4 vector (Promega Corp.) using a 1017 bp PvuII fragment from pT7-

N2DMluI-N2 that contained the primer junction, to yield plasmid pG4-N2dimerPvuII.

This subclone, which contains a single binding site for the primer used in primer extension and sequencing, was used to generate the DNA sequencing ladder for the nuclease mapping analysis.

17 2.3. RNA Isolation and Analysis

Northern blot hybridization analysis. Extraction of total RNA from transformed yeast cells using hot phenol, separation on denaturing formaldehyde-agarose gels, transfer to charged nylon membranes (Nytran, Schleicher & Schuell), and Northern blot hybridization were performed as described previously (30, 40). Strand-specific 32P- labeled RNA probes for NoV subgenomic RNA3 and for GFP expressing RNA2-based replicons were generated by in vitro transcription from PCR products containing opposed bacteriophage T7 and T3 promoters, as described (49). Probes contained nt

2732 to 3204 of the appropriate sense of NoV RNA1 or nt 1 to 17 and 1100 to 1336 of the appropriate sense NoV RNA2, respectively (37). The results were visualized with a

Bio-Rad Personal Molecular Imager and analyzed using Quantity One 1-D Analysis

Software (Bio-Rad Laboratories).

2.4 Nuclease Mapping and Primer Extension Analysis

Nuclease mapping studies were performed on NoV2 in vitro transcripts as described (22, 48, 57). Due to the technical difficulty in mapping an RNA structure within

14 nt of the 3’ end of RNA2, we employed as transcription template a head-to-tail dimer of NoV RNA2, under transcriptional control of a bacteriophage T7 promoter (described above). This plasmid was linearized with EagI for use in in vitro transcription reactions, which were carried out using the AmpliCap™ T7 High Yield Message Maker kit

(Epicentre Biotechnologies). The resulting transcripts were purified from the transcription reaction using an RNeasy Mini Kit (QIAGEN) and eluted in water.

Nuclease digestion of unlabeled in vitro transcripts of the NoV2 dimer was carried out using single- or double-strand specific ribonucleases (RNases) A, T1, V1,

18 and I (Applied Biosystems/Ambion), as described (48, 57); duplicate samples were left undigested. The digested samples were subjected to primer extension analysis, using

Superscript III reverse transcriptase (Invitrogen) and an infrared-labeled primer (IR700;

LI-COR Biosciences), which allowed us to map the sensitive sites for each nuclease.

The primer contained the sequence 5’-GGGGCCGCTCTTCGGCGGCG-3’ which is complementary to nt 57-38 of NoV RNA2. A DNA sequencing ladder was generated with a SequiTherm EXCEL™ II DNA sequencing kit (Epicentre Biotechnologies) using the same IR700-labeled primer and plasmid pG4-N2dimerPvuII (above) as template.

Samples were analyzed on a polyacrylamide sequencing gel (LI-COR Biosciences 3.7% gel matrix) using a LI-COR 4200 DNA Analyzer running e-Seq V2.0 software (LI-COR

Biosciences). For ease of interpretation, the gel images were inverted and reversed so that the complement of the negative sense primer extension products can be read directly from the gel. Similarly, the DNA sequencing ladder was labeled with its complement, and T’s replaced with U’s, so that its correspondence to the computer prediction is readily apparent.

19 CHAPTER 3

RESULTS

3.1. Prediction of a Stem-Loop Structure in NoV RNA2

We wished to map the minimal cis-acting element involved in initiation of negative strand synthesis for NoV RNA2 and wondered whether the specific promoter(s) for negative strand synthesis would involve the formation of extensive RNA secondary structures such as those described for plant viruses (reviewed by (9)). Since many of these viral RNA replication elements have been shown to form pseudoknot structures, we selected software tools that are able to predict these structures, using the capacities of the RNAVLab (RNA Virtual Laboratory) when the RNA structure prediction demanded significant amount of time and resources. We focused on the 3’ terminus of

NoV RNA2 (the last 200 nucleotides) as a target for structural prediction because

Albariño et al. (1) had previously shown that for the related nodavirus FHV a cis-acting signal for RNA2 replication lies within the 3’ terminal 50 nt of this genome segment.

The results of the RNAVLab analysis predicts formation of a stem-loop structure within the 3’ non-coding region of NoV RNA2. Using the 3’ terminal 50 or 100 nt (1237-

1336 or 1287-1336, respectively) as a template for folding, this method predicts a stem- loop structure involving nt 1299-1322, containing a 7 bp stem (nt 1299-1305 base- pairing with nt 1316-1322) and a 10 nt loop (nt 1306-1315), located just 14 nt from the 3’ end (Figures 5 and 6). It remains possible that this structure may participate in other, long-range interactions that might include pseudoknot structures, when a longer 3’- terminal fragment is considered. The length of the 3’ terminal fragment was limited to 50 and 100 nt because the algorithm underlying Pknots-RE has a runtime and memory

20 demand in the order of n6 and n4, respectively, where n is the length of the sequence.

Currently, high-end workstations and clusters cannot predict sequences with lengths longer than approx 200, without taking months of computation. In addition, a database search showed that neither the loop sequence (5’-UACCCAUCUC-3’) nor its complement (5’-GAGAUGGGUA-3’) appears elsewhere within RNA2 (data not shown), making it unlikely that the loop is involved in base-pairing interactions such as those found in most types of pseudoknots.

3.2. Effect of Stem-Loop Deletion on RNA2 Replication

The role of the predicted secondary structure at the 3’ end of NoV RNA2 in the viral life cycle is unknown. Since nucleotides in this region are necessary and sufficient to direct replication of a FHV RNA2 derivative (1), we hypothesize that these sequences might be essential for replication of NoV RNA2. To test this hypothesis, we deleted the nucleotides that comprise the predicted structure and assay for the ability of the resulting mutant to replicate in transfected yeast cells.

We previously described our ability to reproduce the entire NoV replicative cycle in cells of the yeast Saccharomyces cerevisiae from plasmids expressing cDNA copies of NoV RNA1 and RNA2 (plasmids pN1 and pN2, respectively) under transcriptional control of an inducible yeast GAL1 promoter (41). On induction with galactose, the cells produce primary transcripts of RNA1 that are translated to produce the viral RdRp, which initiates RNA1 replication and subgenomic RNA3 synthesis. If yeast cells are co- transformed with RNA1 and RNA2, RNA2 is replicated by the RdRp in trans. Using this system, we showed that a minimal RNA2-based replicon, which expresses the green fluorescent protein (GFP) ORF as a fusion protein with the first 13 aa of the NoV capsid

21 protein, replicates in transformed yeast cells (41). The original replicon contains only

912 nt of NoV RNA2 (nt 1-53 nt and 477-1336), suggesting that these sequences are sufficient to direct its replication. For the present work, we further modified this replicon to replace the capsid protein translation initiation codon with that of GFP, retaining only

17 nt at the 5’ end and eliminating the capsid protein fusion. We also deleted nt 477-

1098, resulting in a replicon containing 254 nt of RNA2, pN2GFP. In this case, the GFP

ORF serves as a common heterologous core region to which we can direct strand- specific probes, providing a uniform assay for testing the activity of different N2GFP deletion variants, as previously described for FHV (1, 13). To test the role of the predicted stem loop in RNA replication, we deleted it from the pN2GFP replicon, resulting in plasmid pN2GFPΔSL1. Plasmids pN2GFP and pN2GFPΔSL1 are shown schematically in Figure 7.

Figure 7. Schematic of plasmids pN2GFP and pN2GFPΔSL1. Both plasmids contain the NoV RNA2 cDNA under transcriptional control of the yeast GAL1 promoter; LpG-N2GFPΔSL1 contains a 23-nt deletion of nt 1299-1322. The yeast selectable marker LEU2 allows nutritional selection for yeast cells that have taken up the plasmid.

Figure 8. Schematic of plasmids TpG-NoV1 (pN1) and TpG-NoV1R59Q (pN1R59Q). Both plasmids contain the NoV RNA1 cDNA under transcriptional control of the yeast GAL1 promoter; TpG-NoV1R59Q contains an arginine-to-glutamine mutation at position 59 in the B2 ORF. The yeast selectable marker TRP1 allows nutritional selection for yeast cells that have taken up the plasmid.

Yeast cells were transformed with wildtype or ΔSL1 versions of pN2GFP, together with pN1 as a source of RdRp. Total yeast RNA was isolated and subjected to

Northern blot hybridization analysis with probes specific for the positive (panel A) or negative strands (panel B) of RNA3 (which also detect RNA1 species) or of the N2GFP replicon (Figures 9 and 10, respectively). As shown in Figure 9, we were readily able to detect positive strands of monomeric and dimeric RNA1 and RNA3 species when cells were transformed with wildtype pN1 (Figure 9A, lane 4). When cells were co- transformed with pN1 and wildtype pN2GFP (Figure 9A, lane 5), levels of the RNA1 and

RNA3 monomers decreased in the presence of RNA2, as observed previously (25, 41).

Co-transformation of yeast cells with pN2GFPΔSL1 and pN1 resulted in no further decrease in levels of RNA1 or RNA3, showing that the deletion of the predicted

23 structure had no effect on RNA1 replication or RNA3 synthesis (Figure 9A, lane 6). We also detected negative strand replication intermediates of RNA1 and RNA3 (Figure 9B, lanes 4 – 6).

Figure 9. ΔSL1 mutant doesn’t affect RNA1 replication or RNA3 synthesis. Yeast cells transformed with wildtype or ΔSL1 versions of NoV2GFP, together with wildtype or R59Q versions of RNA1 as a source of RdRp, were selected and primary transcription induced as described (41). Total yeast RNA was isolated and subjected to Northern blot hybridization analysis using probes specific for the positive (Panel A) or negative (Panel B) strands of RNA 3.

When yeast cells were co-transformed with wildtype pN1 and pN2GFP, the

RNA2-GFP chimera replicated (Figure 10A, lane 5) and negative strand intermediates

24 were detected (Figure 10B, lane 5). However, we were unable to detect positive (Figure

10A, lane 6) or negative strands (Figure 10B, lane 6) of the RNA2-GFP chimera when cells were co-transformed with pN1 and pN2GFPΔSL1, indicating a severe defect in

RNA2 replication in the absence of the predicted stem-loop. These results establish the necessity of the predicted structure for efficient replication of RNA2.

Figure 10. ΔSL1 mutant exhibits a defect in RNA2 replication. Yeast cells were transformed with wildtype or ΔSL1 versions of pN2GFP, together with wildtype or R59Q versions of pN1 as a source of RdRp, as described in the legend to Figure 9. Total yeast RNA was subjected to Northern blot hybridization analysis as before, using probes specific for the positive (Panel A) or negative (Panel B) strands of the RNA2 region within the N2GFP replicon.

Partial rescued of ΔSL1 mutant by over-expression of RNA2 from a mutant form of RNA1. We wondered whether the ability of the N2GFPΔSL1 mutant to replicate

25 could be rescued by co-expression with an RNA1 mutant that up-regulates RNA2 replication in yeast. We reasoned that perhaps up-regulation of RNA2 might compensate for the replication defect seen for N2GFPΔSL1. We therefore tested an existing RNA1 mutant, R59Q, for its ability to rescue the stem-loop deletion; the mutant plasmid is shown schematically in Figure 8.

This naturally occurring RNA1 mutant was discovered when we selected for replication-dependent colony formation in yeast cells transformed with RNA2-based replicons containing the yeast HIS3 coding region. In that case, colony formation was entirely dependent on expression of the HIS3 protein, which in turn could be produced only on replication of the RNA2 derivative that encodes it. Colony formation was a rare event, and was linked to generation of spontaneous mutations in the protein B2 ORF encoded by subgenomic RNA3. One such mutant contained a single point mutation

(RNA1 G2919A) that resulted in replacement of the arginine at position 59 in B2 with a glutamine (R59Q). These results suggest that efficient RNA2 replication had occurred, although RNA2 replication was not directly measured at the time (41).

When yeast cells were transformed with pN1-R59Q, we detected positive strands of both RNA1 and RNA3 at a consistently higher level than with wildtype pN1 (Figure

9A, lane 7), suggesting that RNA replication in general may be up regulated in the absence of functional B2 protein. Interestingly, the R59Q versions of RNA1 and RNA3 appear to be down regulated to a lesser extent than are the wildtype (compare Figure

9A, lanes 7 – 9 with lanes 4 – 6). When we co-transformed yeast cells with pN1-R59Q and pN2GFPΔSL1 mutant, we were still unable to detect RNA2 negative strands (Figure

10B), although their presence can be inferred by our ability to detect low levels of positive strands under these conditions (Figure 10A).

26 3.3. The Predicted Stem-Loop Can Form Under Biological Conditions

Predictions alone will not credibly support our hypothesis since it is possible RNA secondary structures form by pairing through mechanisms not entirely understood. The necessity of experimental verification that these structures do in fact exist under biological conditions and not just in theory is of great importance.

We therefore performed nuclease-mapping studies to determine whether the predicted structure could form in NoV RNA2 in solution; following nuclease treatment as described below, the resulting cleavage products would be analyzed by primer extension. However, the location of the predicted stem-loop within 14 nt of the 3’ end of

NoV RNA2 complicated the primer extension analysis, since there is insufficient room to anneal a primer downstream of the predicted structure. To solve this dilemma, we turned to a possible nodavirus RNA2 replication intermediate, namely a head-to-tail dimer. Previous studies with FHV showed that homodimers of RNA1, RNA2, and RNA3, as well as an RNA2-RNA3 heterodimer, can be readily found in FHV-infected

Drosophila S2 cells or in mammalian cells transfected with FHV cDNA plasmids; these authors suggested a role for the dimeric molecules as replication intermediates (2). Use of NoV RNA2 dimers would allow us to place a primer near the 5’ end of the downstream copy that could be extended across the dimer junction and allow nuclease mapping of the predicted stem-loop at the 3’ end of the upstream copy.

We therefore constructed a plasmid containing a head-to-tail dimer of NoV

RNA2, under transcriptional control of a bacteriophage T7 promoter, for use as a template for in vitro transcription in place of the RNA2 monomer. Dimeric in vitro transcripts were synthesized and subjected to digestion with ribonucleases specific for single- (RNases A, T1, and I) or double-stranded RNA (RNase V1) or left untreated.

27 RNase A cleaves 3’ of single-stranded C and U residues, RNase T1 cleaves 3’ of single-stranded G residues, RNase I cleaves 3’ of all four single-stranded nucleotides, and RNase V1 cleaves 3’ of double-stranded nucleotides. To map the cleavage sites generated by each nuclease, treated samples were subjected to primer extension analysis using an infrared-labeled primer complementary to NoV RNA2 nt 57-38. A DNA sequencing ladder was generated with the same primer and the corresponding plasmid

DNA template. Samples were analyzed on denaturing sequencing gels using a LI-COR

4200 DNA Sequencer running e-Seq V2.0 software (LI-COR Biosciences).

The results of a representative experiment are shown in Figure 11; the nuclease mapping results are shown in panel 11A together with the DNA sequencing ladder and projected onto the predicted structure in panel 11B. Treatment with RNase T1 (lane 1) resulted in a digestion pattern that was identical to a control that lacked RNase treatment (lane 2). The latter being the result of strong stops makes its interpretation difficult; the analysis must be repeated before the work can be submitted for publication.

Single-strand specific RNase A cleaved 3’ of nucleotides C1309, A1311, C1315, all of which lie in the predicted single-stranded loop (Figure 5). Similarly, residues from both sides of the predicted stem were sensitive to digestion with the double-strand specific ribonuclease V1, including A1300, C1301, C1302, C1303, and A1305 from the upstream side of the stem and residues U1316, A1317, G1318, G1319, G1320, and

C1322 from the downstream side (Figure 11).

Figure 11. Nuclease mapping studies show the predicted structure can form in solution. In vitro transcripts of an NoV RNA2 homodimer were synthesized and subjected to digestion with ribonucleases specific for single- (RNases A, T1, and I) or double-stranded RNA (RNase V1), or left untreated. Treated samples were subjected to primer extension analysis using an infrared-labeled primer complementary to NoV RNA2 nt 57-38. A DNA sequencing ladder was generated with the same primer and the corresponding plasmid DNA template. Samples were analyzed on a 3.7% polyacrylamide sequencing gel using a LI-COR 4200 DNA Analyzer running e-Seq V2.0 software (LI-COR Biosciences). Panel A, nuclease map with sequencing ladder; panel B, predicted structure annotated with the results of the nuclease mapping. T1, RNase T1 (lane 1); A, RNase A (lane 3); I, RNase I (lane 5); V1, RNase V1 (lane 12); N, no nuclease (lanes 2, 4, 6, 11); U, G, C, A (lanes 7 – 10), sequencing ladder. The sequencing ladder (lanes 7 – 10) is labeled with the nucleotides complementary to the sequence obtained, and U’s were substituted for T’s, to allow direct comparison with panel B.

We also found some areas of inconsistency between the nuclease mapping and the prediction. Nucleotides C1303 and U1316 were sensitive to digestion with both

RNase A and RNase V1, despite their positions within the predicted double-stranded stem. Residue U1316 is predicted to form a U-A base pair with A1305 at the top of the stem, so it is conceivable that breathing at this position could result in its being sensitive to cleavage by both ribonucleases. Similarly, residues C1309, C1310, and C1315 were

29 sensitive to digestion by RNase V1 although they lie in the predicted single-stranded loop; C1309 and C1315 were also sensitive to digestion by RNase A. Together the results of the nuclease mapping studies are consistent with formation of the predicted stem-loop structure in solution.

30 CHAPTER 4

SUMMARY AND CONCLUSIONS

4.1. Summary

Here we have demonstrated that a predicted RNA secondary structure plays a key role in nodavirus RNA replication. We used three different computer programs to predict the structure adopted by the 3’-terminal 200 nt of NoV RNA2 (Figure 5 shows the structural element identified in the last 50 nt). All three programs consistently predicted the same structure for this region, increasing our confidence in the resulting prediction. We described elsewhere our observation that a similar structure could be predicted in the same position within the RNA2 segments of six other nodaviruses (55).

This phylogenetic conservation prompted us to further examine the structure for its possible involvement in nodavirus RNA replication. To determine the functional role of the predicted structure, we deleted the sequences that comprise the structure from a cDNA clone of RNA2. When this plasmid was used to transform yeast cells, together with a plasmid expressing wildtype or mutant versions of RNA1 as s source of RdRp, we observed abundant RNA1 replication and RNA3 synthesis (Figure 9) and a severe defect in RNA2 replication (Figure 10). Strand-specific Northern blot hybridization analysis was used to show that the primary defect was in negative strand synthesis, suggesting that the predicted stem-loop structure can function as a promoter for minus strand synthesis. We used nuclease mapping to show that the predicted stem-loop could form in solution within in vitro transcribed RNA (Figure 11), supporting our hypothesis that it is the structure rather than the primary RNA sequence that is important for NoV RNA2 replication.

31 4.2. Conclusions

A previously published report used Mfold prediction software to examine the predicted secondary structures of the 3’-noncoding regions of four insect nodaviruses:

BBV, BoV, FHV, and NoV (28). Our prediction methods differed from those of Kaesberg et al. (28) in that we used three computer software programs that are able to predict the formation of pseudoknots – structural elements often found at the 3’ termini of plant virus RNA genomes that have been shown to play important roles in their RNA replication mechanisms. We included three additional members of the nodavirus family discovered since the previous analysis: the insect alphanodavirus Pariacoto virus (PaV) and the fish betanodaviruses striped jack nervous necrosis virus (SJNNV) and greasy grouper nervous necrosis virus (GGNNV). Despite our using software that allowed for the prediction of pseudoknots, our results were similar to those of the previous analysis

(28). This was particularly true for NoV, where our prediction was identical to the previously reported structure. With the exception of NoV and BBV, the predicted stem- loop structure formed a part of a larger pseudoknot structure for all of the other nodavirus RNA2 segments examined (55). Whether too short a length of the sampled 3’ termini in NoV and BBV is the reason for the absence of pseudoknots in the RNA2 structure predictions and whether these structures play a role in viral RNA replication remains to be determined.

The results of the nuclease mapping studies with in vitro transcripts of NoV

RNA2 dimers are consistent with the predicted structure overall, suggesting that the stem-loop can form in solution. We observed some areas of inconsistency between the nuclease mapping and the prediction, as follows. Nucleotides C1303 and U1316 were sensitive to digestion with both RNase A and RNase V1, despite their positions within

32 the predicted double-stranded stem. Residue U1316 is predicted to form a U-A base- pair with A1305 at the top of the stem, so it is conceivable that breathing at this position could result in its being sensitive to cleavage by both ribonucleases. Similarly, residues

C1309, C1310, and C1315 were sensitive to digestion by RNase V1 although they lie in the predicted single-stranded loop; C1309 and C1315 were also sensitive to digestion by RNase A. The observation that both C1309 and C1315 were sensitive to cleavage by both RNase T1 and RNase A might suggest that this region is capable of forming more than one structural isoform.

Deletion of the nucleotides that comprise the predicted structure led to reduction of negative and positive strand RNA2 replication products to levels undetectable by

Northern blot hybridization. We believe the defect in minus strand synthesis to be the primary one, with the reduction in positive strand synthesis a direct effect of the lack of negative strand intermediates. These results pinpoint the stem-loop structure as an essential element of the minus strand promoter. The RNA2 derivatives we tested retained nt 1066-1298 upstream of the predicted structure and nt 1323-1336 immediately downstream. At this point, we are unable to rule out the possibility that sequences upstream of the stem-loop contribute to the promoter, perhaps via long- range structural interaction. However, a database search showed that neither the loop sequence (5’-UACCCAUCUC-3’) or its complement (5’-GAGAUGGGUA-3’) appears elsewhere within RNA2 (data not shown), making it unlikely that the loop is involved in base-pairing interactions such as those found in many types of pseudoknots.

Experiments in progress will determine whether a replicon containing only the stem-loop can confer replicability to an otherwise heterologous RNA, or whether the upstream sequences are also required.

33 Interestingly, the RNA replication defect can be partially rescued by the co- expression of an existing NoV RNA1 mutant. We used the existing RNA1 mutant R59Q, which contains a point mutation within the B2 ORF but is translationally silent in the overlapping RdRp ORF, to support the replication of the stem-loop deletion mutant. In that case, we were still unable to detect minus strands, but their presence was inferred from our ability to detect a low level of N2GFPΔSL1 positive strands (Figure 10). The

NoV1-R59Q mutant was found to greatly increase the level of the wildtype RNA2 chimera detected by Northern blot hybridization (Figure 10A, lane 8). Surprisingly, the

NoV1-R59Q mutant increased levels of RNA1 and RNA3, suggesting that these molecules also replicated better in the presence of the mutant B2 protein (R59Q) than they did in the presence of wildtype B2. The reason for this is unclear. The NoV B2 protein suppresses RNAi in human and insect cells (26, 31, 54), and enhances viral

RNA replication in mammalian and insect cells (26). The NoV1 R59Q mutant was found to be defective in its ability to suppress RNAi in transfected human HEp2 cells (B.

Duane Price and L. Andrew Ball, unpublished observation). However, since the RNAi response does not occur in Saccharomyces cerevisiae due to the lack of critical components in the RNAi pathway (16), these results underline the intriguing idea that

B2 must play an important, albeit unknown, role in nodavirus RNA replication in yeast aside from its function in RNAi suppression. It was shown previously that a similar R-Q change at position 54 in FHV B2 led to a decrease in FHV RNA accumulation in C. elegans (34). The FHV mutant was defective in its ability to bind to double-stranded

RNA. The RNA binding phenotype of the NoV R59Q mutant is not known, although it is conceivable that RNA binding is also a critical component of the B2 function in NoV

RNA replication in yeast.

34 Finally, our results with NoV are consistent with those for FHV described by

Albariño et al. (1), who showed that the 3’ terminal 50 nt of RNA2 was sufficient to direct replication of a heterologous RNA replicon. In the absence of FHV3, replicons that contained the 3’ terminal 50 nt of FHV2 were unable to replicate, demonstrating that the cis-acting RNA2 replication signal was absolutely dependent on the presence of RNA3

(1). Since all of the experiments described here were performed in the presence of

RNA3, provided from the NoV1 plasmids used as a source of RdRp, it remains to be determined whether the NoV RNA element requires RNA3 to function in RNA replication. However, by analogy with the FHV system, we expect that the NoV minus strand promoter will also require the presence of RNA3.

In conclusion, we have shown that a predicted stem-loop structure near the 3’- terminus of NoV RNA2 is essential for negative strand synthesis. The exact role of the structure is, as yet, unknown although it may provide a recognition site for the viral

RdRp during RNA replication. This work highlights for the first time a promoter for negative strand synthesis used by a member of the Nodaviridae. This reagent will greatly assist ongoing studies that utilize NoV RNA2 as an expression vector.

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39 CURRICULUM VITA

John H. Upton III was born in Phoenix, Arizona. The eldest son of Dr. John Upton Jr.,

D.D.S. and Marianne Webb, he graduated from Deer Valley High School in Glendale,

Arizona in the spring of 1994 and entered The University of Arizona in Tucson the following fall. In 1999, while pursuing a bachelor’s degree in biological sciences, John enlisted in the Army Reserve. In 2003, his medical support unit was mobilized to Fort

Sill in Lawton, Oklahoma where he served as a medical laboratory technician at a blood donor center for a year. John voluntarily extended his mobilization for an additional year at William Beaumont Army Medical Center in El Paso, Texas, continuing the efforts of collecting blood for shipment to the troops overseas. In 2005, after the completion of his tour, he continued to work toward completing his bachelor’s degree at the University of

Texas at El Paso (UTEP). He graduated with a B.S. in Biological Sciences and was immediately accepted into the Biology Master’s Program, receiving the Thelma E.

Louise Scholarship for academic excellence. In the fall of 2007, John was elected

President of the recently founded Pre-Dental Society at UTEP. In 2008, he was accepted into the Baylor College of Dentistry in Dallas, Texas, where he now resides with his wife and three children.

Permanent Address: 2213 Loretta Lane

Rowlett, Texas 75088