Ribosome Profiling of Late Adenovirus Infected Cells Reveals Ribosome Stacking on the 5’ Untranslated Region of Late Messenger Rna

RIBOSOME PROFILING OF LATE ADENOVIRUS INFECTED CELLS REVEALS RIBOSOME STACKING ON THE 5’ UNTRANSLATED REGION OF LATE MESSENGER RNA

JASON M. GAGLIANO

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Biology

August 2019

Winston-Salem, North Carolina

Approved By:

James F. Curran, Ph.D., Advisor

David A. Ornelles, Ph.D., Chair

Keith Bonin, PhD.

Erik C. Johnson, Ph.D.

Ke Zhang, Ph.D.

ACKNOWLEDGEMENTS

I would like to thank Dr. Jim Curran, Dr. David Ornelles, and Dr. Keith Bonin for the endless support I received from them on this challenging project. I am grateful for all the time and effort they spent in helping me work through problems in bench chemistry, biological assays and bioinformatics. I would also like to thank my entire committee for their insights and helpful suggestions towards making this project a success. Furthermore,

I thank the entire biology department of Wake Forest University for accepting me and allowing me to embark on a part-time Ph.D. project.

In addition, I would like to thank Dr. Jennifer Weller (UNC Charlotte) for her training in high-throughput sequencing and bioinformatics. Of course, I need to thank my wife Zeynia and my three children, Kayla, Antonio and Alyanna for their patience as I worked endless hours on my project.

Finally, I would like to thank all those who indirectly supported me through this project and whose contributions were important tomy success: Dr. Jed Macosko (WFU physics), Dr. Martin Guthold (WFU physics), Dr. Roger Cubicciotti (NanoMedica LLC),

Kara Libby (NanoMedica LLC), Dr. George Holzwarth (WFU physics), Dr. Nicholas

Ingolia (Berkeley), Dr. Eric Davies (NC State), Dr. Hugh Bender (UC Irvine), and Dr.

David Matthews (University of Bristol).

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... II

TABLE OF CONTENTS ...... III

LIST OF TABLES ...... VII

LIST OF FIGURES ...... VIII

LIST OF ABBREVIATIONS ...... XI

ABSTRACT ...... XIV

INTRODUCTION ...... 1

The Current Understanding of Adenovirus Infection ...... 1

The Early Phase of Adenovirus Infection ...... 3

The Delayed Early and Late Phase of Adenovirus Infection ...... 4

Transcription and Post-Transcription Processing of HAdVC mRNA ...... 5

Cap-Dependent Translation of Host, Viral Early and IX mRNA ...... 9

The Preferential Translation of Tripartite and IVa2 mRNA via Ribosome Shunting .. 13

The L4 100K Protein ...... 14

18S rRNA Complementarity ...... 17

Ribosome Shunting ...... 19

The Translation of Circular mRNA ...... 22

Polyribosome and Ribosome Profiling ...... 23

Hypothesis for the Translation of Late Shunting-mRNA ...... 26

MATERIALS AND METHODS ...... 28

Cell Culture ...... 28

Virus ...... 28

Viral Infection ...... 29

III

Sucrose Gradients ...... 29

Cell Lysis ...... 30

Polysome Profiling ...... 30

Ribosomal RNA Purification from Sucrose Fractions ...... 31

Control Ribosome Protected Fragments ...... 31

RNA Separation by Denaturing Polyacrylamide Gel Electrophoresis ...... 32

RNA Quantification by Gel Densitometry ...... 32

RNA Separation and Quantification by Microchip Electrophoresis ...... 33

Control RNA Precipitation from Polysome Fractions ...... 34

Control RNA Enrichment from Polysome Fractions ...... 36

Control RNA Polyacrylamide Gel Purification ...... 38

Ribosome Profiling ...... 40

Polynucleotide Kinase Modification of Ribosome Protected Fragments ...... 42

Reverse Transcription of Ribosome-protected Fragment ...... 42

Template Preparation for Sequencing ...... 45

The Measured Distribution of Templated vs Non-Templated Ion Sphere Particles . 45

Enrichment of Template-Positive ISPs ...... 46

High Throughput Sequencing of the cDNA library Using the IonTorrent Personal Genome Machine ...... 47

Integrity Check of the FASTQ File After Downloading ...... 47

Read-Length Determination of Sequencing Reads ...... 48

Quality Scoring of Sequencing Output ...... 48

Quality Trimming, Filtering and Mapping ...... 49

RNA-Seq Mapping with CLC ...... 50

The Detection of Periodicity from Mapped Reads ...... 50

RESULTS ...... 51

Separation and Detection of Polyribosomes in Translation ...... 51

Verification of RNA and Ribosomal RNA from the Polysome Profile ...... 53

Separation and Detection of Single 80S Ribosomes in Translation ...... 54

Polyribosome and Ribosome Profile of Adenovirus Infected HeLa Cells ...... 56

High Throughput Sequencing of Ribosome-protected Fragments from Adenovirus Infected Cells ...... 57

Trimming or Filtering ...... 58

Mapping Sequences to the HAdVC Reference Genome AY601635 ...... 61

Mapping of RPFs to HAdVC, Human, rRNA, and HPV-18 References ...... 61

Translational Phenomena ...... 64

Spliced Messages ...... 64

Reading Frames ...... 65

Periodicity ...... 65

Mapping to the Adenovirus Genome to Study Late Translation ...... 66

The Analysis of 80S Ribosomes in the 5’ UTR of Shunting-mRNA ...... 69

Ribosome Protection of 18S rRNA Complementary regions in the 5’ UTR of TPL and IVa2-mRNA Exhibit Hierarchical Density of 80S Ribosomes ...... 70

Mapping to the Individual Adenovirus mRNA to Study Late Translation ...... 71

Comparison of Ribosome Profile vs RNA-Seq of Adenovirus Infected Cells ...... 72

DISCUSSION ...... 74

Models of Canonical Translation and Shunting ...... 74

80S Ribosomes Appear to Stack in the 5’ UTR of Shunting-mRNA ...... 76

Ribosomes in the 5’ UTR of Shunting-mRNA do not Appear to be Translating ...... 78

Hierarchy in RPF Mapping may Indicate an Alternative Cap-Independent Translation Mechanism ...... 80

The 4F/43S Pre-Initiation Complex Appears to be the Shunting Species ...... 81

The Closed-Loop Model for the Selective Translation of Late mRNA by Ribosome Shunting ...... 82

Ribosome Reloading by 100K and Cooperative Binding Facilitates Stacking in the 5’ UTR of Circular Shunting mRNA ...... 83

Stacked Ribosomes on the UTRs of Shunting mRNA Shift 5’ to 3’ via 18S Complementary Regions ...... 85

The Closed-Loop Model of Translation Accounts for Several Features of Late HAdVC Preferential Translation by Ribosome Shunting ...... 86

Work that might resolve the Mechanism of Translation on Late Shunting-mRNA ..... 88

Future Studies: Atomic Force Microscopy on Late HAdVC mRNA ...... 88

Future Studies: Ribosome Profile of 40S and 80S Protected Fragments ...... 89

Future Studies: The Location of 40S and 80S Ribosomes During Translation Initiation ...... 90

CONCLUSION ...... 92

LITERATURE CITED ...... 93

FIGURES ...... 118

APPENDIX I ...... 144

Adenovirus Infection ...... 144

The Early Phase of Infection ...... 145

Viral DNA Replication ...... 148

The Delayed Early and Late Phase of Infection ...... 149

APPENDIX II ...... 151

Eukaryotic Translation ...... 151

APPENDIX III ...... 153

Translation of Circular-mRNA ...... 153

CURRICULUM VITAE ...... 155

LIST OF TABLES

Table I. The read-length frequency distribution of ribosome protected fragments before and after quality trimming or filtering ...... 60

Table II. Overall mapping of RNA protected by ribosomes ...... 64

VII

LIST OF FIGURES

Figure 1. The Mastadenoviridae virus group C serotype 5 ...... 2

Figure 2. The Mastadenoviridae genomic and transcriptomic map with a 600 base-pair deletion and then insertion of foreign DNA in the E3 region to make the dl309 species .. 6

Figure 3.The dl309 mRNA is capped with 7-methylguanosine (m7GpppN), polyadenylated at the 3’ end, and spliced before translation ...... 8

Figure 4. Standard models for translation by scanning and shunting ...... 12

Figure 5. The viral 100K protein mediates the preferential translation of late mRNA via the displacement of Mnk1 and binding to the tripartite leader ...... 16

Figure 6. The tripartite leader and 5' UTR of IVa2 have mRNA regions that are complementary to the 18S rRNA of the small ribosomal subunit ...... 18

Figure 7. Schematic representation of tripartite leader and IVa2 mRNA that are translated by shunting ...... 20

Figure 8. Polysome and ribosome profiling in late infected cells ...... 25

Figure 9. The hypothesized ribosome distribution during late viral translation ...... 27

Figure 10. One volume of isopropyl alcohol is as good as 4 volumes of ethanol at precipitating RNA the size of ribosome protected fragments from sucrose having polysomes ...... 35

Figure 11. RNA the size of ribosome protected fragments is enriched from total RNA in polyribosome fractions from cells infected with dl309 ...... 37

Figure 12. Enriched RNA the size of ribosome protected fragments was purified from PAGE gels ...... 39

Figure 13. The ribosome protected fragments from late infection were enriched and gel purified from a PAGE gel ...... 41

Figure 14. The RNAs protected by ribosomes were successfully converted to cDNA .... 44

Figure 15. The polyribosome profile of uninfected HeLa cells shows the separation and detection of ribosomal species ...... 118

Figure 16. The peaks in the polysome profile, of uninfected cells, are from ribosomes 119

Figure 17. The increasing ratio of 28S/18S ribosomal RNA confirms the presence of ribosomes in the polyribosome profile ...... 120 VIII

Figure 18. Polyribosome and ribosome profile of uninfected HeLa cells shows a decrease in polyribosomes due to RNaseI and a increase in the single ribosome peak .. 121

Figure 19. Ribosome profile shows a clear single ribosome peak upon RNaseI treatment ...... 122

Figure 20. Ribosome profile of adenovirus dl309 infected HeLa cells reveals a distinct peak of ribosome-protected fragments ...... 123

Figure 21. The ion torrent summary report of ribosome protected fragment cDNA sequencing ...... 124

Figure 22. Post-sequencing analysis of ribosome protected fragments shows a mean base-call accuracy of greater than 99.4% and the correct read-length ...... 125

Figure 23. Ribosome-protected fragments of varying Phred quality mapped to adenovirus genome ...... 126

Figure 24. RPF Mapping to rRNA is consistent with 80S ribosomes ...... 127

Figure 25. RPF mapping to the L4 region captured the splicing of 33K consistent with 80S ribosomes in translation ...... 128

Figure 26. Individual viral mRNA transcripts show a dominant reading frame consistent with translation ...... 129

Figure 27. The reading frame change in the L4 transcription unit from 100K translation to 33K translation is consistent with translation ...... 130

Figure 28. Different length MRNA produce a three-nucleotide periodicity in individual transcripts consistent with translation ...... 131

Figure 29. The 5’ position of ribosome protected fragments reveal a 3-nucleotide periodicity consistent with the rigid exit channel of the 80S ribosome in translation .... 132

Figure 30. The ribosome protected fragments mapped uniquely to one transcript, most of which from the late phase unit ...... 133

Figure 31. Adenovirus translation expression normalized for transcript length reveal dramatic ribosome association with the TPL ...... 134

Figure 32. Reads mapping to the tripartite leader and coding regions are essentially identical in length ...... 135

Figure 33. The 80S ribosomes in the tripartite leader show a periodicity of 29-nucleoties ...... 136

Figure 34. The ribosomes in the tripartite leader protected the 18S complementary regions ...... 137

Figure 35. The ribosomes in the 5’ untranslated region of IVa2 protected the 18S complementary regions ...... 138

Figure 36. Mapping of protected fragments to individual viral mRNA show ribosome pausing upstream, upon, and downstream of the start codon ...... 139

Figure 37. Zoomed in view of mapping to individual MRNA transcripts show pausing near the start codon ...... 140

Figure 38. The ribosome fragment data correlates with transcriptome data late in infection ...... 141

Figure 39. Comparison of ribosome fragment mapping and transcriptome mapping from Evans et al. shows pronounced translational expression of the tripartite leader ...... 142

Figure 40. Models of late viral mRNA translation by ribosome shunting ...... 143

LIST OF ABBREVIATIONS

ATP adenosine tri-phosphate

BAM binary alignment mapping

CAR coxsackie and adenovirus receptor

CDS coding sequence

CHX cycloheximide

CRISPR clustered regularly interspaced short palindromic repeats

DBP DNA binding protein

DEPC diethyl pyrocarbonate

DNA deoxyribonucleic acid

DTT dithiothreitol

EDTA ethylenediaminetetracetic acid eIF eukaryotic initiation factor

ERF eukaryotic release factor

ETOH ethanol

GDP guanine di-phosphate

GTC guanidine thiocyanate

GTP guanine tri-phosphate

HAdVC human adenovirus

HDAC histone deacetylase complex

HPV human papilloma virus

HSB high salt buffer

IPA isopropyl alcohol XI

IRES internal ribosome entry site

ISP ion sphere particle

MHC major histocompatibility complex

MLP major late promoter mRNA messenger RNA

NES nuclear export sequence

NLS n-lauryl sarcosine

NLS nuclear localization sequence

NT nucleotide

ORF open reading frame

PABP poly(A) binding protein

PBS phosphate buffer saline

PCR polymerase chain reaction

PFU plaque forming units

PGM personal genome machine

PKR RNA-dependent protein kinase

PNK polynucleotide kinase

RFU relative fluorescent unit

RNA ribonucleic acid rRNA ribosome RNA

RPF ribosome-protected fragment

RPKM reads per kilobase million mapped

RPM revolution per minute

XII

RRM RNA recognition motif

RSB reticulocyte standard buffer

RT room temperature

SAM sequence alignment mapping

SDS sodium dodecyl sulfate

SS single stranded

TAE tris acetate EDTA

TBE tris borate EDTA

TEMED tetramethylethylenediamine

TPL tripartite leader

UTR untranslated region

XIII

ABSTRACT

Adenovirus type 5 is a non-enveloped, icosahedral capsid, containing a linear genome thirty-five thousand base-pairs in length. The genome has 16 genes that, through alternative splicing, code for 31 different proteins. The genes are expressed in groups called transcription units. The units are temporally expressed during the early, delayed early, and late phase of infection.

Each late phase mRNA transcript is spliced into the same tripartite leader. The tripartite leader facilitates the almost exclusive translation of late viral mRNA via the unique translation mechanism called ribosome shunting.

Shunting involves ribosomes leaping around secondary structure in the 5’ untranslated regions of late viral mRNA. Ribosomes land at or near the start codon and translation elongation ensues.

To gain insight into the preferential translation of viral late mRNA via shunting, ribosome profiling was performed during the late phase of infection. Ribosome profiling involves high-throughput sequencing of ribosome-protected mRNA fragments. The subsequent mapping of the protected fragments revealed the location of ribosomes on a transcriptome-wide scale. The profile revealed that ribosomes attach and stack in the 5’

UTR of all shunting-mRNA. As a result, the preferential translation of late-viral mRNA was 500 times that of its host. The data was also consistent with late mRNA being circular and leads to a new model of ribosome shunting.

XIV

INTRODUCTION

Mastadenoviridae is the adenovirus genus that infects humans (Davison et al.,

2003). It can infect the retina (e.g. pink-eye), adenoids, tonsils (e.g. tonsillitis), and several organs (Berk, 2007; Acheson, 2011). Infections have caused significant mortality in people with compromised immune systems (Lion et al., 2014; Prasad et al., 2014; Sandkovsky et al., 2014; Ottaviano et al., 2019). For more information see Appendix I.

The work in this thesis was performed with a modified Mastadenoviridae virus group C serotype 5 (HAdVC). The modification is a 600 base-pair (bp) deletion and then insertion (e.g. salmon sperm DNA) in its E3 region (see below; Bett et al., 1995). This species is named HAdVC_dl309.

The Current Understanding of Adenovirus Infection

HAdVC is an icosahedral capsid with a diameter of about 90 nm. Inside the capsid is the viral DNA genome. The capsid has about 11 proteins but primarily consists of the hexon, penton, and fiber (Fig. 1). Projecting from the penton bases are fiber terminal knobs.

(Fig. 1; Berk, 2007; Acheson, 2011).

Infection begins with fiber proteins binding to cellular receptors, triggering the endocytosis of the viral capsid into an endosome (Berk, 2007; Acheson, 2011; Kumar et al., 2017). Dynein attaches to the endosome and walks it down microtubular tracts near the nucleus (Kumar et al., 2017). There the capsid protein VI (Fig. 1) lyses the endosome releasing the capsid. The capsid attaches to the nucleus and releases the viral genome inside

(Wold, 2007; Acheson, 2011; Kumar et al., 2017).

Figure 1. The Mastadenoviridae virus group C serotype 5 An illustrated cross-section of the viral capsid. The structural proteins that make up the capsid are hexon (II), penton (III) and fiber (IV) proteins. Hexon is the most abundant of the three proteins. The fiber protrudes from the penton base that is in contact with five hexon proteins. The linear double stranded DNA genome is inside the capsid. Reprinted with permission from Acheson, 2011. © John Wiley & Sons, Inc

2 The viral DNA genome is linear, double-stranded, and 35 kilobases (kb) in size

(Fig. 2). Each end of the viral genome has a 55-kDa terminal protein and inverted repeats that both serve as DNA replication origins (Schaack et al., 1990; Schneider, 1995;

Acheson, 2011; Kumar et al., 2017). The genome has 16 genes that are expressed in groups called transcription units. The transcription units are temporally divided into early phase, delayed early phase, and late phase relative to DNA replication (Fig. 2; Schneider, 1995;

Acheson, 2011). The genomic and transcription map in Fig. 2 shows that the expression of the HAdVC genome is well organized, dense in biological information, and completely functional.

There are six early phase transcription units, two delayed early, and one major late transcription unit respectively (Fig. 2; Schneider, 1995; Acheson, 2011). The major late unit is grouped into five families (e.g. L1-L5) defined by their polyadenylation sites (Fig.

2 arrows; Schneider, 1995; Acheson, 2011).

The capsid reaches the nucleus of the host cell and releases the viral genome inside at 2 h post-infection. This event begins the early phase of infection.

The Early Phase of Adenovirus Infection

The early phase of infection includes viral DNA replication and the countering of the antiviral response mechanisms of the host cell. E1A and E2A proteins induce dormant cells to divide and, as the cell prepares, it makes amino acids and nucleic acids for its replication. The viral DNA polymerase (E2B) along with the viral DNA binding protein

(E2A) use nucleic and amino acids to replicate the viral genome (Berk 2005,2007; Zheng,

2010; Acheson, 2011)

3 The cell responds with several antiviral defense processes. The E1B, E3, and E4 transcription units express proteins to combat the cellular antiviral defense (Acheson,

2011). This ensures that the cellular environment stays favorable for infection.

The dl309 virus used in this thesis has a deletion and insertion of foreign DNA in its E3 region (Fig. 2 red box). This alteration prevents it from countering key antiviral defense strategies. As a result, dl309 is phenotypically identical to the wild-type virus but cannot sustain an infection (Jones & Shenk., 1979; Bett et al., 1995).

At the end of the early phase, viral DNA replication continues, and the cellular environment is suitable for the ongoing infection. The next two stages involve the expression of genes from the delayed early and late phase transcription units (Fig 2).

The Delayed Early and Late Phase of Adenovirus Infection

The two delayed-early proteins (i.e. IX and IVa2) aid the viral DNA replication and the transcription of late genes (Fig. 2; Acheson, 2011). In addition, IX and IVa2 remain functional well into the late phase and helps in viral capsid formation prior to the release of progeny (Zhang & Imperiale., 2003; Parks, 2005). For more information on adenovirus infection, see Appendix I.

The late phase is the focus of this thesis. The late proteins form the capsid and aid in packaging the viral genome inside (Fig. 1; Acheson, 2011). Every late mRNA has an identical 5’ UTR called the tripartite leader (Fig. 2; TPL). The same leader results in the coordinated expression of late capsid proteins. The experiments presented in this thesis were performed early in the late phase at 15-16 h post-infection.

4 In Fig. 2 there are several gaps that stand for the splice sites that separate regions within transcripts. The 16 genes of the viral genome code for 31 different proteins via alternative splicing. The IX mRNA is the only exception. The number of gaps illustrate the extensive splicing that occurs during the transcription and post-transcription processing of viral messages.

Transcription and Post-Transcription Processing of HAdVC mRNA

The mRNA from the viral genes are initially transcribed into one large pre-mRNA that is alternatively spliced into several unique transcripts (except IX). Each late mRNA transcript is spliced into the same 5’ UTR called the tripartite leader (Fig. 2; TPL-mRNA).

This leader is tripartite because it itself is made by the splicing of three regions (Fig 2 gaps;

TPL-1, TPL-2, TPL-3).

Figure 2. The Mastadenoviridae genomic and transcriptomic map with a 600 base- pair deletion and then insertion of foreign DNA in the E3 region to make the dl309 species The early, delayed early, and late transcription units with corresponding mRNA are colored black, grey, and red respectively. Solid circles represent the 5’ untranslated regions (UTRs) and the arrows represent the poly(A) tail. The gaps signify the introns that are spliced out to make the final mRNA transcripts. The red box is the region of the deletion and insertion of DNA in the E3 region. R=number of amino acid residues; S = Svedberg units; K = kilodaltons. Adapted from Acheson, 2011 and Zhao et al., 2014.

6 In addition to taking advantage of the cell for its replication, the virus also uses the host transcription and translation machinery. This means that the the viral mRNAs are processed the same as host mRNAs. For example, the host and viral messages have a 5’ 7- methylguanosine (m7GpppN) nucleotide cap and a 3’ poly(A) tail (Fig. 3; Schneider,

2000). The non-coding introns of host and viral mRNA are spliced out and the protein- coding exons are spliced together (Fig. 3). The splicing out of different introns in the same mRNA results in several different transcripts from one gene (Figs. 2 and 3; Jackson, 2010,

Acheson, 2011). The complete exon region, flanked by the 5’ and 3’ UTRs, is called the open reading frame (Fig. 3; ORF).

The ORF may also be referred to as the coding region or coding sequence (CDS;

Jackson, 2010). This is because every 3-nucleotides (i.e codons) inside the ORF code for the amino-acids to make proteins. The ORF begins with a start codon (Fig. 3; AUG) that codes for the amino-acid methionine and the ORF terminates at one of three stop codons

(Fig. 3; UAA, UGA, UAG).

At the end of the post-processing of either host or viral mRNA, the final mature message is transported out of the nucleus and into the cytoplasm for translation by host- ribosomes. The ribosomes, along with transfer RNAs (tRNAs), translate the ORF codon by codon (every 3-nucleotides) to produce proteins.

Figure 3.The dl309 mRNA is capped with 7-methylguanosine (m7GpppN), polyadenylated at the 3’ end, and spliced before translation Adenovirus genes are initially transcribed to a pre-mRNA. The pre-mRNA is capped with 7-methylguanosine (m7GpppN) and polyadenylated at the 3’ end. The 5’ untranslated region (red) and the 3’ untranslated region (green) are labeled. Introns (blue) are removed and the exons (tan) are spliced together to make the ORF or the coding sequence (CDS). The splicing together of different exons produces several unique messages from the same gene.

8 Cap-Dependent Translation of Host, Viral Early and IX mRNA

The cap-dependent scanning mechanism of translation is the mechanism used to translate host mRNA, viral early mRNA, and IX mRNA (Fig. 4 top; Schneider, 2003).

Translation of mRNA occurs in the 80S ribosome that is made up of a large 60S ribosomal subunit and a small 40S ribosomal subunit. The 60S and 40S subunits are composed of several proteins and ribosomal RNA (rRNA). The 60S has the 28S, 5.8S and 5S rRNA and the 40S has the 18S rRNA (Yusupova et al., 2006).

Translation can be divided into three stages: initiation, elongation, and termination

(Jackson 2010). Initiation is performed by a 43S/4F pre-initiation complex. The complex includes the eukaryotic initiation factor 4F (eIF4F) and 40S ribosomal subunit (Jackson,

2010). The eIF4F complex has a 5’ cap-binding protein (i.e.eIF4E), the scaffolding protein

(i.e eIF4G), the RNA helicase (i.e. eIF4A), the map kinase activating kinase (i.e. Mnk1), and the poly(A) binding protein (i.e. PABP; Dever & Green, 2012).

Initiation begins with the binding of the 43S/4F complex to the mRNA 5’ cap via eIF4E (Jackson et al, 2010; Dever & Green, 2012). Mnk1 phosphorylates 4E increasing its affinity for the cap (Yueh & Schneider, 2000). This in turn favors the canonical cap- dependent scanning translation mechanism (Fig. 41; Fukunaga and Hunter., 1997;

Waskiewicz et al., 1997; Xi et al., 2004). The scanning mechanism is cap-dependent because the translation of the ORF is contingent on the successful binding of 4E of the 4F complex to the 5’ cap on mRNA.

After the 43S/4F complex binds to the cap, it scans each nucleotide of the 5’ UTR until it finds a start codon in a favorable context (Fig. 4 top; Kozak, 1991,1992; Yueh &

Schneider, 1996; Andreev et al., 2016). At the start codon the 60S ribosomal subunit joins

9 the 43S/4F complex forming the complete 80S ribosome. Translation elongation now begins.

The 80S ribosome, along with transfer RNAs (tRNAs), translate the mRNA ORF into protein. The tRNAs are covalently linked to amino acids (i.e. aminoacyl-tRNAs) and have 3 nucleotide anticodons that can base pair with codons of the ORF (Jackson, 2010).

The aminoacyl-tRNA delivers the correct amino acid to the ribosome by matching its anticodon with the codon of the ORF.

The 80S ribosome has three binding sites for tRNAs, the amino (A-site), peptidyl

(P-site) and exit (E-site). Each site spans a 3-nucleotide codon. When the 80S ribosome forms at the start codon, it has the starting aminoacyl-tRNA in the P-site. The next codon is in the A-site. The correct aminoacyl-tRNA enters the A-site and the ribosome catalyzes a peptide bond, transferring the amino acid from the P-site to the A-site. The ribosome translocates to the next codon placing the peptide-tRNA in the P-site and the deacylated tRNA in the E-site. The A-site can now accept the aminoacyl-tRNA that is specific for that codon.

The translating ribosome moves down the mRNA codon by codon or every 3- nucleotides. Therefore, the translating ribosome shifts with a 3-nucleotide periodicity

(Ingolia et al., 2009; Dever & Green, 2012).

The ribosome can begin translation on either of the 3-nucleotides of the start codon.

This means that each CDS has three reading frames (Jackson, 2010). In general, the CDS of mRNAs have only one reading frame that codes for proteins (Jackson, 2010;

Vanderprerre et al., 2013). However, as seen in HAdVC, more than one ORF can come from a single mRNA via alternative splicing (Figs. 2 and 3).

10 Termination occurs at one of three stop codons. The stop codons do not code for an amino acid causing the ribosome to pause. Termination factors disassemble the paused 80S ribosome back into the 40S and 60S subunits (Jackson et al, 2010). For more information about translation see Appendix II.

The host, early, and IX mRNA translate in a cap-dependent manner. However, the

TPL-mRNA and IVa2 are translated by a cap-independent manner called ribosome shunting (Fig. 4 bottom). This cap-independent mechanism facilitates the hijacking of the ribosomes so that the TPL-mRNA and IVa2 are almost exclusively translated. The focus of this thesis is to gain more insight into the preferential, cap-independent translation of late messages by shunting.

Current Model of Host Translation: Scanning Current Model of Host Translation: Scanning 60S 5’ Cap-Dependent 60S 5’ Cap-Dependent 40S 40S 80S 80S Stop Codon Stop Codon Start Codon

Start Codon Current Model of Late-Viral Translation: Shunting

Current Model of Late5’ Cap-Viral-Independent Translation: Shunting

40S 5’ Cap-Independent

40S 40S

40S 80S

18S Comp. Stop Codon Regions = 100K Protein Start Codon80S 18S Comp. Stop Codon Regions Yueh & Schneider, 1996,2000 = 100K Protein Start Codon Yueh & Schneider, 1996,2000

Figure 4. Standard models for translation by scanning and shunting

(top) The current model of translation by cap-dependent scanning. The preinitiation complex (e.g. 40S) loads at the 5’ cap and then scans to find the start codon. The 80S ribosome forms and elongation begins. At the stop codon the 80S ribosome is disassembled. (bottom) The current model of translation by cap- independent shunting on late viral mRNA. The initiation complex binds to the cap, performs limited scanning and shunts around the secondary structure landing at or near the start codon. The processes of elongation and termination are identical to the scanning model (Yueh & Schneider, 2000; Xi et al., 2004,2005).

12 The Preferential Translation of Tripartite and IVa2 mRNA via Ribosome Shunting

At the beginning of the late-phase of infection (12-15 h post-infection) TPL-mRNA makes up only a fraction of the cytoplasmic pool (5%). However, these messages make up the vast majority (95%) of mRNAs found associated with 80S ribosomes (Dolph et al.,

1988,1990; Schneider, 2000; Evans et al., 2012).

The preferential translation of late transcripts begins with the nuclear-to- cytoplasmic export of viral mRNA. HAdVC does not shut-down host transcription nor degrade or alter host mRNA in the cytoplasm (Dolph et al., 1988; Schneider 2000). Rather, the HAdVC E4orf6 and E1B-55K (Fig. 2;496R) complex hinders host-mRNA transport from the nucleus and selectively exports late-mRNAs (Logan and Shenk, 1984; Schneider,

2000, 2003). This results in an accumulation of TPL-mRNA in the cytoplasm. While early in the late phase, viral transcripts are only 5% of the total cytoplasmic mRNA, by 24 h post-infection, they make up ~80% of the cytoplasmic transcripts in the cell (Evans et al.,

2012).

It appears that some late transcripts are more efficiently exported. For example, in

HeLa cells at 24 h post-infection, fiber was found to have 50% more of its transcripts in the cytoplasm relative to the nucleus. In contrast, hexon had slightly less of its transcripts in the cytoplasm than the nucleus (Younis et al., 2018). This implies that fiber is transported more efficiently than hexon.

The difference in transport efficiency appears to involve the cellular stress protein

ZC3H11A.This protein is upregulated during viral infection and is stabilized by association with the E4orf6/55K complex. Deletion of the stress protein resulted in a 50% reduction in nuclear-to-cytoplasmic export of total viral mRNA, and a 60% reduction in export of fiber

13 (Younis et al., 2018). The deletion had a minimal effect on hexon. Hence, different viral transcripts may need to take advantage of different export proteins (Younis et al., 2018).

Despite the apparent differential export efficiencies, at 24-36 h post-infection, fiber and hexon have similar proportions of mRNA in the cytoplasm (Younis et al., 2018;

Chrisostomo et al., 2019).

The preferential translation of late mRNA is also due to a unique cap-independent translation mechanism called ribosome shunting. Two factors that aid in the selective translation of TPL and IVa2 mRNA are the 100K protein and 18S complementary regions in the 5’ UTR.

The L4 100K Protein

The 100K protein is one of the first proteins translated late in infection. The 100K protein binds to the same location on eIF4G as Mnk1 removing Mnk1 from the host translation initiation complex (Fig.5; Cuesta et al., 2000,2004; Xi et al., 2005). This results in a reduction in the phosphorylation of the 4E cap-binding protein thereby hindering host cap-dependent translation. Hence, the cap-independent translation of late-mRNA by ribosome shunting is favored (Fig.4 bottom; Dolph et al.,1990; Huang and Schneider.,

1991; Yueh & Schneider., 1996, 2000).

The 100K also has a binding domain that is specific for the tripartite leader. This recruits the host ribosomes to the TPL-mRNA. The modified ribosome (e.g. 100K) also enhances ribosome shunting. How or why it increases shunting is not completely clear

(Yueh & Schneider., 1996; Cuesta et al., 2000,2004; Xi et al., 2005).

14 The delayed early mRNA of IVa2 has been observed to also translate by shunting,

(Yueh & Schneider., 1996; Xi et al., 2005). However, the 100K does not have a binding domain specific for its 5’ UTR. Rather, both IVa2 and TPL-mRNA have 18S complementary regions in their 5’ UTR. These regions also contribute to ribosome shunting (Yueh & Schneider., 2000).

Figure 5. The viral 100K protein mediates the preferential translation of late mRNA via the displacement of Mnk1 and binding to the tripartite leader

(top) The eIF4F/43S complex binds to the 5’ m7G cap of host mRNA via the eIF4E cap-binding protein to initiate translation. Mnk1 (blue arrow) phosphorylates eIF4E (red arrow) which strengthens the binding. The PABP is in contact with the poly(A) tail circularizing the mRNA. (bottom) The 100K protein displaces Mnk1 (blue arrow) preventing the phosphorylation of 4E (red arrow). This reduces host cap- dependent translation in favor of viral cap-independent translation. The PABP and 4A helicase are still associated with the eIF4F/43S complex after 100K modification (black arrows). Reprinted with permission from Schneider & Mohr, 2003. © Elsevier.

16 18S rRNA Complementarity

The TPL regions 1-3 (Fig. 2) and the 5’ UTR of IVa2 contain regions that are complementary to the 18S rRNA of the 40S ribosome (Fig. 6; Yueh & Schneider.,2000;

Ramke et al., 2017). Mutational analysis of these regions in the TPL and IVa2 showed that they are vital for translation by shunting, but their exact function is unclear (Dolph et al.,

1990; Yueh & Schneider., 2000; Xi et al., 2004).

Yueh & Schneider, 1996,2000 found that these regions in the TPL are hierarchical in their importance for translation by shunting (Fig. 7). Mutating TPL-3 had a greater impact on shunting than mutating TPL-2 which had more of an impact than TPL-1 (Yueh

& Schneider., 1996, 2000). The authors did not find the same 3’ to 5’ hierarchy in the 5’

UTR of IVa2.

The data in this thesis reveals the same 3’ to 5’ hierarchy in the TPL in terms of ribosome density. Interestingly, this same 3’ to 5’ pattern was observed to also exist in the

5’ UTR of IVa2 (Fig. 7; see below).

TPL:18S

18S rRNA 3’ (1)AUUACUAGGAAGGCGUCCAA--GUGGAUGCCUUUGGAAGAAU(40) TPL-1 5’ (5)UCUUCCGCA(13)(20)UCUGCGAGGGCC(31)

18S rRNA 3’ (1)AUUACUAGGAAGGCGUCCAA--GUGGAUGCCUUUGGAAGAAU(40) TPL-2 5’ (68)GGUCUUUCC(76) (91)GGAAACC(97)

18S rRNA 3’ (1)AUUACUAGGAAGGCGUCCAA--GUGGAUGCCUUUGGAAGAAU(40)

TPL-3 (143)UCCGCA(148) (160)GGAAAACCUCU(169)

IVa2:18S

18S rRNA 3’ (1)AUUACUAGGAAGGCGUCCAA--GUGGAUGCCUUUGGAAGAAU(40)

IVa2 UTR 5’ (3)AGUGGUCC(10) (20)UACGAGGA(27)

18S rRNA 3’ (1)AUUACUAGGAAGGCGUCCAA--GUGGAUGCCUUUGGAAGAAU(40)

IVA2 UTR 5’ (88)CCUUUU(93) (105)GUGGACACC(113)

Figure 6. The tripartite leader and 5' UTR of IVa2 have mRNA regions that are complementary to the 18S rRNA of the small ribosomal subunit

The 18S complementary regions in the tripartite leader (top) and 5’ UTR of IVa2 (bottom). Each region can dual hybridize in the same region of the rRNA. Data from Yueh & Schneider, 2000.

18 Ribosome Shunting

The TPL and IVa2 mRNA translate by the cap-independent mechanism of ribosome shunting (Fig. 7). The TPL is purported to have extensive secondary structure that has binding sites for the 100K protein (Fig. 5) and hybridization sites for the 18S rRNA

(Fig 6). However, the 5’ UTR of IVa2 mRNA is linear, but has the same 18S complementary regions as the TPL (Fig. 6).

Experiments performed on the TPL determined that the first 25-nucleotides (TPL-

1) are unstructured, the middle 70-nucleotides (TPL-2) have RNA loops, and the last 90- nucleotides (TPL-3) have loops and RNA hairpins (Zhang et al., 1989).

Between the pre-spliced region of TPL-2 and TPL-3, there is also a 440-nucletotide i-leader gene that codes for a 13.6-kDa protein (Fig. 2; Evans et al., 2012; Ramke et al., 2017). In addition, the i-leader is spliced as a 5’ UTR into L1 mRNA transcripts (Ramke et al., 2017).

19 Tripartite Leader (TPL) mRNA

180 120

80 25 40 ORF

1 202 AUG 5’ 3’

TPL-1 TPL-2 TPL-3

IVa2 mRNA ORF AUG 5’ 3’ 1 118

5’ UTR

Figure 7. Schematic representation of tripartite leader and IVa2 mRNA that are translated by shunting

(top) The tripartite leader is the product of the splicing together of three regions (TPL1-3; black) These are spliced into a late transcript. The leader is purported to have extensive secondary structure (Zhang et al., 1989). The ramp below the image stands for the 3’ to 5’ of increasing importance for shunting (Yueh et al., 1996,2000). (bottom) The 5’ UTR of IVa2 is linear. The ORF of IVa2 is the product of one splicing event. The ramp stands for the ribosome density observed from the results in this thesis.

20 The current model for ribosome shunting on TPL-mRNA is depicted in Fig. 4. The mechanism begins with the 43S/4F complex binding to the 5’ cap of TPL mRNAs. The complex then performs limited scanning in the linear region of TPL-1. Upon reaching the secondary structure in TPL-2, the ribosome shunts or translocates around the secondary structure landing on or near the start codon. Here the 80S ribosome forms to begin translation elongation (Yueh & Schneider., 1996; reviewed in Cuesta et al., 2001; Xi et al.,

2005).

The evidence for the current model comes from studies of Yueh & Schneider,

1996,2000. When start codons were placed in TPL-1, the 80S ribosome formed there. This resulted in no evidence of ribosome shunting. Rather, the 80S ribosome translated a protein, unwinding the secondary structure via eIF4A. They only observed shunting in the absence of the start codon, by what was assumed to be the 43S/4F complex (Kozak &

Shatkin., 1978; Yueh & Schneider, 1996,2000).

The unwinding of secondary structure in the TPL, new 43S/4F, loading at the 5’ cap, were now unable to shunt. Therefore, the translation mechanisms of 80S scanning and

4F/43S shunting were mutually exclusive (Yueh & Schneider, 1996,2000). Shunting by the 43S/4F ribosome resumed only after a stop codon was placed before the secondary structure of TPL-2 disassembling the 80S ribosome (Yueh & Schneider, 1996,2000).

The current model of ribosome shunting has limitations. For example, the model does not explain how ribosome shunting is cap-independent while also requiring that the

43S/4F complex first bind to the cap. It does not explain why the 43S/4F complex binds to the cap when it contains the 100K protein that can bind the TPL regions upstream (Fig. 5).

It does not explain the function of the RNA helicase 4A in the 4F/43S complex during

21 shunting. The helicase can unwind the secondary structure of the TPL as shown with the experiments just discussed (Yueh & Schneider, 1996,2000). It does not address the observation that ribosomes are unevenly distributed throughout late message, possibly stacking in the 5’ UTR (Velicer and Ginsberg, 1968; personal communication, Ornelles lab). The experiments used a synthetic TPL-reporter that only resembled the 5’ UTR of

HAdVC pV (L2; Fig. 2). While it is true that each late transcript has the TPL, there are still

UTRs unique to late messages joining the TPL to the viral ORF. Finally, the model does not account for the circularization of mRNA by the binding of the PABP, in the 4F/43S, to the mRNA poly(A) tail.

The Translation of Circular mRNA

Circular mRNAs, coated with polyribosomes, have been directly observed by high resolution cryo-electron tomography (Afonin et al., 2015). Kinetic studies have shown that the translation of circular mRNA is faster and more efficient than the translation of linear mRNA. This was found to be due to the ribosomes, terminated at the 3’ end, reinitiating in the 5’ UTR and not at the cap (Alekhina et al., 2007; Kopeina et al., 2008).

The increase in the translation of circular mRNA has been observed when ribosome availability is limited (Michel et al., 2000). During the stress of some viral infections, both the host and the virus translate circular mRNA (Rogers et al., 2017; Zarai et al., 2017;

Vicens et al., 2018; Keiper, 2019). For more information see Appendix III.

Considering the limitations of the current model of ribosome shunting, the distribution of ribosomes, on all late mRNA in vivo, was obtained. Polysome and ribosome

22 profiling experiments gave a global snapshot of translating ribosomes during the late phase of infection.

Polyribosome and Ribosome Profiling

The general strategy of polysome and ribosome profiling used in this thesis is illustrated in Fig. 8. HeLa cells were infected with dl309. At 15-16 h post-infection, cells were lysed in high salt buffer and cycloheximide (Zylber & Penman., 1970).

Cycloheximide is a elongation inhibitor that stops translating 80S ribosomes in their current location on the mRNA (Ingolia et al., 2011). The high salt breaks up any 80S ribosomes not engaged in translation.

In polysome profiling, the cell extract is layered onto sucrose gradients and centrifuged (Fig. 8 left). The polyribosomes, single 80S ribosomes, and ribosomal subunits are separated. The heavier polysomes move further down the gradient. The different ribosomal species are detected by UV and collected in fractions (Fig 8 right).

In ribosome profiling, RNase is added to the polyribosomes prior to layering onto sucrose gradients and centrifuged (Fig. 8 left). The RNase cuts the inter-ribosomal mRNA of the polyribosomes but is unable to degrade the mRNA protected inside the ribosome.

Therefore, RNase treatment converts the translating polyribosomes into single 80S ribosomes. The ribosome protected mRNA fragments (RPFs) inside the ribosome are protected and are 27-32-nucleotides in length (Wolin & Walter., 1988; Ingolia et al., 2009,

2013).

23 The RPFs are isolated and sequenced by high-throughput sequencing. Mapping of the RPFs to an HAdVC reference genome and transcriptome (Fig. 2) reveals the location of the translating 80S ribosomes on late transcripts.

Infect HeLa Cells UV Detector with HAd-Dl309

Lyse Cells in CHX Top-Down and HSB Fractionation

Polysome Ribosome Profile Profile Rnase-free Rnase-digest Fraction Collector

27-32nt Centrifugation Purify RNA Protected 1.5 hr 3 hr Fragments 10% Sucrose Free RNA Low MW Free RNA 40S 60S 40S 60S cDNA Library Preparation 80S

polysomes

polysomes 80S High-Throughput polysomes Sequencing 50% and Analysis Sucrose High MW

Figure 8. Polysome and ribosome profiling in late infected cells

(left) Hela cells infected with dl309 are treated with cycloheximide to stop translation. The ribosome extract is either treated (ribosome profile) or not treated (polysome profile) with RNase. The ribosomes are separated by centrifugation in sucrose gradients and detected by UV absorbance. The separated ribosomes are collected in fractions and the mRNA protected fragments are purified and sequenced.

25 Hypothesis for the Translation of Late Shunting-mRNA

The hypothesis, made prior to the experiments performed in this thesis, was that the uneven distribution of ribosomes observed on late messages was due to 80S ribosomes stacking in the 5’ UTR of shunting-mRNA (Fig. 9; Velicer and Ginsberg, 1968; personal communication, Ornelles lab).

The results were consistent with this hypothesis. The stacking of ribosomes appeared to be exclusive to mRNA thought to be translated by shunting. The data explain the non-uniform distribution of ribosomes on late-messages and lead to several new models for late-phase translation.

Figure 9. The hypothesized ribosome distribution during late viral translation Ribosome-protected fragments mapped to the HAdVC reference are shown to have deep coverage on the tripartite leader. This result would be consistent with ribosomes stacking in the leader.

27 MATERIALS AND METHODS

Cell Culture

Cell culture media, supplements, and serum were obtained through the Cell and

Viral Vector Core Laboratory of the Comprehensive Cancer Center of Wake Forest

University. HeLa cells (ATCC CCL 2; American Type Culture Collection, Manassas, VA) were maintained as monolayer cultures in Dulbecco-modified Eagle minimal essential medium (DMEM) supplemented with 10% newborn calf serum. HEK 293 cells were kept in DMEM supplemented with 10% fetal bovine serum. Cells were maintained in sub- confluent adherent cultures in a 5% CO2 atmosphere at 37°C by passaging them twice weekly at approximately a 1:10 dilution.

Virus

The dl309 virus was propagated by infecting HEK 293 cells at a low multiplicity of infection. This virus has a 600 bp deletion and insertion of salmon sperm DNA in its E3 region.

The virus was harvested 3 to 5 days post-infection from a concentrated freeze-thaw lysate by sequential centrifugation in discontinuous and equilibrium cesium chloride gradients. The gradient-purified virus was supplemented with 5 volumes of 0.012 M Tris-

Cl (pH 8.0), 0.12 M NaCl, 0.1 mg of bovine serum albumin per ml, 50% glycerol and stored at -20°C. The titer of each stock was determined by plaque assays using 293 cells.

28 Viral Infection

Cells were passaged 16 to 24 h prior to infection to a density of 1-2 x 105 cells per cm2. The cells were washed once with phosphate-buffered saline (PBS). The final wash was replaced with virus (10-25 plaque forming units [PFU] per cell) in infection medium

(PBS supplemented with 0.001 M CaCl2, 0.001 M MgCl2, and 2% calf serum or serum- free medium with 2% calf serum). The virus was added at one-fourth the normal culture volume and the cells were gently rocked for 60 min at 37°C. The viral suspension was then replaced with normal growth medium and the infected cells returned to 37°C.

Sucrose Gradients

Sucrose gradients were prepared in high salt buffer (HSB; 10 mM Tris-Cl, pH 7.4,

50 mM MgCl2, 500 mM NaCl) and cycloheximide (CHX). A 10% sucrose solution was gently layered onto 42% sucrose in RNase-free SW41 polyallomer tubes. The tubes were incubated horizontally for 2.5 h at 4°C and then vertically for 40 min at room temperature

(RT). Sucrose gradients were prepared and used the same day.

29 Cell Lysis

Cycloheximide (Sigma) was added to each cell plate (0.1 mg/ml) at 15-16 h post- infection. The plates were subsequently incubated at 37°C for 10 min and transferred to a cold room (4°C).

The media was exchanged with Dulbecco Phosphate Buffer Saline (PBS) supplemented with CHX (1.5 mM KH2PO4, 8 mM Na2HPO4, 138 mM NaCl, 2.7 mM

KCL, Mg-, Ca-, pH 7.4, 0.1 mg/ml CHX). Cells were harvested from plates with a cell scraper and pelleted in a centrifuge at 400 x g (5 min, 4°C). The pellet was resuspended in

PBS/CHX and transferred to a 2 ml microtube. The cells were pelleted again at 1100 x g

(5 min, 4°C) and swelled in 0.2 ml Reticulocyte Standard Buffer supplemented with the

RNase inhibitor vanadyl ribonucleoside (10 mM) and CHX (0.1 mg/ml). The cells were lysed in 0.2 ml RSB + detergent (RSB;10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2

+ 0.5% sodium deoxycholate, 1% tween 40) and sheared 5X with a 25-gauge needle gently at 4°C. The nuclei and organelles were pelleted at 2000 x g for 10 min at 4°C. The supernatant containing the ribosomes was transferred to a new sterile 2-ml tube.

Polysome Profiling

Polyribosomes were isolated from cells that were either infected or non-infected with dl309. HSB and CHX (0.1 mg/ml) were added to the polyribosome extracts prior to loading onto sucrose gradients (Fig. 8). The sucrose gradients were centrifuged using a

SW41 rotor at 37,000 RPM for 3 h at 4°C. The sucrose fractions (700 ul) were removed from the top. The sucrose fraction passed through a UV detector at a wavelength of 280 nm and was collected into 2-ml sterile tubes.

30 The collection and detection system consisted of the Bio-Rad LP UV system coupled to the Labconco Auto Density-Flow device. The system was sterilized with 0.2 N

NaOH, 0.2% SDS, DEPC-treated water.

Ribosomal RNA Purification from Sucrose Fractions

One volume of isopropanol with the coprecipitate, glycoblue (Fisher AM9515), was added to the fractions having ribosomes (Fig. 10). The rRNA was precipitated at -20°C overnight. The RNA was pelleted (16,000 x g, 30 min, 4°C) and washed with 75% ethanol.

Each pellet was resuspended in 150 ul 1% N-Lauryl Sarcosine (N-LS in DEPC H2O) with proteinase K (20 mg/ml) and incubated (30 min, 37°C). After incubation, each fraction received 150 ul of 4 M guanidine thiocyanate (GTC), 20 mM sodium acetate (pH 5.2),

0.5% N-LS, DEPC H2O with 0.7% 2-mercaptoethanol. The pellets were recovered and resuspended in 10 uM sodium acetate (pH 5.5). The ribosomal RNA was detected using a non-denaturing 1% agarose gel.

The 1% agarose gel (Fisher BP 165-25) was prepared in Tris-Acetate-EDTA (TAE) buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.6) supplemented with 1X

SYBR gold (Fisher S11494). Gels were run for 10 min at 200 V.

Control Ribosome Protected Fragments

Control RNA that was 28 and 30-nucleotides in length was used in experiments to test each step of the processing of small RNA for sequencing. The 28-nucleotide RNA standard was 5-AUGUACACGGAGUCGACCCGCAACGCGA-3’ (IDT). The 30-

31 nucleotide standard was 5’-AGCUGGGAUGAUCAGUCAGGAUCGUCCAUG-3’

(IDT).

RNA Separation by Denaturing Polyacrylamide Gel Electrophoresis

The 17.5% polyacrylamide gels (40%, 19:1) were prepared as follows: 4.38 ml of

40% (w/v) polyacrylamide (19:1 Acrylamide/Bis), 4.2 g Urea (mw 60), 5.62 ml of 0.5X

Tris-Borate-EDTA (TBE) and incubated in a water bath (60°C) until all the Urea dissolved.

40 ul of 25% Ammonium Persulfate and 13 ul TEMED were added to polymerize the gel and they were cast for 1 hour at room temperature.

The RNA was denatured loading buffer consisting of 8 M Urea, 20 mM EDTA, 5 mM Tris and 0.5% bromophenol blue. Prior to loading in the gel, the RNA was incubated at 70°C for 10 min and immediately placed on ice. The gels were run for 1 h 15 min at 256

V followed by staining with 1X SYBR gold (Fisher S11494).

RNA Quantification by Gel Densitometry

The RNA from polysome and ribosome profiling separated polyacrylamide gels was quantified using the using the gel densitometry (Berardi et al., 2019) function in

ImageJ (https://imagej.nigh.gov/ij/). Gel images were uploaded (File > Open) and converted to 8-bit greyscale images (Image>Type>8-bit). The gel bands intensity, with background, was measured using the gel analysis tool (Analyze > Gels). The intensity from each gel band was plotted (Analyze > Gels > Plot Lanes) and a baseline was drawn using the straight-line tool. Each peak was integrated using the wand tool and clicking within

32 each enclosed peak. The reported concentration of the RNA molecular weight standards was used to calculate the concentration of RNA. RNA Quantification by Fluorescence

The RNA from polysome and ribosome profiling was quantified using the Qubit fluorimeter (Fisher). The working solution was prepared with supplied buffer and RNA specific dye. The RNA was mixed with the working solution in assay tubes, vortexed and incubated for 2 min at room temperature and read.

RNA Separation and Quantification by Microchip Electrophoresis

The RNA from polysome and ribosome profiling was separated by size and quantified by microchip gel electrophoresis using the Bioanalyzer 2100 (Agilent). The chip priming station was prepared by installing a fresh syringe, adjusting the syringe clip to the top position, and adjusting the base plate to position C. The Agilent RNA ladder was denatured at 70°C for 2 min, placed on ice and diluted with DEPC-water. The RNA gel- dye mix was prepared by first centrifuging the RNA denaturing polyacrylamide gel matrix

(1500 x g, 10 min, RT) and then adding the RNA dye concentrate. The gel-dye mix was infused into the microchip prior to loading RNA. The RNA conditioning solution, RNA marker, denatured RNA ladder, and RNA samples were added to the chip and vortexed at

2400 rpm for 1 min at room temperature (IKA Vortex).

The sizes and concentration of all detected RNA are recorded by the instrument.

The 18S and 28S rRNA were detected and reported by the instrument.

33 Control RNA Precipitation from Polysome Fractions

The large rRNA was extracted from sucrose using 1 volume IPA with glycoblue.

A control experiment nucleotide was performed to test if IPA can successfully extract RNA the size of RPFs along with rRNA.

Polysome sucrose fractions from non-infected cells were spiked with an RPF control RNA and precipitated with 1 volume IPA or 4 volumes of ETOH (overnight at -

20°C). Purification of RNA was performed using 1% N-Lauryl Sarcosine (N-LS in DEPC

H2O) with proteinase K (20 mg/ml), 30 min at 37°C. The RNA was separated and detected in a 17.5% denaturing polyacrylamide gel (Fig. 10). The IPA was found to be as good as

ETOH at precipitating small RNA from polysome fractions at ¼ the volume. The RNA precipitation using 1 volume IPA was used in this thesis.

34 IPA ETOH ETOH

28 nt

Figure 10. One volume of isopropyl alcohol is as good as 4 volumes of ethanol at precipitating RNA the size of ribosome protected fragments from sucrose having Figure polysomes 8: fffffffffffffffdfdfd Polysome sucrosefdfdfdfdfdfdfdfdfdfdfdfdfdf fractions were spiked with an RPF control RNA of 28- nucleotides. The total RNA was precipitated with either 1 volume IPA or 4 volumes of ETOH. RNA precipitation with IPA was as good as ETOH at ¼ the volume. The RNA was separated in a 17.5% denaturing gel run at 200 V for 1 hr.

35 Control RNA Enrichment from Polysome Fractions

Polysome sucrose fractions, from infected cells, were spiked with an RPF control

RNA of 30-nucleotides. The total RNA was precipitated as described previously and enrichment of the small RNA was performed using the Mirvana kit (Fisher). The 30 nucleotide RNA was enriched from the total RNA (Fig. 11; lane 6). Therefore, the small

RNA enrichment protocol was used during the ribosome profiling experiments. The RNA standards used were the low range ssRNA standard (Ambion) and the microRNA ssRNA standard that consisted of 25, 21, and 17-nucleotides (Ambion).

36 RNA_STND TOTAL_RNA RNA_STND RNA_STND ENRICHED_RNA BLANK RNA_STND 1 2 3 4 5 6 7

80 80

30 25 * 21

Figure 11. RNA the size of ribosome protected fragments was enriched from total RNA in polyribosome fractions from cells infected with dl309 Polysome sucrose fractions, from dl309 infected cells, were spiked with an RPF control RNA of 30-nucleotides. The total RNA was purified (lane 4). The control RNA was enriched from the total RNA (lane 6; asterisk). The 30 nucleotide RNA was identified using RNA standards (lanes 1,3,5,7). The RNA was separated in a 17.5% denaturing gel run at 200 V for 1 hr.

37 Control RNA Polyacrylamide Gel Purification

Ribosome-protected Fragments (RPFs) needed to be extracted from polyacrylamide gels prior to cDNA conversion and sequencing. Consequently, a small

RNA PAGE purification protocol was tested (Zymo). The supplied buffers and columns are proprietary.

The control RNA (30 nt), used in Fig. 11, was separated in a 17.5% denaturing polyacrylamide gel (Fig. 12). The gel band was quantified by densitometry and had a concentration of 10 ng/uL (Fig. 12; lane 5)

The gel slice having the RPF control RNA (Fig. 12 box) was crushed in a spin IV column. The RNA recovery buffer was added, and the sample was incubated at 65°C for

15 min and then -80°C for 5 min. The spin IV column was centrifuged at 1600 x g for 30 s. The flow-through was added to a spin IIIC column and centrifuged at 1600 x g for 30 s.

The RNA-Max buffer (2 volumes) was added to the flow-through and transferred to a a spin IC column. The column was centrifuged at 12000 x g for 30 s and the flow-through discarded. The RNA Wash Buffer was added to the column and centrifuged at 12,000 x g for 30 s (2X). The RNA was recovered by adding 10 ul of DEPC-water to the column, incubating for 1 min at room temperature, and centrifuging at 10,000 x g for 1 min.

The RNA purified from the PAGE gel was quantified using the Qubit fluorimeter. The

RNA concentration was 6 ng/uL or a 60% recovery. This purification protocol was used to purify RPFs in this thesis.

38 Small_Stnd Small_Stnd Total_RNA No_Enrichment Total_RNA After_Enrichment Enriched_RNA RNA_Stnd RNA_Stnd Blank

1 2 3 4 5 6 7 8

30 28 30

25 21 17

Figure 12. Enriched RNA the size of ribosome protected fragments can be purified from PAGE gels The 30-nucleotide control RNA used in Fig. 11(asterisk) was cut out and purified with a PAGE purification protocol. The integrity of the RNA (box) was checked using electrophoresis. The gel was a 17.5% denaturing gel run at 200 V for 1 hr.

39 Ribosome Profiling

RNaseI was added (2.3 U/ul) to the polyribosome supernatant and incubated for 45 min at room temperature with gentle rocking (Fig. 8). To stop the reaction, the RNaseI inhibitor SUPERase-in (200 U) was added after 10 min at 4°C. The sucrose gradients were centrifuged using a SW41 rotor at 37,000 RPM for 3 h at 4°C and fractionated from the top-down. The sucrose fractions passed through a UV detector at a wavelength of 280 nm and was collected into 2-ml sterile tubes.

The total RNA was precipitated with 1 volume IPA and the small RNA enriched

(Mirvana). The small RNA was next treated with Polynucleotide Kinase (PNK; NEB).

PNK was needed because RNaseI leaves a 2’,3’ cyclic monophosphate and a 5’ hydroxyl group. The RPFs needed to have a 5’ phosphate and 3’ hydroxyl group prior to reverse transcription.

PNK transfers the gamma-phosphate of ATP to the 5’ hydroxyl groups of RNA making a 5’ phosphate. The enzyme also cleaves 2’, 3’ cyclic monophosphate rings leaving a 3’ hydroxyl group.

The PNK reaction consisted of 5 ul 10X PNK buffer, 5 ul 10 mM ATP, 2 ul 10 U/ul

PNK, 38 ul RPFs, 1.96 mM DTT. Then it was incubated at 37°C for 30 min and then 65°C for 20 min. After the reaction was complete the samples were analyzed on a 17.5% denaturing polyacrylamide gel (Fig. 13 top). The gel bands in the size range of RPFs were sliced out of the gel (Fig. 13 bottom) and the RNA purified using the PAGE purification protocol (see above).

Figure 13. The ribosome protected fragments from late infection were enriched and gel purified from a PAGE gel (Top) The enriched small RNA from the ribosome profile (lanes 4-9). (Bottom) RNA, the size of RPFs, were cut out of the gel and purified. The gel was a denaturing 17.5% polyacrylamide gel run at 200 V for 1 hr.

41 Polynucleotide Kinase Modification of Ribosome Protected Fragments

The PAGE purified small RNA was treated with PNK again to ensure the proper

5’ and 3’ ends. After the reaction, a modified PAGE recovery protocol was used to recover

RNA in solution.

The sample was brought to 50 ul in DEPC-water. Then RNA recovery buffer (3 volumes) and RNA-Max-buffer (2 volumes) were added. The sample was added to a spin

IIIC column and centrifuged at 1600 x g for 30 s. RNA-Max buffer (2 volumes) was added to the flow-through and transferred to a spin IC column that was centrifuged at 12,000 x g for 30 s. Following the removal of the flow-through, the RNA Wash Buffer was added to the column and the column was centrifuged at 12,000 x g for 30 s (2X). The PNK modified

RNA was eluted off the column with 10 ul of DEPC-water, incubated at room temperature for 1 min. The column was centrifuged at 10,000 x g for 1 min and the flow-through was recovered. The RNA was 30 ng (6 ng/ul).

Reverse Transcription of Ribosome-protected Fragment

The RNA in the size range of RPFs was converted to cDNA using a reverse transcription protocol according to the manufacturer instructions (Ion Total RNA-Seq Kit v2: catalog number 4475936, publication number 4476286 RevB). The RNA and DNA primers, enzymes and buffers used are proprietary.

The PNK-treated RNA (12 ng) was hybridized and ligated into the supplied strand specific adapters. The adaptors are flanked with the primers necessary for amplification and sequencing. The adaptors also have a unique barcode sequence that allows the post-

42 sequencing software of the PGM to parse out cDNA correctly prepared from incorrect species.

The hybridization reaction consisted of hybridization solution, adapter mix and two reaction temperatures of 65°C (10 min) and 16°C (5 min). The ligation of the RNA to the adapters was performed using a ligation enzyme and buffer. The ligation reaction in the protocol was supposed to be done at 16°C for 16 h. However, the ligation reaction used for the RPF conversion was done for 16 h at room temperature by mistake. The higher temperature may have increased the rate of the ligation reaction without harming the enzyme.

The ligated RNA library was converted to cDNA using the supplied reverse transcriptase at 42°C for 30 min. The cDNA library was size selected and amplified by

PCR for 16 cycles. The resultant cDNA was analyzed using the bioanalyzer (Fig. 14). With the adapters used, the final cDNA should be close to 105 bp if the reaction was successful.

The cDNA was measured to be 104 bp which is within the error of the system.

A smear analysis, using the bioanalyzer was performed to determine the distribution of the correct cDNA products in the sample. The program integrates the area of desired cDNA products (86-106 bp) divided by the area of all ligation products (50-300 bp). The smear analysis for the cDNA was 68% consistent with the recommended > 50% value (Ion Total RNA-Seq Kit v2). The cDNA was therefore bead templated and sequenced.

Figure 14. The RNAs protected by ribosomes were successfully converted to cDNA The length of the cDNA library was measured three times by the bioanalyzer. The three assays gave identical peaks. The cDNA peak was 104 bp indicating a successful cDNA conversion. The flanking peaks, at 35 and 10380, are internal standards. The y-axis is in fluorescent units (FU) and the x-axis is in base-pairs (bp).

44 Template Preparation for Sequencing

The cDNA library was clonally amplified onto Ion Sphere Particles (ISPs) via the

Ion One Touch 2 system according to the manufacturer instructions (Ion Template OT2

200 Kit protocol catalog number 4482006, publication number MAN0007273). The

OneTouch recovery tubes, amplification plate, oil, recovery solution, and amplification solution were prepared and inserted into the system. The reagent mix, PCR reagent B, enzyme mix, cDNA library, and ISPs were prepared and added to the OneTouch reaction filter assembly. The reaction oil was added to the filter and inserted into the system. After the reaction was completed, the templated and non-templated ISPs were centrifuged and recovered. The distribution of both species of ISPs was assessed by fluorescence.

The Measured Distribution of Templated vs Non-Templated Ion Sphere Particles

The quality of the template-positive ISPs was assessed using the Qubit fluorimeter

(Ion Sphere Quality Control assay, catalog number 4482006, publication number

MAN0007273). Alexa-Fluor 488 and Alexa-Fluor 647 primer probes were annealed (95°C

2 min, 37°C) to a sample of ISPs. The ISPs were washed 3X with wash buffer to remove unbound probes.

The 488 value measures the total bead population and the 647 value is a measure of the total templated population. The ratio of the two signals gives a qualitative assessment of the percent templated beads. The percent templating for the cDNA library was 14% which is within the manufacture’s specifications. The templated ISPs were next enriched from the total population.

45 Enrichment of Template-Positive ISPs

The template-positive ISPs were enriched using the Ion OneTouch Enrichment

System (ES) according to the manufacturer instructions (catalog number 4482006, publication number MAN0007273). The following were added to each well of the enrichment 8-well strip in order: templated and untemplated bead sample, MyOne streptavidin C1 beads, wash solution, and melt-off solution. The melt-off solution consisted of 125 mM NaOH and 0.1% Tween20. Following enrichment, the ISP population was

97.8% templated (Fig. 21). The template-positive ISPs were recovered and sequenced.

46 High Throughput Sequencing of the cDNA library Using the IonTorrent Personal

Genome Machine

The enriched template-positive ISPs were sequenced using the Ion Personal

Genome Machine (PGM) according to the manufacturer instructions (Ion PGM 200

Sequencing Kit catalog number 4480974, publication number MAN0007220). The wash solutions and nucleotide reagent bottles were prepared and the PGM initialized to a pH of

7.4. The PGM sequencing primer was annealed to the enriched template-positive ISPs

(95°C 2 min, 37°C). The DNA polymerase was added and incubated at room temperature for 5 min. The templated-ISPs were loaded onto a 318 chip and sequenced.

Integrity Check of the FASTQ File After Downloading

High throughput DNA sequencing data is reported in the FASTQ format (Cock et al., 2010) The FASTQ format consists of 4 lines per sequence. The first line is the header and begins with an “@” sign followed by a name and the Y, X coordinates of the sequencing well on the chip. The second line has the DNA sequence. The third line is just a placeholder “+” and the fourth line has the Sanger quality scores of each base in ASCII format. The FASTQ file had 1,633,229 reads (6,532,916 lines) that passed the inherent quality filtering performed by the PGM software (Fig. 21).

Due to the large size of the file, an integrity check was performed after downloading and transferring it to make sure no data was lost. Using the UNIX command line, the total number of sequences in the downloaded FASTQ file was checked with the following command: $ echo "$(wc -l < FASTQ_2)/4" | bc

47 The first command returns (i.e. echo) the number of lines (i.e. wc -l) of the FASTQ file and divides the lines by 4. The output is then passed (i.e. piped) to the Unix basic calculator (i.e. bc). This gives the number of sequences in a FASTQ file. There was no data lost from the FASTQ.

Read-Length Determination of Sequencing Reads

The read-length distribution of each sequence was determined with the following command (Table I):

$ cat FASTQ_2 | awk 'NR%4==2'|awk '{print length($1)}' | sort | uniq -c

| sort -rn

The cat command extracts each line and pipes the output to the awk command. The option NR%4==2 prints every second line in the FASTQ. This is the line that has the sequences. The read-length of each sequence was determined by the length function of awk and the different lengths were sorted by size (sort). Finally, the frequency of each unique read-length was calculated (uniq -c) and sorted in decreasing order according to number

(sort -rn).

Quality Scoring of Sequencing Output

The measurement of sequencing quality (Q) is in what is known as the phred format. This format estimates the probability (P) that each base call was wrong

(Korpelainen et al., 2014). The Sanger quality scores in the FASTQ file are converted to phred quality (Q) scores by subtracting an offset of 33. The phred score is converted to a 48 probability that the base call is incorrect by using the equation: Q = -10 log10P (Cock et al.,

2010). The phred score for each base was obtained using the program FASTQC and the results opened using firefox (Andrews, 2010; Fig. 22).

$ fastqc FASTQ_file

$ firefox

Quality Trimming, Filtering and Mapping

After determining the quality of each base using FASTQC, the sequences were trimmed using a minimumpPhred quality score of Q20 (99% accuracy). The trimming was done using the CLC Genomics Workbench (Qiagen). The sequences were filtered using the FASTX program (http://hannonlab.cshl.edu/fastx_toolkit/). The command used was:

$ fastq_quality_filter -q 20 -Q33 -p 100 -i –o

The minimum phred quality score of Q20 was used (i.e. -q 20) for 100% of the bases (i.e. -p 100). The option -Q33 in the command informs the program that the sequencing file is in the Sanger quality format. The reads that were below an average of

Q20 were removed.

Reads not trimmed, trimmed, and filtered were mapped against an adenovirus reference (AY601635) to assess if the increased quality of the bases/reads resulted in a higher mapping efficiency (Fig. 23). The mapping criteria was as follows: Masking mode

= No masking, Match score =1, Mismatch cost = 2, Linear gap cost, Insertion cost = 3,

Deletion cost = 3, Length fraction = 1, Similarity fraction = 0.8, Global alignment = No,

Non-specific match handling = Map randomly.

49 RNA-Seq Mapping with CLC

Reads were mapped to the adenovirus reference genome AY601635 using the

RNA-Seq tool in the CLC Genomic Workbench with the following parameters: No spike- in control, Mismatch cost = 2, Insertion cost = 3, Deletion cost = 3, Length Fraction = 0.8,

Similarity Fraction = 0.8, Strand Specific: Forward, Maximum Number of Hits: 10.

The Detection of Periodicity from Mapped Reads

RPFs were mapped and the alignment recorded in a sequencing alignment map

(SAM) file. The presence of periodicity from the mapped reads was detected using the 5’ position of each read reported in the SAM file and the algorithm developed by Sokolove and Bushnell (Sokolove and Bushnell, 1978; Thieler et al., 2016). The algorithm was implemented using R. The statistics were generated by a periodogram that tests the trial periods in order to identify periodically varying signals by either chi-square statistics or a robust curve-fitting method. The R code parameters were: pgram(x, periods, method = c("chisq", "robust"), alpha = 0.05, p.adj = "BH", regression = c("huber", "L2"), model = c("step", "sine", "splines", "fourier(2)")

50 RESULTS

Translating 80S ribosomes protect mRNA sequences 27-32-nucleotides in length

(Wolin & Walter, 1988; Ingolia et al., 2014,2016). Several experiments were done to confirm that the mRNA purified for sequencing came from 80S translating ribosomes in the late phase of infection.

Separation and Detection of Polyribosomes in Translation

RNA-containing components in cellular extracts sediment in sucrose gradients under a centrifugal force at different rates depending on their mass. The mRNA transcripts, with the largest number of ribosomes, move at the greatest rate and therefore are closer to the bottom of the gradient. Lighter molecular weight material is found towards the top of the gradient (Fig. 8).

To detect the different ribosomal species, the polyribosome profile of uninfected

HeLa cells was performed. The polyribosomes were stalled by cycloheximide. The extracts from nine plates (i.e. 3.6 x 107 cells) were layered on linear sucrose gradients and centrifuged for 1.5 h. Sucrose gradients were prepared and used the same day. The gradients were extracted and collected from the top-down to avoid contamination by messenger ribonucleoproteins (mRNP) at the bottom of the gradient (Juntawong et al.,

2014). The mRNPs control mRNA processing, function, translation, localization and turnover (Maquat, 2004).

A characteristic polyribosome profile was observed (Fig. 15 black curve; Qiu et al.,

2009). The largest peak is from the top of the gradient (left). This contains low density

51 cellular material. The next peak is the 40S ribosome followed by the 60S, 80S, and a large polyribosome peak (Fig. 15 black curve; Zybler et al., 1970; Ingolia et al., 2019).

The large polysome peak, relative to the single 80S ribosome peak, is consistent with reports that, on average, most mRNAs are attached to more than one ribosome during translation (Rogers et al., 2017). Direct counts of ribosomes, by atomic force microscopy

(AFM), found that there are 8.7 ribosomes per transcript in human HEK293T cells and 8.3 ribosomes in human MCF-7 cells (Lauria et al, 2015). Real-time imaging of the individual mRNAs in HeLa cells (e.g. cells used in this thesis), were found to have an average of 12 ribosomes per transcript (Wang et al., 2016). The polyribosome and ribosome profiles in this thesis were performed using human HeLa cells.

The small single 80S peak is also the result of using high-salt-buffer. The high salt is known to disassemble any ribosomes that are not engaged to a message (Zylber &

Penman., 1970). The amount of 80S ribosomes in cells that are not attached to transcripts can distort the results of polysome and ribosome profiling experiments (Liu & Qian, 2016).

Therefore, prior to loading the cellular extract onto sucrose gradients, high-salt-buffer

(HSB) was added. Therefore, the HSB increases the likelihood that the proteins detected and collected are ribosomes translating mRNA.

The wavelength used to detect the ribosomal species was 280 nm. Proteins, RNA, and DNA absorb at this wavelength. To verify that the peaks in the profile represent ribosomes, experiments that can detect general RNA and specifically rRNA were performed.

52 Verification of RNA and Ribosomal RNA from the Polysome Profile

The RNA from the first 15 fractions of the gradient were purified and quantified using the qubit fluorimeter. Though not having the same level of resolution as the polysome profile, the total RNA-specific pattern was consistent (Fig. 15 red curve). The most abundant peak from the RNA- specific assay corresponded to the polyribosome peak (Fig

15 black and red curve). This result confirmed that much of the absorbance at 280 nm in the polyribosome peak was indeed from protein and RNA.

The human 60S ribosomal subunit has the 28S (5070 nt), 5.8S (156 nt) and 5S rRNA (121 nt). The 40S ribosomal subunit has the 18S rRNA (1869 nt). Therefore, the detection of the 28S, 5.8S, and 5S rRNA would be indicative of the 60S ribosome and 18S rRNA the 40S ribosome. All four rRNAs in the same fraction would be consistent with the complete 80S ribosome.

The first 11 of the fractions from the polysome profile checked for ribosomal RNA using microfluidic electrophoresis via the bioanalyzer (Fig. 16). The internal standards used in the bioanalyzer allows the program to automatically detect and report the 28S and

18S rRNA. This assay would confirm that the proteins and RNAs in the polyribosome profile were in fact from ribosomes (i.e. rRNA).

The digital gel from the bioanalyzer assay is in Fig. 16. Lanes 2-4, from the top of the gradient, did not have rRNA but low molecular weight material. Lane 5 showed only the presence of 18S rRNA of the 40S ribosomal subunit. Lanes 7-12 had both the 18S and

28S rRNA of the complete 80S ribosome.

The program does not automatically identify and report the 5.8S and 5S rRNA of the 60S subunit. However, the digital gel showed two RNA bands that were around the

53 right size of these rRNA species (Fig. 16). These bands are only found in the fractions that have both the 28S and 18S rRNA. This is consistent with these bands being from the 5.8S and 5S rRNA.

Another bioanalyzer assay was performed on the first 12 fractions. The 28S and

18S rRNA were detected and their ratio plotted in Fig. 17. The ratio increased down the gradient consistent with an increase in the number of polyribosomes on mRNA (Fig. 17).

The polyribosome profile (Fig. 15 black curve), the RNA-specific qubit assay (Fig.

15 red curve) and the bioanalyzer assays (Figs. 16, 17) confirmed that the polyribosome profiling method was suitable for isolating and detecting ribosomal species.

The ribosome profile requires the release of 80S ribosomes from polyribosomes

(Fig. 8). Therefore, polyribosome profiling was performed in the presence of RNase I.

Separation and Detection of Single 80S Ribosomes in Translation

RNaseI from E. coli cuts the inter-ribosomal mRNA in polyribosomes releasing single ribosomes (Ingolia et al., 2009, 2019). It degrades all dinucleotide bonds of RNA nonspecifically reducing bias. In addition, the enzyme has been found to not degrade rRNA in ribosome profiling experiments (Gerashchenko & Gladyshev., 2016).

RNaseI can be controlled by inhibitors. One inhibitor, SUPERase-in, has been successfully used in several ribosome profiling experiments (Ingolia et al., 2009, 2011;

Guo et al., 2010). After cutting polyribosomes with RNase, the single ribosome peak in the profile increases as the polyribosome peak in the profile decreases (Ingolia et al.,

2009,2011; Guo et al., 2010). The increase in intensity of the single ribosome peak is a clear indicator of 80S ribosomes (Ingolia et al., 2009,2011; Guo et al., 2010).

54 Extracts from non-infected cells, having polyribosomes, were treated with RNaseI prior to centrifugation. Following RNase treatment there was a clear decrease in the polyribosome peak (Fig. 18 black curve). This is consistent with the RNase cleaving the inter-ribosomal RNA, releasing the 80S ribosomes. However, there was only a slight rise in intensity for the single 80S peak (Fig. 18 red curve).

The RNA-specific assay was performed on all fractions from both profiles. The most abundant peak in the non-RNase sample matched the polyribosome peak (Fig 18 inset; black curve). The fluorescent signal of this peak decreased with RNase consistent with the releasing of ribosomes (Fig. 18 inset; red curve). Interestingly, the RNA-specific assay showed a corresponding increase in a new peak that may correspond to the release of 80S ribosomes.

The apparent shift in the RNA-specific assay (Fig. 18 inset) comes from only one data point and may be an artifact. To better resolve the single 80S ribosomes, RNase concentration and centrifugal time were explored.

Polysome extracts, from non-infected cells, were non-treated or treated with three different RNaseI concentrations (i.e. units: 0.3, 0.8, 2.4) and centrifuged for 3 h. The polyribosome profile that was not treated with RNase showed the characteristic small single ribosome peak and larger polysome peak (Fig. 19 purple curve). Treatment with

RNase showed a clear increase in the released 80S ribosome peak (Fig. 19). The increase in the 80S ribosome concentration was the direct result of the decrease in polyribosomes

(Fig. 19). Performing ribosome profiling, with the addition of HSB, made the RNase induced single ribosome peak clearly distinguishable.

55 When the RNase concentration was increased from 0.3 units to 0.8 units there was no clear difference in the number of single ribosomes (Fig. 19 red and green curve).

However, increasing the RNase to 2.4 units clearly showed that more 80S ribosomes were released (Fig. 19 cyan peak). It is not clear why increasing the RNase a factor of 2.7X (i.e.

0.3 to 0.8) had no discernible effect but increasing by 3X (0.8 to 2.4) did.

The slight leftward shift at 2.4 units of RNase is likely due to variation in the sucrose gradients and not degradation of rRNA. It has been previously determined that

RNaseI does not degrade rRNA at this concentration (Gerashchenko & Gladyshev., 2016).

The single ribosomes were released from polyribosomes in HSB. Therefore, the single ribosomes came from polyribosomes in translation. The increase of a single peak, as the polysome peak decreased, is a clear indicator of the fractions that have the ribosome protected fragments (RPFs; Guo et al., 2010; Ingolia et al., 2019). These conditions were applied to cells infected with dl309 late in infection.

Polyribosome and Ribosome Profile of Adenovirus Infected HeLa Cells

The polyribosome and ribosome profiles were performed late in infection (Fig. 20).

Unlike non-infected profiles, where there was always a small 80S peak, all the polyribosome profiles of infected cells only showed the low absorbing material and a large polyribosomes peak (Fig. 20 purple curve). As a result, a mock infected sample was included to define the 80S ribosome location (Fig. 20 red curve).

RNase treatment of the polyribosomes from infected cells showed a clear peak arising from the release of 80S ribosomes (Fig. 20. green and cyan curve). The single ribosome peak also increased with added RNase. A more careful preparation of sucrose

56 gradients minimized variation between samples. The fractions containing the RNA protected by ribosomes were collected (Fig. 20 blue bar) and RPFs were purified and subjected to high-throughput sequencing.

High Throughput Sequencing of Ribosome-protected Fragments from Adenovirus

Infected Cells

The ribosome-protected fragments were converted to cDNA, clonally amplified onto ion sphere particles (ISPs), and templated-ISPs were enriched prior to sequencing.

The enrichment for clonal ISPs resulted in 97.8% (6,281,428) templated (i.e. live) beads loaded onto the sequencing chip (Fig. 21). After sequencing, reads that were of low quality, primer dimers, or polyclonal ISPs were filtered out. Consequently, there were 2,450,647 final reads passing the inherent filters of the system (Fig. 21).

Of the 2,450,647 reads, there were 1,633,229 reads that had a detectable barcode from the adapters used in preparing the library (Fig. 21 RNA_Barcode). The barcode identifies the cDNA that was correctly made from the RPF library. These reads were used for analysis.

The quality and read-length of each base in the sequencing file was analyzed using

FASTQC (Fig. 22). The box plot (top) shows the quality (Phred score) as a function of base position in all reads. The red line across each base position is the mean quality score.

Reads in the range of RPFs (27-32 nt) had a mean score greater than Q22 or 99.4% base- call accuracy.

The x-axis shows that reads over 120 bases were detected. These are likely amplification and/or sequencing artifacts (Rothberg et al., 2011; Korpelainen et al., 2014).

57 These longer reads were minimal in comparison to the reads within the length of RPFs

(Fig. 22 bottom; Table I)

The minimum quality scores in the RPF range were Q12-15. This amounts to a base-call accuracy of 94-97%. One standard approach of post-sequencing data processing is to trim bases or filtered reads based on a minimum value of Q20 (99% base-call accuracy;

Korpelainen et al., 2014).

Trimming involves removing lower quality bases, usually from the 3’ end, resulting in shorter reads of higher quality. Filtering involves taking the average quality of the entire read and discarding the read if its average is below Q20. The value of trimming or filtering the RPFs prior to analysis was explored.

Trimming or Filtering

The read-length of RPFs are a characteristic 27-32-nucleotides (Ingolia et al.,

2019). Out of the 1,633,229 reads, there were 1,053,356 reads within this range (Table I

Column I).

Trimming resulted in a reduction of over 500,000 total reads that were in the read- length of RPFs (Table I, Columns I, II). Hence trimming produced false negatives (RPFs trimmed to lower lengths). In addition, trimming may have produced false positives (longer reads trimmed to 27-32-nucleotides). The identification of false positives can only be obtained by mapping the reads to a reference.

In contrast to trimming, filtering RPFs does not alter the read-length. This drops the potential for false positives and negatives. However, quality filtering did reduce the

58 number of reads (Table I). The filtering of RPFs resulted in a reduction of over 700,000 reads within the read-length of the RPFs.

Trimming or filtering, prior to analysis, is often found to increase the accuracy of reads mapping to a reference (Korpelainen et al., 2014). This is due to the higher quality base-calls and/or reads. Therefore, the reads that were trimmed, filtered, or neither trimmed nor filtered were mapped against a reference genome.

59 Table I. The read-length frequency distribution of ribosome protected fragments before and after quality trimming or filtering

60 Mapping Sequences to the HAdVC Reference Genome AY601635

RPFs from the original file, trimmed, or filtered were mapped against the HAdVC reference genome AY601635 (Fig. 23). Trimming did not improve the mapping efficiency in terms of location or depth. (Fig. 23 red and green). Filtering the reads did not affect the location of mapping (Fig. 23 red and blue). However, filtering did greatly reduce the depth of mapping in some regions (e.g. position 6000-12000).

Neither approach increased the accuracy of mapping. Rather trimming resulted in false negatives and filtering caused a significant reduction in total reds. Therefore, the RPFs were not trimmed or filtered prior to mapping.

Since the reads came from 80S ribosomes, the RPFs are expected to contain sequences from the human genome, rRNA, HAdVC, and possibly human papillomavirus virus (HPV-18). The HPV-18 reference was used because HeLa cells are from a woman that had cervical cancer from HPV infection.

Mapping of RPFs to HAdVC, Human, rRNA, and HPV-18 References

The RPFs were mapped to the human rRNA reference. About 66% of the reads mapped to rRNA (Table II). This is due to rRNA fragmenting during the preparation of

RPFs and is a common result in ribosome profiling (Ingolia et al., 2009,2019; Guo et al.,2010).

The reads mapped to each species of rRNA (Fig. 24). The 28S rRNA is larger than the 18S by a factor of 2.7 (Fig. 16). The larger size is thought to be the reason for the observation that the 28S is less stable than the 18S resulting in more 28S fragments found

61 in RNA sequencing experiments (Bahar et al., 2007;Palmer & Prediger.,Ambion

TechNote)

Fig. 24 shows the mapping to rRNA. Prior to mapping, the rRNAs were normalized for their differences in length. The number of normalized reads that mapped to the 28S were 2X as much as the reads that mapped to the 18S. The 2X difference is consistent with the 28S being 2.7X the size of 18S rRNA (Palmer & Prediger.,Ambion TechNote). This

2:1 ratio of 28S:18S rRNA is also considered to be a characteristic of high quality intact

RNA (Palmer & Prediger.,Ambion TechNote).

The 5.8S rRNA is annealed to the 3’ end of the 28S rRNA. As a result, there is often a 1:1 ratio of the two species even after fragmentation (Walker et al., 1983; Keller et al., 2009). Consistent with this fact, the number of reads that mapped to both the 28S and

5.8S rRNA were essentially identical (Fig. 24).

The 5S rRNA is associated with the 28S, and 5.8S rRNA in the 60S ribosome. The

5S rRNA has been observed to be the most stable of the four rRNAs. It is often used as a control RNA in reverse transcription RT-PCR due to its stability (Takamizawa et al., 2004;

Pineles et al., 2007). Heat shock experiments on HeLa cells (used in this thesis) showed that the 5S rRNA was significantly less degraded than other rRNA species (Sadis et al.,

1988).

The stability of the 5S rRNA suggests that it would fragment less during the preparation of RPFs and therefore not have many cDNA sequences compared to the other rRNAs. Fig. 24 shows that the reads mapping to the 5S rRNA were lower than the reads mapping to the 28S and 5.8S by a factor of six. This is consistent with the higher stability of the 5S rRNA.

62 Interestingly, the 5.8S and 5S rRNA are similar in size, differing by only 30- nucleotides (Fig. 16). The 6X difference of the reads that mapped to the 5.8S rRNA would suggest that stability of the 5S rRNA is not due to its size This result is consistent with earlier reports that state that the stability of the 5S rRNA is due to its secondary structure

(Hadjiolov, 2012).

The mapping to rRNA showed that the fractions collected from the ribosome profile peak had both the 40S and 60S subunits (Fig. 20). These results are another confirmation that the mRNA that was sequenced came from 80S ribosomes (Fig. 24).

The reads that did not map to rRNA were mapped to the female human (hg19),

HAdVC (AY601635), and Human Papillomavirus (HPV-18) genomes. The human and

HAdVC genome had a total of 86,014 and 52,647 mapped reads respectively (Table II).

Only 2 reads mapped to HPV-18 (Table II).

When the number of mapped reads were normalized for exon length, rRNA was 10 times more abundant than HAdVC, and the expression of HAdVC was 500 times greater than its host. The difference in viral expression over its host is consistent with HAdVC ability to use the ribosomes for its own translation during late infection (Schneider 2000,

2004).

63 Table II. Overall mapping of RNA protected by ribosomes

Translational Phenomena

RNA protected by translating ribosomes would be expected to contain certain features that are exclusive to translatable mRNA (Ingolia et al., 2009). These features include spliced RNA, the translation of distinct reading frames, and triplet periodicity. The data in this section show the power of ribosome profiling to detect and study translational phenomena

Spliced Messages

The mRNA from the late transcription unit is differentially spliced resulting in several ORFs from a single mRNA (Fig. 2). The distinct messages share some of the mRNA, but in different frames.

The L4 transcription unit contains the ORFs for 100K, 33K and 22K (Figs. 2-3).

The first 300-nucleotides of the 33K and 22K ORFs overlap with the end of the 100K ORF.

Moreover, the ORFs from the 33K and 22K transcripts have the same start site, sharing the

64 first 100 N-terminal amino acids. However, they differ at their C-terminus via alternative splicing of 33K, making two unique proteins (Lan et al., 2016).

The overall read coverage of the L4 unit is shown in Fig. 25. The start sites of 33K and 22K are shown to be 2000-nucleotides downstream from the start site of 100K. The data show that the RPFs captured the splicing event of 33K consistent with translating ribosomes (Fig. 25).

Another feature of translation is the ribosome translating one reading frame in the coding region. Hence, individual codon frames were investigated.

Reading Frames

For several, but not all HAdVC mRNA transcripts, there was one dominant frame found consistent with translation elongation (Fig. 26; Jackson et al., 2010; Dinman, 2012).

Interestingly, when looking at the framing for the 100K transcript, the RPF mapping was able to capture a switch in reading frames 2000 nucleotides downstream of the start codon.

This location corresponds to the start site of 33K (Fig. 27). The change in reading frame further demonstrates that RPFs were from translating ribosomes.

Reading frames are made up of 3-nucleotide codons. Each codon is specific for an amino-acid. The ribosome translates by shifting every 3-nucleotides down the ORF.

Therefore, individual codons were examined for a 3-nucleotide periodicity.

Periodicity

During elongation, ribosomes translate one reading frame by successive triplet codons, therefore their movement is thus phased. In ribosome profiling, a three-nucleotide

65 periodicity is often observed (Ingolia et al., 2009, 2019; Guo et al., 2010; Andreev et al.,

2016). A statistically significant three nucleotide periodicity was detected for several, but not all, individual HAdVC mRNA transcripts (Fig. 28). The periodicity is consistent with the RPFs coming from 80S ribosomes performing elongation.

Interestingly, different size mRNA transcripts produced periodicity for different viral mRNA (Fig. 29). This may mean that varying ribosomal conformations correlate to certain HAdVC transcripts (Fig. 28; Afonina et al., 2014; Lareau et al., 2014; Hussman et al. 2015; Ingolia et al., 2019). Changes in ribosomal conformations during translation is a known phenomenon (Hussman et al. 2015).

The 3-nucleotide periodicity was also exclusive to the 5’ rather than the 3’ end of the transcript (Fig. 29). The ribosome has a 5’ exit channel that is rigid, but a 3’ entry channel that is dynamic (Weissman et al., 2013;Lareau et al., 2014; Andreev et al., 2016).

Hence, a 5’ periodicity, but not a 3’ periodicity has been observed in other ribosome profiling experiments (Lareau et al., 2014; Andreev et al., 2016)

Mapping to the Adenovirus Genome to Study Late Translation

The RPFs were mapped directly to the HAdVC reference genome and ORF. The number of reads that mapped to the references were 52,647 with over 90% mapping uniquely to one location in the reference genome (Table II; Fig. 30).

Normalizing the unique mapping for read-length showed that very few reads mapped to the early phase compared to the late transcription unit (Fig. 31). The early genes that were associated with ribosomes are known to be active in the beginning of the late phase of infection (Dolph et al., 1988, 1990; Fessler & Young., 1998).

66 The E1B 19K small T-antigen, E3 10.5 KD protein, and the E2A single-stranded

DNA binding protein (DBP) had significant RPF mapping late in infection (Fig. 31 red).

The 19K protein functions to counter the cellular apoptosis pathway. The 10.5 kD protein is a membrane glycoprotein that, with 19K, functions to counter the immune response from the cell (Patwary et al., 2016). These proteins continue to be translated to ensure the viability of infection (Cleghon et al., 1989; Radko et al., 2015).

Since the late phase of infection begins after the onset of viral DNA replication, it is not surprising to find that DBP is still being translated. (Schneider, 2000). The DBP, along with the viral DNA polymerase (POL), is essential for replication (Fig. 2; Acheson,

2011) However, for each POL, there are several DBPs that are needed to coat the displaced strand during replication (Van Breukelen et al., 2003; Acheson, 2011). Consistent with the need for more DBP, the mapping data show that DBP has a significantly higher ribosome density than POL.

The early genes that had the lowest expression, were from the E3 region that was disrupted to make HAdVC dl309. These include the E3-CR1-alpha, E3-RID-alpha, E3-

RID-Beta, and E3-14.7K (Fig. 2). These E3 proteins function to inhibit various host antiviral response mechanisms (Acheson, 2011; Patwary et al., 2016). Therefore, dl309 cannot sustain an infection.

The two delayed proteins, IX and IVa2, had significant expression (Fig. 31 blue).

Both proteins aid in capsid formation. The capsid has 240 IX proteins that act as glue to hold together the major structural proteins (Fig. 1; Parks, 2005; Velinga et al., 2005). The

IVa2 protein aids in genome packaging and transfer into the capsid (Morris et al., 2010)

67 Several late messages had an abundance of mapped reads consistent with the use of ribosomes for high expression (Fig. 31 green). The ribosome density of the top 8 transcripts differed only by 1.5X showing that they had similar numbers of ribosomes.

The pX, and pVII, from the L2 late unit, (Fig. 2) assist to condense the HAdVC genome for packaging into the capsid. Proteins, such as IVa2, help transfer pX, pVII, and the genome into the capsid. Hence, pX and pVII (and L2_pV) are genomic core proteins.

They are located in the interior of the capsid (Fig. 1; Parks, 2005; Ahi & Mittal., 2016).

When progeny viruses infect new cells, the pVI (L3) protein is responsible for lysing the endosome, releasing the capsid at the nucleus, (Gros & Guedan, 2010; Ahi &

Mittal, 2016). In addition, pVI shuttles the hexon (L3) protein into the nucleus for capsid formation (Ahi et al., 2017).

The capsid is made from hexon, penton (L2), and fiber (L5). Hexon is the largest and most abundant protein (Fig. 1). The capsid has 720 hexons (240 trimers), 60 pentons

(12 pentamers), and 36 fiber proteins (12 trimers; Barry, 2016).

The 33K and 100K proteins are from the L4 unit (Fig. 2). The 100K is vital for the preferential translation of late mRNA by shunting (Fig. 5). The 33K is involved in activating late gene expression and is an RNA splicing factor for late messages (Wu et al.,

2013; Zhao et al., 2014; Lan et al., 2016).

Interestingly, the RPFs on late shunting-mRNA, were found largely on their 5’

UTR leader regions relative to coding sequences (Fig. 31 inset). This was not true for non- shunting-mRNA. (see below)

68 The Analysis of 80S Ribosomes in the 5’ UTR of Shunting-mRNA

The TPL and IVa2-mRNA have been found to translate by ribosome shunting

(Fig.7; Yueh & Schneider, 1996,2000). The other messages found late in infection translate by the canonical scanning mechanism (Fig. 4; Yueh & Schneider, 1996,2000).

The highest density of mapping RPFs was found in the TPL of late messages (Fig.

31 inset, green). This is a fascinating result, because it is evidence that the 80S ribosomes are in the 5’ UTR of shunting mRNAs. This is also true for the 5’ UTR of IVa2 relative to its coding region (Fig. 31 inset, blue). The other messages found late in infection (i.e. host, viral early, IX) did not have evidence of 80S ribosomes in their 5’ UTR (see below).

The highest frequency read-length that mapped to the ORFs of late messages was

28-nucleotides consistent with 80S ribosomes (Fig. 32; Ingolia et al.,2009,2019).

Interestingly, the most abundant read-lengths mapping to the TPL were 28-29 nucleotides

(Fig. 32). This is further evidence that the RPFs mapping to the 5’ UTR are from 80S ribosomes and no other species.

The 80S ribosome translates the coding regions with a 3-nucletode periodicity. The coding regions, of several late transcripts, were found to have this feature (Fig. 28,29).

Intriguingly, the mapping data showed a 29-nucleotide periodicity in the TPL (Fig. 33).

Hence, the 80S ribosomes in the 5’ UTR of shunting mRNA do not appear to be translating.

Rather, a 29-nucleotide periodicity has been observed to be a characteristic of stacking 80S ribosomes (Wolin & Walter, 1988; Simms et al., 2017).

Yueh & Schneider, (2000) observed that the late shunting messages have 18S rRNA complementary regions (Fig. 6). Deleting these complementary regions in the TPL showed

69 that there is a 3’-5’ hierarchy in terms of shunting efficiency (Fig. 7). The authors did not observe the same hierarchy in the 5’ UTR of IVa2.

The RNA protected by 80S ribosomes had the same 18S complementary regions

(Figs. 34 and 35). Furthermore, these regions showed the same hierarchy, but in terms of ribosome density. This pattern was found in the TPL and 5’ UTR of IVa2.

Ribosome Protection of 18S rRNA Complementary regions in the 5’ UTR of TPL and

IVa2-mRNA Exhibit Hierarchical Density of 80S Ribosomes

The mapping of RPFs showed that the 80S ribosomes protected the 18S complementary regions in the TPL (Fig. 34). The number of RPFs mapping to these areas were greater than the reads that mapped in-between these regions (data not shown). This is consistent with the conclusion that the 18S complementarity is important for shunting

(Yueh & Schneider, 2000).

The data from the TPL mapping also showed a 3’ to 5’ hierarchy in relation to the fragments mapping to the TPL (Figs. 31 and 34). The coverage for TPL-3 was 3 times greater than TPL-2, and TPL-2 was 11 times greater than TPL-1 (Figs. 32 and 35). This means that there were about 33X more ribosomes on the 3’ end of the leader relative to the

5’ end.

The mapping data revealed the same 18S complementary regions in the 5’ UTR of

IVa2 (Fig. 35). The ribosome density in the 3’ end of the UTR was about 29X the density in the 5’ end. Therefore, the same 3’ to 5’ hierarchy was discovered.

The data in this thesis is consistent with the 18S complementary regions being involved in HAdVC translation by ribosome shunting. The data is also consistent with the

70 3’ to 5’ hierarchy in these regions. However, how and why these features are involved in shunting is still not resolved.

Mapping to the Individual Adenovirus mRNA to Study Late Translation

The RPFs were from 80S ribosomes performing translation on late mRNA. The late mRNAs are produced from extensive splicing events. Therefore, the RPFs were mapped to late mRNA transcripts (Figs. 36-37).

Each transcript from the major late transcription unit has the TPL in its first 200- nucleotides. Because the TPL is common to all messages there is no way to know which mRNA had a specific TPL sequence (see below). Therefore, the CLC mapping algorithm distributed the TPL fragments in proportion to each genes mapping density.

The reads mapping to the transcripts show pronounced peaks (i.e. ribosome density) in the 5’ UTR of TPL and IVa2 mRNA prior to the start codon (red line; Figs. 36-37) This is consistent with the earlier data (Figs. 30-35). The mapping also showed significant peaks at the start codon and about 200-nucleotides downstream.

Ribosome pausing around the start codon is often observed when cycloheximide

(CHX) is used in ribosome profiling experiments (Ingolia et al., 2009,2019; Andreev et al.,

2016). Cycloheximide binds to the E-site of the 80S ribosome preventing translocation.

Therefore, it has been observed to only impede ribosomes performing elongation (Ennis &

Lubin, 1964, Schneider-Poetsch et al., 2010). The start codon is where elongation begins and therefore where cycloheximide starts to take effect.

To identify the possible reasons for the peaks (e.g. pause sites) throughout the late messages, the mapping data was checked against codon frequencies, secondary structure,

71 and additional 18S complementarity. No correlation was found (data not shown). A curious pronounced peak is seen at the end of the penton transcript (Fig. 36). This would suggest a pile up of ribosomes at the end of the transcript. The reason for this increased ribosome density is uncertain, but it is worth noting that translational termination has been shown to be a slower step than elongation, at least for some messages (Bertram et al., 2001).

However, the reason that this would be unique to penton is unknown.

Other mRNAs that are not associated with shunting do not show evidence of ribosomes in their 5’ UTR (e.g. IX; Fig. 37; DBP not shown). This result would suggest that 80S ribosomes in the 5’ UTR during infection is a characteristic exclusive to messages that are translated by ribosome shunting.

Comparison of Ribosome Profile vs RNA-Seq of Adenovirus Infected Cells

The RPF data was compared to the RNA-seq data of Evans et al., (2012). Evans et al. (2012) purified Poly(A)-tail mRNA, randomly sheared the transcripts, and sequenced the transcriptome from 0, 9, and 24 h post-infection. The RPF data in this thesis was from

15-16 h post-infection.

The RPF data was found to correlate well with the data from Evans et al., in terms of what messages are present late in infection (24 h post-infection; Fig. 38). This shows that much of the viral mRNA late in infection is being translated by ribosomes.

The transcriptome data of Evans et al. (2012) randomly captured some reads that mapped back to the TPL. This is not surprising since the 18 mRNAs from the late transcription unit all have the same leader (Fig.2). Therefore, the transcriptional expression

72 of the TPL would be expected to be the most abundant since it is associated with all late mRNA.

Interestingly however, it was found that translational expression, measured by mapping of RPFs, was highest for the TPL (Fig. 39). The RPF data showed about 3000 times greater mapping to the leader region than Evans et al. (Fig. 39). This shows that the

RPF mapping to the TPL could not be attributed to its transcription or by chance. Rather there were 80S ribosomes in the TPL that protected leader fragments in the presence of

RNase. The transcriptome data of Evans et al. (2012) was also checked for periodicity and framing. As expected, the transcriptome data showed no evidence of periodicity or framing

(data not shown).

73 DISCUSSION

In this thesis, ribosomal profiling was performed on late HAdVC infected HeLa cells (Fig.8). The analysis of the results leads to several significant conclusions. One major result is that 80S ribosomes appear to be associated with the 5’ UTR of late HAdVC mRNA transcripts that are thought to be translated by ribosomal shunting, a process in which ribosomes load near the 5’ end and then bypass a section of the 5’ UTR to reach the start site (Fig. 4). Such shunting is thought to occur in TPL and IVa2 mRNA (Fig. 7). Shunting appears to be linked with the preferential translation of late transcripts. Several features of the data in this thesis are consistent with shunting but reveal more information on the translation of HAdVC late mRNA.

Models of Canonical Translation and Shunting

Several models for eukaryotic translation on HAdVC late mRNA have been proposed. The first standard model of eukaryotic translation is the scanning model (Fig.4 top). The data in this thesis, along with the known eIF4F modification by the 100K protein

(Fig. 5), do not support this translation model for late mRNA (Yueh & Schneider., 1996;

Schneider & Mohr., 2003, Xi et al., 2005). The standard model has support for host mRNA, early HAdVC mRNA, and the delayed early IX mRNA (Dolph et al., 1988; Cuesta et al.,

2000, 2001). However, the mechanism on late genes of HAdVC infection are qualitatively different than canonical translation.

The standard shunting model proposed by Cuesta et al., 2001 (Figs. 4 bottom) has the 4F/43S initiation complex preferentially forming at the 5’ cap of late TPL-mRNAs

(Yueh & Schneider, 2000; Cuesta et al., 2001). After limited scanning, the 43S initiation

74 complex ‘leaps’ (i.e. shunts) around the secondary structure, landing at or near the start codon. Finally, the 80S ribosome forms and begins translating (Yueh & Schneider., 1996;

Cuesta et al., 2001).

The evidence in this thesis, however, suggest that 80S ribosomes are binding or forming in the 5’ UTR of late shunting-mRNA (Figs. 30-37). The evidence for this begins with the RNA that was sequenced. The RNA came from the peak in the ribosome profile derived from RNase treatment of polyribosomes in high-salt buffer (Fig. 20). The collected fractions contained the 18S, 28S, 5.8S and 5S ribosomal RNA of 80S ribosomes. (Figs. 16 and 24). The small RNA enriched and purified from the peak were predominantly 27-32- nucleotides in length (Table I). This is characteristic of RPFs from 80S ribosomes as has been shown by several groups (Guo et al., 2010; Ingolia et al., 2011,2019; Andreev et al.,

2016).

The mapping data of the RPFs showed features of 80S translating ribosomes late in infection. The reads that mapped to the L4 transcription unit were able to capture the splicing of the 33K mRNA that made the final transcript for translation (Fig. 25). The mapping showed one dominant reading frame and was able to distinguish the change in reading frames from 100K to 33K (Figs. 26,27). The reads also showed 3-nucleotide periodicity on several messages consistent with 80S translation ribosomes (Figs. 28 and

29). The periodicity was seen on the 5’ (trailing) end of the message in contrast to the 3’ end.

The RPF read-length in the TPL was essentially identical to the read-length found in the coding regions, where 80S ribosomes are translating mRNA to protein (Fig. 32).

This read-length of RPFs, in 5’ UTRs, was only observed in late messages that translate by

75 shunting (Figs. 36 and 37). The mapped reads in the TPL had a 29-nucleotide periodicity in contrast to the 3-nucleotide periodicity found in the coding regions, (Fig. 33).

The model in Fig. 40A reflects these results. The 60S and 40S ribosomal subunits bind to the 5’ cap forming the 80S ribosome. The 80S ribosomes shunt around the secondary structure landing at or near the start codon. At the start codon the 80S ribosome begins translation elongation.

The totality of these results leads to the conclusion that during the late phase of infection, when the virus is known to hijack the ribosomes for its translation, the 80S ribosomes are binding or forming in the 5’ UTR of late shunting-mRNA

The 29-nucleotide periodicity in the 5’ UTR of shunting-mRNA has two implications. The 80S ribosomes seem to be stacking in the 5’ UTR (Simms et al, 2017) and the 80S ribosomes are not translating the UTR into protein.

80S Ribosomes Appear to Stack in the 5’ UTR of Shunting-mRNA

Two observations suggest that ribosomes are stacked in the 5’ UTR of shunting- mRNA. First, the length of the protected fragments mapping throughout the 5’ UTR of shunting-mRNA is consistent with 80S ribosomes and no other known particles (Figs. 24 and 32; Ingolia et al., 2014). Second, the 29-nucleotide periodicity in the TPL is a distinctive feature of stacked ribosomes (Fig. 33; Simms et al, 2017).

The stacking of 80S ribosomes on late messages is consistent with earlier observations from experiments performed late in infection. Velicer and Ginsberg (1968) tried to estimate the molecular weight of the hexon protein by the number of 80S ribosomes throughout the mRNA. They reasoned that if the 80S ribosomes were evenly distributed,

76 the number of ribosomes should be proportional to the mRNA length and therefore molecular weight.

From the observed ribosomal distribution, they concluded that the maximum molecular weight of hexon must be 25 kDa. (Velicer & Ginsberg, 1968). However, the molecular weight is actually 125 kDa. Hexon either utilizes a small number of 80S ribosomes or the ribosomes are not evenly distributed, or both.

Several polysome profiles performed in the Ornelles lab (personal communcation) have shown similar results for hexon. Comparing the total number of polysomes on hexon and host mRNA, it was observed that the hexon-mRNA appeared to have a lower number of ribosomes than the host. However, subsequent protein analysis showed that the hexon- mRNA produced a similar amount of protein as host-mRNA (data not shown). Hence, the hexon-mRNA had similar translation efficiency with fewer 80S ribosomes. The findings in this thesis are consistent with these observations.

The model in Fig. 40A shows 80S ribosomes stacking. After the 80S forms at the

5’ cap, the rate of 80S formation becomes faster than the rate of shunting. Therefore, the

80S ribosomes stack. Stacking ribosomes are not evenly distributed throughout the mRNA and offer an explanation for the observations from other labs.

The 29-nucletode periodicity is consistent with ribosomes stacking in the 5’ UTR.

It is also consistent with the 80S ribosome not performing translation in the 5’ UTR of late shunting-mRNA.

77 Ribosomes in the 5’ UTR of Shunting-mRNA do not Appear to be Translating

Ingolia et al., 2014 performed ribosome profiling using mouse embryonic stem cells. They observed that 80S ribosomes were found in both coding (i.e. translating) and noncoding (non-translating) regions (e.g. 5’UTRs, lncRNAs). The coding regions always exhibited RPFs of 28-nucleotides and exhibited a 3-nucleotide periodicity. Ribosomes associated with non-coding regions had RPFs below and above 28-nucleotides (Ingolia et al., 2014). The RPFs of 28-nucleotides was a characteristic of ribosomes performing translation. However, it was uncertain whether or not this applied to all systems.

The HeLa cells infected with HAdVC in this thesis also showed RPFs that were predominantly 28-nucleotides (Table I; Figs. 22,32). However, these reads mapped to both coding and noncoding regions (i.e. 5’ UTRs; Figs. 31,37). Therefore, the read-length of

28-nucleotides does not appear to uniquely define translating ribosomes, at least in HAdVC late-mRNA. In addition, the RPFs had a triplet periodicity in the coding regions but a 29- nucleotide periodicity in the TPL. This implies that the 80S ribosomes in the 5’ UTR are not translating mRNA to protein.

If the 80S ribosomes stack and eventually shunt, this mechanism would require that the ribosomes shift down the mRNA (5’ to 3’), a ribosome length at a time, until it gets into position to shunt (Fig.40A). This mechanism may be similar to some features of the ribosome-bypassing mechanism observed in the translation of bacteriophage T4 gene 60

(Weiss et al. 1990; Agirrezabala et al., 2017).

The mRNA of gene 60 contains a 50-nucleotide hairpin that the 70S ribosome

(homologue to the 80S ribosome) bypasses. The bypass mechanism involves a 5’ nascent peptide, 17 to 34 amino acids (51-102 nts) long, that uniquely interacts with the exit-

78 channel of the complete (i.e. 70S) ribosome (Maldonado & Herr, 1998) This interaction results in a ribosomal pause at the ‘take off’ codon (i.e. GGA; glycine). The pause induces a rotation in the ribosome resulting in disengagement. The complete ribosome then bypasses the hairpin, and then re-engages downstream. After re-engagement, the 70S ribosome scans down the mRNA, 5’ to 3’, until finding another glycine codon (i.e. GGA) in a Shine-Dalgarno-like context (Maldonado & Herr, 1998; Chen et al., 2015;

Agirrezabala et al., 2017). At this location, the 70S ribosome begins translation elongation.

The mechanism of ribosome bypassing in the T4 gene 60 has three implications that are relevant to the model in Fig. 41A. The 4F/43S is not the only species that can scan mRNA without translating. The 80S ribosome (homologue to the 70S) may be able to scan the mRNA, 5’ to 3’, without producing a protein. After getting into position, the 80S ribosome may be bypassing (shunting) the secondary structure in late-mRNAs. Following shunting, the 80S binds the mRNA, like the 70S, in and around the start codon.

It would be interesting to see what a ribosome profile of bacteriophage T4 infection would reveal about the bypassing mechanism in gene 60. Perhaps a 3-nucleotide periodicity would be found prior to disengagement (i.e. translating the nascent peptide) but a 29-nucleotide periodicity (i.e. not translating) subsequent to re-engagement. There may be some evidence of ribosome stacking prior to the bypass.

Whether or not the 5’ UTR of shunting-mRNA contains a small peptide signal, like the nascent peptide signal of gene 60, is unclear. The 29-nucleotide periodicity in the 5’

UTRs appear to make translation of a signal peptide unlikely. However, the large peaks about 100-nucleotides downstream of the start codon in several shunting-mRNAs, are similar in nucleotide length to the nascent peptide signal. Perhaps these are regions are

79 pause-sites due to the interaction with the exit channel of a preceding peptide (Figs. 36 and

37). Further studies would need to be done to see if this was the case.

The model in Fig. 40A has the 80S ribosome forming at the 5’ cap of late messages.

However, the data in this thesis showed the read density increasing 3’ to 5’ in the 5’ UTR, consistent with earlier findings (Fig. 31; Yueh & Schneider, 2000). This hierarchy leads to further alternative models for ribosome shunting. This may indicate that rather than forming at the 5’ cap, the 80S ribosomes are forming at the 3’ end of the UTRs. This may explain how late messages translate in a cap-independent manner (Schneider, 2000).

Hierarchy in RPF Mapping may Indicate an Alternative Cap-Independent

Translation Mechanism

Fig. 40B shows another alternative model for ribosomal loading, stacking, and shunting that does not require ribosomal movement. Instead, the high relative frequency of ribosomes at the 3’ end of the TPL suggests that they are most likely to bind there preferentially. Then, other ribosomes may bind toward the 5’end, stepwise, possibly via an allosteric loading mechanism, similar to that observed for the DNA binding repressor protein of phage lambda (Bell et al., 2000; Sarkar-Banerjee et al., 2018).

The lysogenic pathway of a lambda phage infection in E. coli is controlled by a repressor protein. Lambda repressor is a dimer with a N-terminal DNA binding domain and a C-terminal domain that interacts with other repressor proteins. After the repressor binds to the DNA via its N-terminus, the C-terminus interacts with other repressor proteins facilitating their attachment to the DNA. As a result, the repressor proteins block the host

80 proteins needed for cell lysis, in favor of the proteins necessary for lysogeny (Bell et al.,

2000; Sakar-Banerjee et al., 2018).

Perhaps as the ribosomes bind to the 3’ end of the leader, there is some interaction with other ribosomes to facilitate their binding toward the 3’ end (Fig. 40B). If the loading is faster than shunting, then stacking of ribosomes would occur. This simple model of cooperative binding of ribosomes to the TPL, explains the 29-nucleotide phasing (i.e. stacking) as well as the high relative differences in ribosome coverage within the 5’ UTR of shunting-mRNA.

The two models presented so far have two limitations. The models do not account for the findings of other groups concerning the shunting species nor the circular nature of mRNA.

The 4F/43S Pre-Initiation Complex Appears to be the Shunting Species

Several experiments were performed that showed that the 4F/43S pre-initiation complex is the shunting species. (Yueh & Schneider., 1996, 2000; Cuesta et al., 2001).

When the 80S ribosome was formed in the TPL by introducing a start codon no shunting was observed. Rather, the 80S ribosome unwound the secondary structure of the 5’ UTR and translated a protein (Yueh & Schneider., 1996, 2000).

Shunting was restored only after putting in a stop codon, prior to TPL-2, to disassemble the 80S ribosome. These results were consistent with the 4F/43S, and not the

80S, being the shunting species. The removal of the secondary structure prevented the

4F/43S complex from shunting. Therefore, shunting and scanning in the TPL were observed to be mutually exclusive (Yueh & Schneider., 1996, 2000).

81 To account for the 4F/43S complex being the shunting species (Yueh & Schneider.,

1996, 2000), the possible circular nature of late mRNA (Lopez-Lastra., 2010; Keiper, 2019;

Brambilla et al., 2019), and the data in this thesis another model is proposed. Figs. 40C and

D show a ‘closed loop’ model for late translation by ribosome shunting.

The Closed-Loop Model for the Selective Translation of Late mRNA by Ribosome

Shunting

The ‘closed-loop’ model for translation states that the 3’ poly(A) tail of the mRNA is ‘circularized’ by its association with the PABP (Fig. 5; Jackson et al., 2010; Zarai et al.,

2017). This brings the stop codon of the 3’ end of the message close to the 5’ UTR (Fig.

5). When an 80S ribosome reaches a stop codon and is disassembled, the 43S initiation complex is recycled onto the 5’ UTR in a cap-independent manner. The 80S ribosome scans to the start codon for another round of translation (Marshall et al., 2014; Rogers et al., 2017; Vicens et al., 2018; Keiper, 2019).

The late mRNA and 100K modified ribosomes have been found to have a poly(A) tail and the PABP respectively (Fig. 5; Cuesta et al., 2000,2004). Consequently, late

HAdVC mRNAs have been hypothesized, but not observed, to be circular (Fig. 5; Cuesta et al., 2004; Lopez-Lastra et al., 2010). Perhaps the translation of late mRNA begins with the standard model of ribosome shunting (Fig 40C; Yueh & Schneider 1996, 2000; Cuesta et al., 2001). The 4F/43S initiation complex binds to the 5’ cap via the 4E binding protein

(i.e. eIF4F complex), scans the first 25-nucleotides, then shunts around the secondary structure, landing at or near the start codon. At the start codon, the 60S ribosome joins the initiation complex making the 80S ribosome and translation elongation ensues.

82 The first 80S ribosome that reaches the 3’ end of the transcript is now close to the

5’ end of the circular late mRNA (Fig 40D). The 80S ribosome is released at the stop codon and is recycled back onto the 3’ end (not the 5’ cap) of the UTR. The recycling of ribosomes onto 5’ UTRs is an observed cap-independent translation mechanism (Yang et al, 2017;

Zarai et al., 2017). During late infection, the recycling may involve the 100K protein and cooperative binding.

Ribosome Reloading by 100K and Cooperative Binding Facilitates Stacking in the 5’

UTR of Circular Shunting mRNA

The 100K protein contains a binding domain that is specific for the tripartite leader

(Cuesta et al., 2000,2004). The 80S ribosomes that are disassembled at the stop codon can reform at the 3’ end of the TPL via 100K. The newly bound 80S ribosome, along with

100K, can now facilitate the binding of other 80S ribosomes toward the 5’ end of the TPL.

This cooperative loading might be similar to the allosteric loading of the lambda repressor protein (Bell et al., 2000; Sakar-Banerjee et al., 2018).

If the cooperative loading of 80S ribosomes is faster than elongation, then the ribosomes would begin to stack in the 5’ UTR (Fig. 40D). Since the 80S ribosome is capable of unwinding the secondary structure in the TPL, subsequent shunting is apparently eliminated (Yueh & Schneider, 1996, 2000).

There would seem to be a delicate balance between disassembly at the stop codon, reloading in the 5’ UTR, stacking, and elongation. If elongation were too slow compared to reloading, then disassembled ribosomes would be prevented from forming in the already occupied 5’ UTR. This could result in a reduction of the local ribosome concentration due

83 to diffusion. The same result could conceivable occur if elongation were too fast resulting in the disassembly rate being higher than the recycling rate. An increasing number of disassembled ribosomes that cannot reload to the 5’ UTR could diffuse away

Real-time fluorescent imaging, of non-infected live HeLa cells, showed that single mRNA transcripts had an average of twelve 80S ribosomes attached. These ribosomes were, on average, about 86 codons apart (i.e. 258 nts; Wang et al., 2016).

The data from the Ornelles lab (see above) suggests that the TPL-mRNA may have fewer ribosomes than HeLa transcripts. This may mean that the late transcripts have, on average, less than 12 ribosomes. The lower number of ribosomes may increase the distance between them (Fig. 40D). The increased distance may allow time for translation, disassembly, reassembly, stacking, and then scanning to get into position for another round of translation.

The 100K protein does not have a binding domain specific for the 5’ UTR of IVa2.

However, similar to the TPL, the 5’ UTR of IVa2 has 18S complementary regions. These locations may facilitate the rebinding of disassembled ribosomes to the 3’ end of the 5’

UTR. The first reformed 80S ribosome, along with these regions, could facilitate more ribosome attachment, toward the 5’ end, by cooperative binding. If the rebinding is faster than elongation, the ribosomes may stack in the 5’ UTR of IVa2.

The reforming of ribosomes, 3’ to 5’, in the UTRs allow the ribosomes to scan in a

5’ to 3’ direction to get back into position for translation. This is the natural direction for ribosome scanning and translation (Jackson, 2010). The 5’ to 3’ scanning of 80S ribosomes may be like the scanning mechanism of the 70S ribosome on the T4 gene 60 mRNA (see

84 above). How the 70S or 80S ribosomes stays attached during scanning is unresolved (Todd

& Walter, 2013; Agirrezabala et al., 2017).

The T4 70S ribosome scans 50-nucleotides before again translating protein (Todd

& Walter, 2013). The 5’ UTRs of TPL and IVa2 mRNA are 201 and 131-nucleotides, respectively. The recycled ribosomes, in 5’ UTRs of late messages, would have to travel a significantly longer distance than the T4 ribosome. Therefore there is a greater need for the recycled 80S ribosomes to remain attached to the mRNA until reaching the start codon.

The late transcripts have several 18S complementary regions that span the entire 5’

UTR in messages that translate by shunting (Fig. 6). These regions hybridize to the same location in the 18S rRNA. It is possible that these regions assist in keeping the 80S ribosome tethered to the 5’ UTR as it shifts into position for elongation.

Stacked Ribosomes on the UTRs of Shunting mRNA Shift 5’ to 3’ via 18S

Complementary Regions

The stacked 80S ribosomes may shift, 5’ to 3’, down the UTRs of late mRNA by using the 18S rRNA complementary regions (Fig. 6). This may explain why each section of the TPL (1-3), and two sections of the 5’ UTR of IVa2, contain complementary regions

(Fig. 6). The complementary regions hybridize to the exact same location in the 18S rRNA

(Fig. 6). Furthermore, these regions are the size of ribosome-protected fragments (i.e. size of 80S ribosomes; Figs. 6, 34-35). The use of the same 18S region would allow for a coordinated, 5’ to 3’, shifting down the TPL and 5’ UTR of IVa2.

85 The Closed-Loop Model of Translation Accounts for Several Features of Late

HAdVC Preferential Translation by Ribosome Shunting

The closed-loop model for the preferential translation of late-mRNA by ribosome shunting, accounts for the data in this thesis and several observations of other groups (Figs.

40C and 40D). The model accounts for circular mRNA via the PABP and poly(A) tail

(Vicens et al., 2018; Keiper, 2019). The 4F/43S complex binding to the mRNA 5’ cap via

4E to start translation (Dolph et al., 1988; Yueh & Schneider, 1996). The 4F/43S, not the

80S, performing shunting (Yueh & Schneider, 1996,2000). The 80S forming at the start codon as in several systems (Jackson, 2010).

The observed cap-independent translation, of shunting mRNA, is accounted for by the reloading of disassembled ribosomes to the 5’ UTRs (Dolph et al., 1988, 1990; Xi et al., 2005). The ribosomes are recycled back onto the TPL using the 100K protein. The ribosomes are recycled onto the 5’ UTR of IVa2 using the 18S complementary regions.

The 3’ to 5’ hierarchy of mapped RPFs, is the result of reloading at the 3’ end of the UTRs and subsequent cooperative binding (Fig. 31). This is consistent with the 3’ to 5’ importance for translation found in these regions. The 80S ribosomes, via 4A, unwind the

TPL secondary structure thereby hindering future translation by shunting (Yueh and

Schneider, 1996, 2000).

Since the reloading of disassembled ribosomes is faster than elongation, stacking in the 5’ UTR results. The stacking means fewer ribosomes per late mRNA (Velicer &

Ginsberg., 1968; personal communication, Ornelles lab). The fewer ribosomes increases the distance between translating ribosomes. This coordinates the kinetics of disassembly, recycling, 5’ to 3’ scanning, and elongation.

86 Recycling ribosomes in translation has been found to be associated with increased translation efficiency. This is especially true in stress conditions, when ribosome availability is limited (Alekhina et al., 2007; Kopeina et al., 2008 Keiper, 2019). This accounts for the observation by the Ornelles lab that hexon mRNA had fewer ribosomes than the host-mRNA but similar translation efficiency.

Recycling also accounts for the hijacking of ribosomes by viral mRNA for the almost exclusive translation of late messages. After the ribosomes initially attach to late mRNA via 100K and the 18S complementary regions, the ribosomes are sequestered for the continued translation of late transcripts. This sequestration may also deprive host messages of ribosomes.

The reloaded ribosomes scan 5’ to 3’ down the mRNA to get into position for further elongation. The ribosomes remain attached to the mRNA by the 18S complementary regions. The use of these regions by 80S ribosomes explains why they are the size of a ribosome protected fragment (Figs. 6, 34,35). It also explains why these regions were protected by 80S ribosomes (Figs. 34,35).

The 3’ to 5’ stacking of 80S ribosomes and subsequent 5’ to 3’ shifting down the

UTRs, results in several mapped reads of 28-29-nucleotides, and a 29-nucleotide periodicity (Figs. 32 and 33). The shifting to get into position for further translation explains why these regions were found to be important for late translation by ribosome shunting (Yueh & Schneider, 1996, 2000).

The models in Fig. 40 vary in features of translation and complexity. There is a need for several experiments to exclude some models in favor of others.

87 Work that might resolve the Mechanism of Translation on Late Shunting-mRNA

The ribosome profile in this thesis was performed on the late HAdVC mRNA.

During late infection, ribosomes are redirected to translate late viral mRNA that translate by a unique mechanism called ribosome shunting. An interesting result was that 80S ribosomes appear to be forming and stacking in the 5’ UTR of only shunting-mRNA (Figs.

31, 37). These results have led to new models of ribosome shunting (Fig. 40).

Each of the new models for shunting have distinctive characteristics that can be tested through future experiments. For example, the model in Figs. 40C and 40D requires circular mRNA and the presence of the 40S and 80S ribosomes in the 5’ UTR. The models in Figs. 40A and 40B have linear mRNA, and require only the 80S in the 5’ UTR. A r loading (3’ to 5’) might distinguish models A and B.

The following experiments can be done to see which model is more consistent with translation of late shunting-mRNA during HAdVC infection.

Future Studies: Atomic Force Microscopy on Late HAdVC mRNA

An atomic force microscope (AFM) is a high-resolution instrument that can produce a 3-dimensional image of biomolecules in an aqueous environment. AFM has already been used to visualize polyribosomes and to count the number of ribosomes per mRNA transcript (Mikamo et al., 2005; Viero et al., 2015’ Lauria et al, 2015). Additionally,

AFM has been used to resolve single ribosomes (Vanzi et al., 2003), circular mRNA,

(Wells et al., 1998; Alverez et al., 2005) 40S, and 60S ribosomal subunits (Wu et al., 1997;

Matsuura et al., 2004). Therefore, the AFM can visually reveal whether or not the mRNA is circular and the location of different ribosome species (Wells et al., 1998; Mikamo et al.,

88 2005). If the data showed some circular mRNA with only the 40S ribosome in the 5’ UTR and other circular mRNA with the 40S and 80S, this would be consistent with the closed- loop model for translation (Fig 40C,D). However, if the images showed linear mRNA with only 80S ribosomes in the 5’ UTR this would suggest that the 80S ribosome forms in the 5’ UTR rather than at the start-codon without any looping mechanism (Fig 40A,B). An observed 3’ to 5’ hierarchy of 80S ribosome density on different transcripts would eliminate the model in Fig. 40A. If there are no 80S ribosomes in the 5’ UTR but only 40S then I would have to deeply investigate my ribosome profiling technique and preparatory step in AFM imaging to see if the ribosome profile gave a false positive or the AFM gave a false negative.

Proper sample preparation is critical to avoid artifacts in AFM imaging (Fritzche &

Henderson, 1998; Gaczynska & Osmulski, 2008). Therefore, to verify the results from

AFM imaging and further challenge the models proposed in this thesis several ribosome profiles can be done. The profiles should include a plasmid expressing a modified TPL- hexon protein (i.e. hexon2), 40S and 80S protected fragments, and a region of amino-acids that can be controlled by amino acid deprivation.

Future Studies: Ribosome Profile of 40S and 80S Protected Fragments

The 40S ribosome can also protect mRNA from RNase during a ribosome profile.

However, the length of the protected fragment is different than the 27-32-nucleotides fragments of 80S ribosomes. The 40S protected RNA is about 40-70-nucleotides in length depending on the system (Kozak and Shatkin, 1978,1989; Lytle et al., 2001; Ingolia et al.,

2011) Hence, the 40S and 80S protected fragments can be distinguished. A ribosome

89 profile capturing the 40S and 80S protected fragments may reveal the mRNA locations of both species on late mRNA.

Knowing these locations may verify the AFM data and shed more light on which translation model is feasible. For example, the ribosome profile may show only 80S RPFs mapping to the 5’ UTR of shunting-mRNA in a 3’ to 5’ hierarchical manner,. In addition the AFM revealed linear mRNA containing only 80S ribosomes in that location. These results would instill confidence that each experiment worked as intended and the feasibility of the models in Fig. 40.

On the other hand, the AFM data may show that late-mRNA is circular and that both the 40S and 80S are in the 5’ UTR. The different read-length characteristics of the

40S or 80S ribosome may both map to the 5’ UTR of shunting-mRNA. This result would favor the ‘closed-loop’ model of late translation (Figs. 40C and 40D). The ‘closed-loop’ model suggests that only the 40S ribosome is in the 5’ UTR during translation initiation

(Fig. 40C). Translation inhibitors that act on initiation, rather than elongation (i.e. cyclohexamide), can test this aspect of the closed-loop model.

Future Studies: The Location of 40S and 80S Ribosomes During Translation

Initiation

The canonical model for shunting in Fig. 4 (bottom) and in 40C,D have the 40S ribosome initiating translation. The models in 40A,B show the 80S possibly initiating. To locate the 40S and 80S ribosomes during translation initiation ribosome profiles can be done with lactimidomycin, harringtonine, and flash freezing (Ingolia et al., 2011; Stern et al., 2012).

90 Lactimidomycin binds to the empty E-site of 80S ribosomes blocking the initiating tRNAMet from entering. This halts the 80S ribosome at the start codon during initiation

(Schneider-Poetsh., 2010; De Loubresse et al., 2014).

Harringtonine binds to free 60S subunits before 80S formation at the start codon. It blocks the formation of the first peptide-bond (Liu & Qian, 2016). Like lactimidomycin this halts the ribosome at the start codon. The binding of these chemicals prevents the 80S ribosome from performing one translocation (i.e. next codon) step. Thus, ribosome profiles using these chemicals have been used to find ribosome initiation events (Stern et al., 2012;

Ingolia 2019). This may lead to discovering novel ORFs or 80S initiation events in the 5’

UTR via non-canonical start codons.

The most common and efficient non-AUG start codons in eukaryotes are CUG and

CUU (e.g. Lysine; Sasaki & Nakashima, 1999; Corcellette et al., 2000; Kearse and Wilusz,

2017). There are no AUG start codons in the 5’ UTR of shunting-mRNA, but there are several CUG and CUU codons (data not shown). RPFs from 80S ribosomes stopped with initiation inhibitors may reveal initiation events in the 5’ UTR.

Lactimidomycin and harringtonine appear to be only effective on 80S ribosomes during translation initiation. They would not be effective on the 40S ribosome or possibly the 80S in the 5’ UTR moving by some unknown mechanism.

Flash freezing the cells, along with the translational inhibitors, may capture all the ribosomal species on the mRNA (Harding et al., 2000; Fernandez et al., 2016; McGlincy

& Ingolia, 2017).

Under these various conditions, ribosome profiles of the 40S and 80S ribosomes should reveal which ribosome binds to the 5’ UTR at the start of translation.

91 CONCLUSION

Adenovirus uses ribosomes to preferentially translates late viral mRNA by a unique mechanism called ribosome shunting. To gain insight into this mechanism, ribosome profiling on late infected cells was performed. Based on previous research, I hypothesized that the 80S ribosomes are uniformly stacking in the 5’ UTR of late shunting-mRNA.

Several lines of evidence point to the conclusion that I was able to isolate, purify, and sequence viral mRNA protected by 80S ribosomes engaged in translation. The data also showed that this mRNA was being preferentially translated during the late phase of infection, possibly by ribosome shunting.

One key finding in this thesis is, during viral preferential translation, the 80S ribosomes appear to be stacking in the 5’ UTR of shunting-mRNA. This result was consistent with my hypothesis. Not consistent with my hypothesis was the fact that the ribosomal density of stacked ribosomes increased dramatically toward the 3’ end. These results led to several interesting models to explain late preferential translation. Plausible, but time-consuming experiments can be done to filter down to the best model.

Three surprising results were discovered in the course of this research.

• the 29-nucleotide periodicity in the 5’ UTR of shunting mRNA.

• the ability to capture splicing events.

• the ability to capture the shift in reading frames between 100K and 33K proteins.

92 LITERATURE CITED

Acheson, N. H. (2011). Fundamentals of molecular virology. John Wiley & Sons, Inc.

Afonina, Z. A., Myasnikov, A. G., Shirokov, V. A., Klaholz, B. P., & Spirin, A. S. (2014). Conformation transitions of eukaryotic polyribosomes during multi-round translation. Nucleic acids research, 43(1), 618-628.

Ahi, Y. S., & Mittal, S. K. (2016). Components of adenovirus genome packaging. Frontiers in microbiology, 7, 1503.

Ahi, Y. S., Hassan, A. O., Vemula, S. V., Li, K., Jiang, W., Zhang, G. J., & Mittal, S. K. (2017). Adenoviral E4 34K protein interacts with virus packaging components and may serve as the putative portal. Scientific reports, 7(1), 7582.

Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S. I., Itoh, N., Fukami, Y. (1987). Genistein, a specific inhibitor of tyrosine-specific protein kinases. Journal of Biological Chemistry, 262(12), 5592-5595.

Akusjarvi, G., C. Svensson and O. Nygard (1987). A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay. Mol. Cell. Biol. 7(1): 549-551

Alvarez, D. E., Lodeiro, M. F., Luduena, S. J., Pietrasanta, L. I., & Gamarnik, A. V. (2005). Long-range RNA-RNA interactions circularize the dengue virus genome. Journal of virology, 79(11), 6631-6643.

Andreev, D. E., O’Connor, P. B., Loughran, G., Dmitriev, S. E., Baranov, P. V., & Shatsky, I. N. (2016). Insights into the mechanisms of eukaryotic translation gained with ribosome profiling. Nucleic acids research, 45(2), 513-526.

Andrews, S. (2010). FastQC: a quality control tool for high-throughput sequence data.

Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O., & Herschlag, D. (2003). Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 100(7), 3889-3894.

Archer, S. K., Shirokikh, N. E., Hallwirth, C. V., Beilharz, T. H., & Preiss, T. (2015). Probing the closed-loop model of mRNA translation in living cells. RNA biology, 12(3), 248-254.

Archer, S. K., Shirokikh, N. E., Beilharz, T. H., & Preiss, T. (2016). Dynamics of ribosome scanning and recycling revealed by translation complex profiling. Nature, 535(7613), 570.

93 Ashwal-Fluss, R., Meyer, M., Pamudurti, N. R., Ivanov, A., Bartok, O., Hanan, M., … & Kadener, S. (2014). circRNA biogenesis competes with pre-mRNA splicing. Molecular cell, 56(1), 55-66.

Baek, D., J. Villen, C. Shin, F.D. Camargo, S.P. Gygi, and D.P. Bartel. (2008). The impact of microRNAs on protein output. Nature. 455:64-71.

Bahar, B., Monahan, F. J., Moloney, A. P., Schmidt, O., MacHugh, D. E., & Sweeney, T. (2007). Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues. BMC molecular biology, 8(1), 108.

Balachandran, S., Roberts, P. C., Brown, L. E., Truong, H., Pattnaik, A. K., Archer, D. R., & Barber, G. N. (2000). Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. Immunity, 13(1), 129-141.

Barry, M. A. (2016). Adenoviral Vector Targeting via Mitigation of Liver Sequestration. In Adenoviral Vectors for Gene Therapy (pp. 293-317). Academic Press.

Bartsch, D., Zirkel, A., & Kurian, L. (2018). Characterization of circular RNAs (circRNA) associated with the translation machinery. In Circular RNAs (pp. 159-166). Humana Press, New York, NY.

Bell, C. E., Frescura, P., Hochschild, A., & Lewis, M. (2000). Crystal structure of the λ repressor C-terminal domain provides a model for cooperative operator binding. Cell, 101(7), 801-811.

Bencun, M., Klinke, O., Hotz-Wagenblatt, A., Klaus, S., Tsai, M. H., Poirey, R., & Delecluse, H. J. (2018). Translational profiling of B cells infected with the Epstein-Barr virus reveals 5′ leader ribosome recruitment through upstream open reading frames. Nucleic acids research, 46(6), 2802-2819.

Berardi, A., Bombelli, F. B., Thuenemann, E. C., & Lomonossoff, G. P. (2019). Viral nanoparticles can elude protein barriers: exploiting rather than imitating nature. Nanoscale, 11(5), 2306-2316.

Berk, A. (2007). Adenoviridae: The Viruses and Their Replication Fields Virology. D. M. K. a. P. M. Howley. Philadelphia, Lippincott Williams & Wilkins. Volume II: 2355-2398. Berk, A. J. (2005). Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus. Oncogene 24(52): 7673-7685.

Bertram, G., Innes, S., Minella, O., Richardson, J. P., & Stansfield, I. (2001). Endless possibilities: translation termination and stop codon recognition. Microbiology, 147(2), 255-269.

Bett, A. J., Krougliak, V., & Graham, F. L. (1995). DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309. Virus research, 39(1), 75-82.

94 Binnig, G., Quate, C. F., & Gerber, C. (1986). Atomic force microscope. Physical review letters, 56(9), 930.

Bonneau, A. M., & Sonenberg, N. (1987). Involvement of the 24-kDa cap-binding protein in regulation of protein synthesis in mitosis. Journal of Biological Chemistry, 262(23), 11134-11139.

Brambilla, M., Martani, F., Bertacchi, S., Vitangeli, I., & Branduardi, P. (2019). The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology.

Brimacombe, R. (1991). RNA-protein interactions in the Escherichia coli ribosome. Biochimie. 73:927-36.

Budkevich, T. V., Giesebrecht, J., Behrmann, E., Loerke, J., Ramrath, D. J., Mielke, T., … & Sanbonmatsu, K. Y. (2014). Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement. Cell, 158(1), 121-131.

Bugaut, A., & Balasubramanian, S. (2012). 5′-UTR RNA G-quadruplexes: translation regulation and targeting. Nucleic acids research, 40(11), 4727-4741.

Burger, L. L., Vanacker, C., Phumsatitpong, C., Wagenmaker, E. R., Wang, L., Olson, D. P., & Moenter, S. M. (2018). Identification of genes enriched in GnRH neurons by translating ribosome affinity purification and RNASeq in mice. Endocrinology, 159(4), 1922-1940.

Bushell, M., & Sarnow, P. (2002). Hijacking the translation apparatus by RNA viruses. J Cell Biol, 158(3), 395-399.

Chandramouli, P., Topf, M., Ménétret, J. F., Eswar, N., Cannone, J. J., Gutell, R. R., … & Akey, C. W. (2008). Structure of the mammalian 80S ribosome at 8.7 Å resolution. Structure, 16(4), 535-548.

Chen, G., J.D. Wen, and I. Tinoco, Jr. (2007). Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA. Rna. 13:2175-88.

Chen, J., Tsai, A., O’Leary, S. E., Petrov, A., & Puglisi, J. D. (2012). Unraveling the dynamics of ribosome translocation. Current opinion in structural biology, 22(6), 804-814.

Christensen, A. K., Kahn, L. E., & Bourne, C. M. (1987). Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. American journal of anatomy, 178(1), 1-10.

Cladaras, C., & Wold, W. S. (1985). DNA sequence of the early E3 transcription unit of adenovirus 5. Virology, 140(1), 28-43.

95 Cleghon, V., Voelkerding, K., Morin, N., Delsert, C., & Klessig, D. F. (1989). Isolation and characterization of a viable adenovirus mutant defective in nuclear transport of the DNA-binding protein. Journal of virology, 63(5), 2289-2299.

Cock, P. J., Fields, C. J., Goto, N., Heuer, M. L., & Rice, P. M. (2010). The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic acids research, 38(6), 1767-1771.

Corcelette, S., Massé, T., & Madjar, J. J. (2000). Initiation of translation by non-AUG codons in human T-cell lymphotropic virus type I mRNA encoding both Rex and Tax regulatory proteins. Nucleic acids research, 28(7), 1625-1634.

Crappé, J., Ndah, E., Koch, A., Steyaert, S., Gawron, D., De Keulenaer, S., … & Menschaert, G. (2014). PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration. Nucleic acids research, 43(5), e29-e29.

Cuatrecasas, P., S. Fuchs, and C.B. Anfinsen. (1967). Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus. J Biol Chem. 242:1541-7.

Curran, J.F., and M. Yarus. (1989). Rates of aminoacyl-tRNA selection at 29 sense codons in vivo. J Mol Biol. 209:65-77.

Curran, J.F. (1993). Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site. Nucleic Acids Res. 21:1837-43.

Cuesta, R., Xi, Q., & Schneider, R. J. (2000). Adenovirus‐specific translation by displacement of kinase Mnk1 from cap‐initiation complex eIF4F. The EMBO journal, 19(13), 3465-3474.

Cuesta, R., Xi, Q., & Schneider, R. J. (2001). Preferential translation of adenovirus mRNAs in infected cells. In Cold Spring Harbor symposia on quantitative biology (Vol. 66, pp. 259-268). Cold Spring Harbor Laboratory Press.

Cuesta, R., Xi, Q., & Schneider, R. J. (2004). Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein. Journal of virology, 78(14), 7707-7716.

Davison, A. J., M. Benko and B. Harrach (2003). Genetic content and evolution of adenoviruses. J. Gen. Virol. 84(Pt 11): 2895-2908.

De Loubresse, N. G., Prokhorova, I., Holtkamp, W., Rodnina, M. V., Yusupova, G., & Yusupov, M. (2014). Structural basis for the inhibition of the eukaryotic ribosome. Nature, 513(7519), 517-522.

DeRisi, J., Penland, L., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., … & Trent, J. M. (1996). Use of a cDNA microarray to analyze gene expression. Nat. genet, 14, 457-460.

Dever, T. E., & Green, R. (2012). The elongation, termination, and recycling phases of translation in eukaryotes. Cold Spring Harbor perspectives in biology, 4(7), a013706.

Dinman, J. D. (2012). Mechanisms and implications of programmed translational frameshifting. Wiley Interdisciplinary Reviews: RNA, 3(5), 661-673.

Doerfler, W., & Böhm, P. (Eds.). (2003). Adenoviruses: Model and Vectors in Virus-Host Interactions: Virion-Structure, Viral Replication and Host-Cell Interactions (Vol. 272). Springer Science & Business Media.

Dolph, P. J., Racaniello, V., Villamarin, A., Palladino, F., & Schneider, R. J. (1988). The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation. Journal of virology, 62(6), 2059-2066.

Dolph, P. J., Huang, J. T., & Schneider, R. J. (1990). Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex. Journal of virology, 64(6), 2669-2677.

Duncan, R., Milburn, S. C., & Hershey, J. W. (1987). Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. Journal of Biological Chemistry, 262(1), 380-388.

Duncan, R. F. (1996). Translational control during heat shock. Translational control, 271 294.

Epicentre. (2009). CircLigase ssDNA Ligase Product Literature. Epicentre Biotechnologies. Lit. #222, 1-4.

Ennis, H. L., & Lubin, M. (1964). Cycloheximide: aspects of inhibition of protein synthesis in mammalian cells. Science, 146(3650), 1474-1476.

Enright, J. T. (1965). The search for rhythmicity in biological time-series. Journal of theoretical Biology, 8(3), 426-468.

Evans, V. C., Barker, G., Heesom, K. J., Fan, J., Bessant, C., & Matthews, D. A. (2012). De novo derivation of proteomes from transcriptomes for transcript and protein identification. Nature methods, 9(12), 1207.

Ermolenko, D. N., Majumdar, Z. K., Hickerson, R. P., Spiegel, P. C., Clegg, R. M., & Noller, H. F. (2007). Observation of intersubunit movement of the ribosome in solution using FRET. Journal of molecular biology, 370(3), 530-540.

Feigenblum, D., Walker, R., & Schneider, R. J. (1998). Adenovirus induction of an interferon-regulatory factor during entry into the late phase of infection. Journal of virology, 72(11), 9257-9266.

Fernandez, J., Yaman, I., Mishra, R., Merrick, W. C., Snider, M. D., Lamers, W. H., & Hatzoglou, M. (2001). Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability. Journal of Biological Chemistry, 276(15), 12285-12291.

Fessler, S. P., & Young, C. S. H. (1998). Control of adenovirus early gene expression during the late phase of infection. Journal of virology, 72(5), 4049-4056

Fessler, S. P., & Young, C. S. H. (1999). The role of the L4 33K gene in adenovirus infection. Virology, 263(2), 507-516.

Florescu, D. F., Pergam, S. A., Neely, M. N., Qiu, F., Johnston, C., Way, S., … & van der Horst, C. (2012). Safety and efficacy of CMX001 as salvage therapy for severe adenovirus infections in immunocompromised patients. Biology of Blood and Marrow Transplantation, 18(5), 731-738.

Florescu, D. F., & Keck, M. A. (2014). Development of CMX001 (Brincidofovir) for the treatment of serious diseases or conditions caused by dsDNA viruses. Expert review of anti-infective therapy, (0), 1-8.

Frisch, S. M. and J. S. Mymryk (2002). Adenovirus-5 E1A: paradox and paradigm. Nat. Rev. Mol. Cell Biol. 3(6): 441-452.

Fritzsche, W., & Henderson, E. (1998). Ribosome substructure investigated by scanning force microscopy and image processing. Journal of Microscopy, 189(1), 50-56.

Fukunaga, R., & Hunter, T. (1997). MNK1, a new MAP kinase‐activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates. The EMBO journal, 16(8), 1921-1933.

Fütterer, J., Kiss-László, Z., & Hohn, T. (1993). Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA. Cell, 73(4), 789-802.

Gaczynska, M., & Osmulski, P. A. (2008). AFM of biological complexes: what canIlearn?. Current opinion in colloid & interface science, 13(5), 351-367.

Gallie, D. R. (1991). The cap and poly (A) tail function synergistically to regulate mRNA translational efficiency. Genes & development, 5(11), 2108-2116.

Gallie, D. R. (1996). Translational control of cellular and viral mRNAs. Plant molecular biology, 32(1-2), 145-158.

Gambke, C., & Deppert, W.,(1981). Late nonstructural 100,000-and 33,000-dalton proteins of adenovirus type 2. I. Subcellular localization during the course of infection. Journal of virology, 40(2), 585-593.

Gandin, V., et al. (2008). Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation. Nature, 455, 684-688.

Gandin, V., Sikström, K., Alain, T., Morita, M., McLaughlan, S., Larsson, O., & Topisirovic, I. (2014). Polyribosome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale. JoVE (Journal of Visualized Experiments), (87), e51455-e51455.

Gerashchenko, M. V., & Gladyshev, V. N. (2016). Ribonuclease selection for ribosome profiling. Nucleic acids research, 45(2), e6-e6.

Ginsberg, H. S. (Ed.). (2013). The adenoviruses. Springer Science & Business Media.

Godet, A. C., David, F., Hantelys, F., Tatin, F., Lacazette, E., Garmy-Susini, B., & Prats, A. C. (2019). IRES Trans-Acting Factors, Key Actors of the Stress Response. International journal of molecular sciences, 20(4), 924.

Gratia, M., Sarot, E., Vende, P., Charpilienne, A., Baron, C. H., Duarte, M., … & Poncet, D. (2015). Rotavirus NSP3 is a translational surrogate of the poly (A) binding protein-poly (A) complex. Journal of virology, 89(17), 8773-8782.

Green, L., C.H. Kim, C. Bustamante, and I. Tinoco, Jr. (2008). Characterization of the mechanical unfolding of RNA pseudoknots. J Mol Biol. 375:511-28.

Grimley, M., Prasad, V. K., Kurtzberg, J., Chemaly, R. F., Brundage, T. M., Wilson, C., & Mommeja-Marin, H. (2014). Twice-Weekly Brincidofovir (CMX001) Shows Promising Antiviral Activity in Immunocompromised Transplant Recipients with Asymptomatic Adenovirus Viremia. Biology of Blood and Marrow Transplantation, 20(2), S93-S93.

Groft, C. M., & Burley, S. K. (2002). Recognition of eIF4G by rotavirus NSP3 reveals a basis for mRNA circularization. Molecular cell, 9(6), 1273-1283.

Guo, H., Ingolia, N. T., Weissman, J. S., & Bartel, D. P. (2010). Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature, 466(7308), 835.

Guttman, M., Russell, P., Ingolia, N. T., Weissman, J. S., & Lander, E. S. (2013). Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Cell, 154(1), 240-251.

Hadjiolov, A. A. (2012). The nucleolus and ribosome biogenesis (Vol. 12). Springer Science & Business Media.

99 Haghighat, A., Mader, S., Pause, A., & Sonenberg, N. (1995). Repression of cap‐dependent translation by 4E‐binding protein 1: competition with p220 for binding to eukaryotic initiation factor‐4E. The EMBO journal, 14(22), 5701-5709.

Hardesty, B., T. Tsalkova, and G. Kramer. (1999). Co-translational folding. Curr Opin Struct Biol. 9:111-4.

Harding, H. P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., & Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Molecular cell, 6(5), 1099-1108.

Hartz, D., D.S. McPheeters, L. Green, and L. Gold. (1991). Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA. J Mol Biol. 218:99- 105.

Hayden, F. G. (2013). Advances in antivirals for non‐influenza respiratory virus infections. Influenza and other respiratory viruses, 7(s3), 36-43.

Heyer, E. E., & Moore, M. J. (2016). Redefining the translational status of 80S monosomes. Cell, 164(4), 757-769.

Huang, J. T., & Schneider, R. J. (1990). Adenovirus inhibition of cellular protein synthesis is prevented by the drug 2-aminopurine. Proceedings of the National Academy of Sciences, 87(18), 7115-7119.

Huang, J., & Schneider, R. J. (1991). Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein. Cell, 65(2), 271-280.

Huang, J. T., Chen, J. N., Gong, L. P., Bi, Y. H., Liang, J., Zhou, L., … & Shao, C. K. (2019). Identification of virus-encoded circular RNA. Virology.

Hussmann, J. A., Patchett, S., Johnson, A., Sawyer, S., & Press, W. H. (2015). Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast. PloS genetics, 11(12), e1005732.

Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., & Weissman, J. S. (2009). Genome- wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324(5924), 218-223.

Ingolia, N. T., Lareau, L. F., & Weissman, J. S. (2011). Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell, 147(4), 789-802.

Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M., & Weissman, J. S. (2012). The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nature protocols, 7(8), 1534.

100

Ingolia, N. T. (2014). Ribosome profiling: new views of translation, from single codons to genome scale. Nature Reviews Genetics, 15(3), 205-213.

Ingolia, N. T., Brar, G. A., Stern-Ginossar, N., Harris, M. S., Talhouarne, G. J., Jackson, S. E., … & Weissman, J. S. (2014). Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes. Cell reports.

Ingolia, N. T., Hussmann, J. A., & Weissman, J. S. (2019). Ribosome profiling: Global views of translation. Cold Spring Harbor perspectives in biology, 11(5), a032698.

Jacks, T., H.D. Madhani, F.R. Masiarz, and H.E. Varmus. (1988). Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 55:447-58.

Jackson, R. J., Hellen, C. U., & Pestova, T. V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nature reviews Molecular cell biology, 11(2), 113.

Jalili, N., & Laxminarayana, K. (2004). A review of atomic force microscopy imaging systems: application to molecular metrology and biological sciences. Mechatronics, 14(8), 907-945.

Jalkanen, A. L., Coleman, S. J., & Wilusz, J. (2014, October). Determinants and implications of mRNA poly (A) tail size–Does this protein make my tail look big?. In Seminars in cell & developmental biology (Vol. 34, pp. 24-32). Academic Press.

James, S. H., & Prichard, M. N. (2014). Current and future therapies for herpes simplex virus infections: mechanism of action and drug resistance. Current opinion in virology, 8, 54-61.

Ji, Z., Song, R., Regev, A., & Struhl, K. (2015). Many lncRNAs, 5’UTRs, and pseudogenes are translated and some are likely to express functional proteins. Elife, 4, e08890.

Jiang, Z., Yang, J., Dai, A., Wang, Y., Li, W., & Xie, Z. (2017). Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress. BMC genomics, 18(1), 638.

Jones, N., & Shenk, T. (1979). An adenovirus type 5 early gene function regulates expression of other early viral genes. Proceedings of the National Academy of Sciences, 76(8), 3665-3669.

Joshi-Barve, S., Rychlik, W., & Rhoads, R. E. (1990). Alteration of the major phosphorylation site of eukaryotic protein synthesis initiation factor 4E prevents its association with the 48 S initiation complex. Journal of Biological Chemistry, 265(5), 2979-2983.

101 Juntawong, P., Girke, T., Bazin, J., & Bailey-Serres, J. (2014). Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proceedings of the National Academy of Sciences, 111(1), E203-E212.

Kahvejian, A., Roy, G., & Sonenberg, N. (2001, January). The mRNA closed-loop model: the function of PABP and PABP-interacting proteins in mRNA translation. In Cold Spring Harbor symposia on quantitative biology (Vol. 66, pp. 293-300). Cold Spring Harbor Laboratory Press.

Kahvejian, A., Svitkin, Y. V., Sukarieh, R., M’Boutchou, M. N., & Sonenberg, N. (2005). Mammalian poly (A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes & development, 19(1), 104-113.

Karlsen, J., Asplund-Samuelsson, J., Thomas, Q., Jahn, M., & Hudson, E. P. (2018). Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation. mSystems, 3(5), e00126-18.

Kaufman, R. J., & Murtha, P.,(1987). Translational control mediated by 102ukaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells. Molecular and cellular biology, 7(4), 1568-1571.

Kaufman, R. J., Davies, M. V., Pathak, V. K., & Hershey, J. W. (1989). The phosphorylation state of 102ukaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Molecular and cellular biology, 9(3), 946-958.

Kearse, M. G., & Wilusz, J. E. (2017). Non-AUG translation: a new start for protein synthesis in eukaryotes. Genes & development, 31(17), 1717-1731.

Keiper, B. (2019). Cap-Independent mRNA Translation in Germ Cells. International journal of molecular sciences, 20(1), 173.

Keller, A., Schleicher, T., Schultz, J., Müller, T., Dandekar, T., & Wolf, M. (2009). 5.8 S- 28S rRNA interaction and HMM-based ITS2 annotation. Gene, 430(1-2), 50-57.

Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., & Salzberg, S. L. (2013). TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome biology, 14(4), R36.

Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S., & Ban, N. (2011). Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science, 334(6058), 941-948.

Komar, A.A., T. Lesnik, and C. Reiss. (1999). Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation. FEBS Lett. 462:387-91.

102 Kopeina, G. S., Afonina, Z. A., Gromova, K. V., Shirokov, V. A., Vasiliev, V. D., & Spirin, A. S. (2008). Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA. Nucleic acids research, 36(8), 2476-2488.

Korpelainen, E., Tuimala, J., Somervuo, P., Huss, M., & Wong, G. (2014). RNA-seq data analysis: a practical approach. Chapman and Hall/CRC.

Kostura, M., & Mathews, M. B. (1989). Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI. Molecular and Cellular Biology, 9(4), 1576-1586.

Kozak, M., & Shatkin, A. J. (1978). Migration of 40 S ribosomal subunits on messenger RNA in the presence of edeine. Journal of Biological Chemistry, 253(18), 6568-6577.

Kozak, M. (1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 12:857-72.

Kozak, M. (1986a). Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 83:2850-4.

Kozak, M. (1986b). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 44:283-92.

Kozak, M. (1987). At least six-nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol. 196:947-50.

Kozak, M., (1988). Leader length and secondary structure modulate mRNA function under conditions of stress. Molecular and cellular biology, 8(7), 2737-2744.

Kozak, M. (1989). Circumstances and mechanisms of inhibition of translation by secondary structure in 103ukaryotic mRNAs. Mol Cell Biol. 9:5134-42.

Kozak, M. (1991). An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 115:887-903.

Kozak, M. (1992). Regulation of translation in eukaryotic systems. Annu Rev Cell Biol. 8:197-225.

Kwan, T., & Thompson, S. R. (2018). Noncanonical Translation Initiation in Eukaryotes. Cold Spring Harbor perspectives in biology, a032672.

Lan, S., Kamel, W., Punga, T., & Akusjärvi, G. (2016). The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding. Nucleic acids research, 45(4), 1731-1742.

103 Lareau, L. F., Hite, D. H., Hogan, G. J., & Brown, P. O. (2014). Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. Elife, 3, e01257.

Lauria, F., Tebaldi, T., Lunelli, L., Struffi, P., Gatto, P., Pugliese, A., ... & Quattrone, A. (2015). RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes. Nucleic acids research, 43(22), e153-e153.

Lee, E. C. S., Elhassan, S. A. M., Lim, G. P. L., Kok, W. H., Tan, S. W., Leong, E. N., … & Candasamy, M. (2019). The roles of circular RNAs in human development and diseases. Biomedicine & Pharmacotherapy, 111, 198-208.

Lee, T. W. R., Lawrence, F. J., Dauksaite, V., Akusjärvi, G., Blair, G. E., & Matthews, D. A. (2004). Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins. Journal of general virology, 85(1), 185-196.

Lee, S., Liu, B., Lee, S., Huang, S. X., Shen, B., & Qian, S. B. (2012). Global mapping of translation initiation sites in mammalian cells at single nucleotide resolution. Proceedings of the National Academy of Sciences, 109(37), E2424-E2432.

Leppard, K. N. (1993). Selective effects on adenovirus late gene expression of deleting the E1b 55K protein. J. Gen. Virol. 74 ( Pt 4): 575-582.

Leppard, K. N. (1997). E4 gene function in adenovirus, adenovirus vector and adeno- associated virus infections. J. Gen. Virol. 78 ( Pt 9): 2131-2138.

Leppard, K. N. and T. Shenk (1989). The adenovirus E1B 55 kd protein influences mRNA transport via an intranuclear effect on RNA metabolism. 186 EMBO J. 8(8): 2329-2336.

Leppek, K., Das, R., & Barna, M. (2018). Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nature Reviews Molecular Cell Biology, 19(3), 158.

Li, H., Xiao, L., Zhang, L., Wu, J., Wei, B., Sun, N., & Zhao, Y. (2018). FSPP: A Tool for Genome-Wide Prediction of smORF-Encoded Peptides and Their Functions. Frontiers in genetics, 9, 96.

Liao, P.Y., P. Gupta, A.N. Petrov, J.D. Dinman, and K.H. Lee. (2008). A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting. Nucleic Acids Res.

Lim, V.I., and J.F. Curran. (2001). Analysis of codon:anticodon interactions within the ribosome provides new insights into codon reading and the genetic code structure. Rna. 7:942-57.

104 Lindsley, D., J. Gallant, and G. Guarneros. (2003). Ribosome bypassing elicited by tRNA depletion. Mol Microbiol. 48:1267-74.

Lion, T. (2014). Adenovirus Infections in Immunocompetent and Immunocompromised Patients. Clinical microbiology reviews, 27(3), 441-462.

Liu, B., & Qian, S. B. (2016). Characterizing inactive ribosomes in translational profiling. Translation, 4(1), e1138018.

Liu, L., D. Dilworth, L. Gao, J. Monzon, A. Summers, N. Lassam, and D. Hogg. (1999). Mutation of the CDKN2A 5’ UTR creates an aberrant initiation codon and predisposes to melanoma. Nat Genet. 21:127-32.

Locker, n., Easton, l. e., & Lukavsky, P. J. (2006). Affinity purification of eukaryotic 48S initiation complexes. Rna, 12(4), 683-690.

Logan, J., & Shenk, T. (1984). Adenovirus tripartite leader sequence enhances translation of mRNAs late after infection. Proceedings of the National Academy of Sciences, 81(12), 3655-3659.

Loman, N. J., Misra, R. V., Dallman, T. J., Constantinidou, C., Gharbia, S. E., Wain, J., & Pallen, M. J. (2012). Performance comparison of benchtop high-throughput sequencing platforms. Nature biotechnology, 30(5), 434.

Lozano, G., & Martínez-Salas, E. (2015). Structural insights into viral IRES-dependent translation mechanisms. Current opinion in virology, 12, 113-120.

López‐Lastra, M., Ramdohr, P., Letelier, A., Vallejos, M., Vera‐Otarola, J., & Valiente‐ Echeverría, F. (2010). Translation initiation of viral mRNAs. Reviews in medical virology, 20(3), 177-195.

Lorsch, J. (2007). Translation Initiation: Cell Biology, High-throughput and Chemical- based Approaches (Vol. 431). Academic Press.

Lytle, J. R., Wu, L., & Robertson, H. D. (2001). The ribosome binding site of hepatitis C virus mRNA. Journal of virology, 75(16), 7629-7636.

Malys, N., & McCarthy, J. E. (2011). Translation initiation: variations in the mechanism can be anticipated. Cellular and molecular life sciences, 68(6), 991-1003.

Marzi, S., A.G. Myasnikov, A. Serganov, C. Ehresmann, P. Romby, M. Yusupov, and B.P. Klaholz. (2007). Structured mRNAs regulate translation initiation by binding to the platform of the ribosome. Cell. 130:1019-31.

105 Matsuura, T., Tanaka, H., Matsumoto, T., & Kawai, T. (2006). Atomic force microscopic observation of Escherichia coli ribosomes in solution. Bioscience, biotechnology, and biochemistry, 70(1), 300-302.

Maquat, L. E. (2004). Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nature reviews Molecular cell biology, 5(2), 89.

Marguerat, S., & Bähler, J. (2010). RNA-seq: from technology to biology. Cellular and molecular life sciences, 67(4), 569-579.

Marshall, E., Stansfield, I., & Romano, M. C. (2014). Ribosome recycling induces optimal translation rate at low ribosomal availability. Journal of the Royal Society Interface, 11(98), 20140589.

Mata, J., Marguerat, S., & Bähler, J. (2005). Post-transcriptional control of gene expression: a genome-wide perspective. Trends in biochemical sciences, 30(9), 506-514. Matthews, M.B., and T. Shenk. (1991). Adenovirus virus-associated RNA and translation control. J Virol. 65:5657-62.

McGlincy, N. J., & Ingolia, N. T. (2017). Transcriptome-wide measurement of translation by ribosome profiling. Methods, 126, 112-129.

Michel, Y. M., Poncet, D., Piron, M., Kean, K. M., & Borman, A. M. (2000). Cap-poly (A) synergy in mammalian cell-free extracts investigation of the requirements for poly (a)- mediated stimulation of translation initiation. Journal of Biological Chemistry, 275(41), 32267-32276.

Mikamo, E., Tanaka, C., Kanno, T., Akiyama, H., Jung, G., Tanaka, H., & Kawai, T. (2005). Native polyribosomes of Saccharomyces cerevisiae in liquid solution observed by atomic force microscopy. Journal of structural biology, 151(1), 106-110.

Moore, K. S., & von Lindern, M. (2018). RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis. Frontiers in physiology, 9.

Mordstein, Christine, Rosina Savisaar, Robert S. Young, Jeanne Bazile, Lana Talmane, Juliet Luft, Michael Liss, Martin S. Taylor, Laurence D. Hurst, and Grzegorz Kudla. “Splicing buffers suboptimal codon usage in human cells.” BioRxiv(2019): 527440.

Morris, S. J., & Leppard, K. N. (2009). Adenovirus serotype 5 L4-22K and L4-33K proteins have distinct functions in regulating late gene expression. Journal of virology, 83(7), 3049-3058.

Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008). Lehninger principles of biochemistry. Macmillan.

106 Nelson, E. M., & Winkler, M. M. (1987). Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes. Journal of Biological Chemistry, 262(24), 11501-11506.

O’Connel, R. M., (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature, 516, 263-266.

Olson, V. A., Smith, S. K., Foster, S., Li, Y., Lanier, E. R., Gates, I., … & Damon, I. K. (2014). In Vitro Efficacy of Brincidofovir against Variola Virus. Antimicrobial agents and chemotherapy, 58(9), 5570-5571.

O’Malley, R. P., Duncan, R. F., Hershey, J. W., & Mathews, M. B. (1989). Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. Virology, 168(1), 112-118.

Ornelles, D. A. and T. Shenk (1991). Localization of the adenovirus early region 1B 55- kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein. J. Virol. 65(1): 424-429.

Ottaviano, G., Chiesa, R., Feuchtinger, T., Vickers, M. A., Dickinson, A., Gennery, A. R., … & Todryk, S. (2019). Adoptive T Cell Therapy Strategies for Viral Infections in Patients Receiving Haematopoietic Stem Cell Transplantation. Cells, 8(1), 47.

Palmer, M., Prediger, E. Assessing RNA quality. Available at: http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rna- isolation/tech-notes/assessing-rna-quality.html. Accessed July 23, 2019.

Panda, A. C., Grammatikakis, I., Munk, R., Gorospe, M., & Abdelmohsen, K. (2017). Emerging roles and context of circular RNAs. Wiley Interdisciplinary Reviews: RNA, 8(2), e1386.

Panniers, R. (1994). Translational control during heat shock. Biochimie, 76(8), 737-747.

Park, Y.S., S.W. Seo, S. Hwang, H.S. Chu, J.H. Ahn, T.W. Kim, D.M. Kim, and G.Y. Jung. (2007). Design of 5’-untranslated region variants for tunable expression in Escherichia coli. Biochem Biophys Res Commun. 356:136-41.

Parks, R. J. (2005). Adenovirus protein IX: a new look at an old protein. Molecular therapy, 11(1), 19-25.

Patwary, N. I. A., Islam, M. S., Sohel, M., Ara, I., Sikder, M. O. F., & Shahik, S. M. (2016). In silico structure analysis and epitope prediction of E3 CR1-beta protein of Human Adenovirus E for vaccine design. biomedical journal, 39(6), 382-390.

107 Paulin, F.E., S.A. Chappell, and A.E. Willis. (1998). A single nucleotide change in the c- myc internal ribosome entry segment leads to enhanced binding of a group of protein factors. Nucleic Acids Res. 26:3097-103.

Pelletier, J., & Sonenberg, N. (1985). Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell, 40(3), 515-526.

Pelletier, J., & Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature, 334(6180), 320.

Pineles, B. L., Romero, R., Montenegro, D., Tarca, A. L., Han, Y. M., Kim, Y. M., ... & Hassan, S. S. (2007). Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia. American journal of obstetrics and gynecology, 196(3), 261-e1.

Prasad, V. K., Grimley, M., Papanicolaou, G., Yu, L. C., Florescu, D. F., Brundage, T. M., … & Kurtzberg, J. (2014). Brincidofovir (CMX001) Is Well Tolerated in Highly Immunocompromised Pediatric Patients. Biology of Blood and Marrow Transplantation, 20(2), S93-S93.

Proudfoot, N. J., Furger, A., & Dye, M. J. (2002). Integrating mRNA processing with transcription. Cell, 108(4), 501-512.

Proweller, A., & Butler, J. S. (1997). Ribosome concentration contributes to discrimination against poly (A)− mRNA during translation initiation in Saccharomyces cerevisiae. Journal of Biological Chemistry, 272(9), 6004-6010.

Purvis, I.J., A.J. Bettany, T.C. Santiago, J.R. Coggins, K. Duncan, R. Eason, and A.J. Brown. (1987). The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis. J Mol Biol. 193:413-7.

Qiu, C., Ma, Y., Wang, J., Peng, S., & Huang, Y. (2009). Lin28-mediated post- transcriptional regulation of Oct4 expression in human embryonic stem cells. Nucleic acids research, 38(4), 1240-1248.

Radko, S., Jung, R., Olanubi, O., & Pelka, P. (2015). Effects of adenovirus type 5 E1A isoforms on viral replication in arrested human cells. PloS one, 10(10), e0140124.

Ramke, M., Lee, J. Y., Dyer, D. W., Seto, D., Rajaiya, J., & Chodosh, J. (2017). The 5′ UTR in human adenoviruses: leader diversity in late gene expression. Scientific Reports, 7(1), 618.

Reynoso, M. A., Juntawong, P., Lancia, M., Blanco, F. A., Bailey-Serres, J., & Zanetti, M. E. (2015). Translating Ribosome Affinity Purification (TRAP) followed by RNA

108 sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes. In Plant Functional Genomics (pp. 185-207). Humana Press, New York, NY.

Rogers, D. W., Böttcher, M. A., Traulsen, A., & Greig, D. (2017). Ribosome reinitiation can explain length-dependent translation of messenger RNA. PloS computational biology, 13(6), e1005592.

Ronaghi, M., M. Uhlen, and P. Nyren. (1998). A sequencing method based on real-time pyrophosphate. Science. 281:363, 365.

Ronaghi, M. (2001). Pyrosequencing sheds light on DNA sequencing. Genome Res. 11:3- 11.

Rothberg, J. M., Hinz, W., Rearick, T. M., Schultz, J., Mileski, W., Davey, M., … & Bustillo, J. (2011). An integrated semiconductor device enabling non-optical genome sequencing. Nature, 475(7356), 348-352.

Rousseau, B. A., Hou, Z., Gramelspacher, M. J., & Zhang, Y. (2018). Programmable RNA cleavage and recognition by a natural CRISPR-Cas9 system from Neisseria meningitidis. Molecular cell, 69(5), 906-914.

Rowe, W. P., R. J. Huebner, L. K. Gilmore, R. H. Parrott and T. G. Ward (1953). Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med. 84(3): 570-573.

Rowlands, A. G., R. Panniers and E. C. Henshaw (1988). The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2. J. Biol. Chem. 263(12): 5526-5533.

Sachs, A. (2000). Physical and functional interactions between the mRNA cap structure and the poly (A) tail. COLD SPRING HARBOR MONOGRAPH SERIES, 39, 447-466.

Sadis, S., Hickey, E., & Weber, L. A. (1988). Effect of heat shock on RNA metabolism in HeLa cells. Journal of cellular physiology, 135(3), 377-386.

Safer, B. (1983). 2B or not 2B: regulation of the catalytic utilization of eIF-2. Cell 33(1): 7-8.

Saikia, M., Wang, X., Mao, Y., Wan, J., Pan, T., & Qian, S. B. (2016). Codon optimality controls differential mRNA translation during amino acid starvation. RNA, 22(11), 1719- 1727.

Sarkar-Banerjee, S., Goyal, S., Gao, N., Mack, J., Thompson, B., Dunlap, D., … & Finzi, L. (2018). Specifically bound lambda repressor dimers promote adjacent non-specific binding. PloS one, 13(4), e0194930.

109 Sambrook, J., and D.W. Russell. (2001). Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis. In Molecular Cloning: A Laboratory Manual. Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

Sanders, C.L., and J.F. Curran. (2007). Genetic analysis of the E site during RF2 programmed frameshifting. Rna. 13:1483-91.

Sandkovsky, U., Vargas, L., & Florescu, D. F. (2014). Adenovirus: Current Epidemiology and Emerging Approaches to Prevention and Treatment. Current infectious disease reports, 16(8), 1-8.

Sasaki, J., & Nakashima, N. (1999). Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. Journal of virology, 73(2), 1219-1226.

Schaack, J., W. Y. Ho, P. Freimuth and T. Shenk (1990). Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA. Genes Dev. 4(7): 1197-1208.

Scheper, G.C., M.S. van der Knaap, and C.G. Proud. (2007). Translation matters: protein synthesis defects in inherited disease. Nat Rev Genet. 8:711-23.

Schwartz, R., and J.F. Curran. 1997. Analyses of frameshifting at UUU-pyrimidine sites. Nucleic Acids Res. 25:2005-11.

Schneider, R. J., B. Safer, S. M. Munemitsu, C. E. Samuel and T. Shenk (1985). Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection. Proc. Natl. Acad. Sci. U. S. A. 82(13): 4321-4325.

Schneider, R. J. (1995). Cap-independent translation in adenovirus infected cells. In Cap- Independent Translation (pp. 117-129). Springer Berlin Heidelberg.

Schneider, R. J. (2000). Adenovirus inhibition of cellular protein synthesis and preferential translation of viral mRNAs. COLD SPRING HARBOR MONOGRAPH SERIES, 39, 901- 914.

Schneider, R. J., & Mohr, I. (2003). Translation initiation and viral tricks. Trends in biochemical sciences, 28(3), 130-136.

Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., … & Liu, J. O. (2010). Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nature chemical biology, 6(3), 209.

110

Selbach, M., B. Schwanhausser, N. Thierfelder, Z. Fang, R. Khanin, and N. Rajewsky. (2008). Widespread changes in protein synthesis induced by microRNAs. Nature. 455:58- 63.

Shatkin, A.J. (1985). mRNA cap binding proteins: essential factors for initiating translation. Cell. 40:223-4.

Shen, L.X., and I. Tinoco, Jr. (1995). The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus. J Mol Biol. 247:963-78.

Shenk, T.E. 2001. Adenoviridae: The Viruses and Their Replication. In Fundamental Virology. D.M. Knipe, editor. Lippincott, Williams & Wilkins, Philadelphila.

Shine, J., and L. Dalgarno. 1974. The 3’-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 71:1342-6.

Simms, C. L., Yan, L. L., & Zaher, H. S. (2017). Ribosome collision is critical for quality control during no-go decay. Molecular cell, 68(2), 361-373.

Soderlund, H., U. Pettersson, B. Vennstrom, L. Philipson and M. B. Mathews (1976). A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2. Cell 7(4): 585-593.

Sogorin, E. A., Shirokikh, N. E., Ibragimova, A. M., Vasiliev, V. D., Agalarov, S. C., & Spirin, A. S. (2012). Leader sequences of eukaryotic mRNA can be simultaneously bound to initiating 80S ribosome and 40S ribosomal subunit. Biochemistry (Moscow), 77(4), 342-345.

Sokolove, P. G., & Bushell, W. N. (1978). The chi square periodogram: its utility for analysis of circadian rhythms. Journal of theoretical biology, 72(1), 131-160.

Somogyi, P., A.J. Jenner, I. Brierley, and S.C. Inglis. (1993). Ribosomal pausing during translation of an RNA pseudoknot. Mol Cell Biol. 13:6931-40.

Sonenberg, N., Guertin, D., & Lee, K. A. (1982). Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells. Molecular and cellular biology, 2(12), 1633-1638.

Sonenberg, N., and A.G. Hinnebusch. (2007). New modes of translational control in development, behavior, and disease. Mol Cell. 28:721-9.

Soppe, J. A., & Lebbink, R. J. (2017). Antiviral goes viral: harnessing CRISPR/Cas9 to combat viruses in humans. Trends in microbiology, 25(10), 833-850.

111 Spahr, P., and B. Hollingworth. (1961). Purification and mechanism of action of ribonuclease from Escherichia coli ribosomes. Journal of Biological Chemistry. 236:823- 831.

Staring, J., Raaben, M., & Brummelkamp, T. R. (2018). Viral escape from endosomes and host detection at a glance. J Cell Sci, 131(15), jcs216259.

Sullivan, C.S., and D. Ganem. (2005). MicroRNAs and viral infection. Mol Cell. 20:3-7. Takahashi, K., and S. Moore. (1982). The Enzymes. Vol. 15, Part B. P. Boyer, editor. Academic Press, Orlando, FL. 435-468.

Steitz, T. A. (2008). A structural understanding of the dynamic ribosome machine. Nature Reviews Molecular Cell Biology, 9(3), 242.

Stern-Ginossar, N., Weisburd, B., Michalski, A., Le, V. T. K., Hein, M. Y., Huang, S. X., … & Weissman, J. S. (2012). Decoding human cytomegalovirus. Science, 338(6110), 1088-1093.

Stephens, C. J., Lauron, E. J., Kashentseva, E., Lu, Z. H., Yokoyama, W. M., & Curiel, D. T. (2019). Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. Journal of Controlled Release.

Strutt, S. C., Torrez, R. M., Kaya, E., Negrete, O. A., & Doudna, J. A. (2018). RNA- dependent RNA targeting by CRISPR-Cas9. Elife, 7, e32724.

Takahashi, Y., Hirayama, S., & Odani, S. (2005). Ribosomal proteins cross-linked to the initiator AUG codon of a mRNA in the translation initiation complex by UV- irradiation. Journal of biochemistry, 138(1), 41-46.

Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, H., Endoh, H., ... & Mitsudomi, T. (2004). Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer research, 64(11), 3753-3756.

Takyar, S., R.P. Hickerson, and H.F. Noller. (2005). mRNA helicase activity of the ribosome. Cell. 120:49-58.

Terns, M. P. (2018). CRISPR-based technologies: impact of RNA-targeting systems. Molecular cell, 72(3), 404-412.

Thanaraj, T.A., and P. Argos. (1996a). Protein secondary structural types are differentially coded on messenger RNA. Protein Sci. 5:1973-83.

Thanaraj, T.A., and P. Argos. (1996b). Ribosome-mediated translational pause and protein domain organization. Protein Sci. 5:1594-612.

112 Thieler, A. M., Fried, R., & Rathjens, J. (2016). RobPer: An R Package to Calculate Periodograms for Light Curves Based on Robust Regression. Journal of Statistical Software, 69(9), 1-36.

Todd, G. C., & Walter, N. G. (2013). Secondary structure of bacteriophage T4 gene 60 mRNA: implications for translational bypassing. RNA (New York, N.Y.), 19(5), 685–700. doi:10.1261/rna.037291.112

Tollefson, A. E., Ying, B., Doronin, K., Sidor, P. D., & Wold, W. S. (2007). Identification of a new human adenovirus protein encoded by a novel late l-strand transcription unit. Journal of virology, 81(23), 12918-12926.

Tuller, T., Carmi, A., Vestsigian, K., Navon, S., Dorfan, Y., Zaborske, J., … & Pilpel, Y. (2010). An evolutionarily conserved mechanism for controlling the efficiency of protein translation. Cell, 141(2), 344-354.

Tyc, K., Konarska, m., Gross, h. j., & Filipowicz, W. (1984). Multiple ribosome binding to the 5′‐terminal leader sequence of tobacco mosaic virus RNA: Assembly of an 80S ribosome· mRNA complex at the AUU codon. European journal of biochemistry, 140(3), 503-511.

Uemura, S., M. Dorywalska, T.H. Lee, H.D. Kim, J.D. Puglisi, and S. Chu. (2007). Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome. Nature. 446:454- 7.

Valášek, L., Szamecz, B., Hinnebusch, A. G., & Nielsen, K. H. (2007). In vivo stabilization of preinitiation complexes by formaldehyde cross-linking. Methods in enzymology, 429, 163-183.

Van Breukelen, B., Brenkman, A. B., Holthuizen, P. E., & van der Vliet, P. C. (2003). Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin. Journal of virology, 77(2), 915-922.

Van Der Vliet, P. C. and A. J. Levine (1973). DNA-binding proteins specific for cells infected by adenovirus. Nat New Biol 246(154): 170-174.

Vanderperre, B., Lucier, J. F., Bissonnette, C., Motard, J., Tremblay, G., Vanderperre, S., … & Roucou, X. (2013). Direct detection of alternative open reading frames translation products in human significantly expands the proteome. PloS one, 8(8), e70698.

Vanzi, F., Vladimirov, S., Knudsen, C. R., Goldman, Y. E., & Cooperman, B. S. (2003). Protein synthesis by single ribosomes. Rna, 9(10), 1174-1179.

Velicer, L. F., & Ginsberg, H. S. (1968). Cytoplasmic synthesis of type 5 adenovirus capsid proteins. Proceedings of the National Academy of Sciences of the United States of America, 61(4), 1265.

113 Vellinga, J., Van der Heijdt, S., & Hoeben, R. C. (2005). The adenovirus capsid: major progress in minor proteins. Journal of General Virology, 86(6), 1581-1588.

Vicens, Q., Kieft, J. S., & Rissland, O. S. (2018). Revisiting the Closed-Loop Model and the Nature of mRNA 5′–3′ Communication. Molecular cell, 72(5), 805-812.

Viero, G., Lunelli, L., Passerini, A., Bianchini, P., Gilbert, R. J., Bernabò, P., … & Quattrone, A. (2015). Three distinct ribosome assemblies modulated by translation are the building blocks of polyribosomes. J Cell Biol, 208(5), 581-596.

Virtanen, A., P. Gilardi, A. Naslund, J. M. LeMoullec, U. Pettersson and M. Perricaudet (1984). mRNAs from human adenovirus 2 early region 4. J. Virol. 51(3): 822-831.

Wahid, A. M., Coventry, V. K., & Conn, G. L. (2009). The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures. Nucleic acids research, 37(17), 5830-5837.

Wakiyama, M., Imataka, H., & Sonenberg, N. (2000). Interaction of eIF4G with poly (A)- binding protein stimulates translation and is critical for Xenopus oocyte maturation. Current Biology, 10(18), 1147-1150.

Walker, T. A., Endo, Y., Wheat, W. H., Wool, I. G., & Pace, N. R. (1983). Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA. Journal of Biological Chemistry, 258(1), 333-338.

Wang, Y., & Wang, Z. (2015). Efficient backsplicing produces translatable circular mRNAs. Rna, 21(2), 172-179.

Wang, C., Han, B., Zhou, R., & Zhuang, X. (2016). Real-time imaging of translation on single mRNA transcripts in live cells. Cell, 165(4), 990-1001.

Waskiewicz, A. J., Flynn, A., Proud, C. G., & Cooper, J. A. (1997). Mitogen‐activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. The EMBO journal, 16(8), 1909-1920.

Weinberg, D. E., Shah, P., Eichhorn, S. W., Hussmann, J. A., Plotkin, J. B., & Bartel, D. P. (2016). Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation. Cell reports, 14(7), 1787-1799.

Weingarten-Gabbay, S., Elias-Kirma, S., Nir, R., Gritsenko, A. A., Stern-Ginossar, N., Yakhini, Z., … & Segal, E. (2016). Systematic discovery of cap-independent translation sequences in human and viral genomes. Science, 351(6270), aad4939.

Weinmann, R., H. J. Raskas and R. G. Roeder (1974). Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection. Proc. Natl. Acad. Sci. U. S. A. 71(9): 3426-3439.

114

Weissman, J., O'Connor, P. B. F., Li, G. W., Weissman, J. S., Atkins, J. F., & Baranov, P. V. (2013). RRNA: mRNA pairing alters the length and the symmetry of mRNA-protected fragments in ribosome profiling experiments.

Weitzman, M.D., and D.A. Ornelles. (2005). Inactivating intracellular antiviral responses during adenovirus infection. Oncogene. 24:7686-96.

Wells, S. E., Hillner, P. E., Vale, R. D., & Sachs, A. B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Molecular cell, 2(1), 135-140.

Wen, J.D., L. Lancaster, C. Hodges, A.C. Zeri, S.H. Yoshimura, H.F. Noller, C. Bustamante, and I. Tinoco. (2008). Following translation by single ribosomes one codon at a time. Nature. 452:598-603.

Whalen, S. G., Gingras, A. C., Amankwa, L., Mader, S., Branton, P. E., Aebersold, R., & Sonenberg, N. (1996). Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins. Journal of Biological Chemistry, 271(20), 11831-11837.

Whyte, P., K. J. Buchkovich, J. M. Horowitz, S. H. Friend, M. Raybuck, R. A. Weinberg and E. Harlow (1988). Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature 334(6178): 124- 129.

Winz, M. L., Peil, L., Turowski, T. W., Rappsilber, J., & Tollervey, D. (2019). Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control. Nature communications, 10(1), 563.

Wintermeyer, W., F. Peske, M. Beringer, K.B. Gromadski, A. Savelsbergh, and M.V. Rodnina. (2004). Mechanisms of elongation on the ribosome: dynamics of a macromolecular machine. Biochem Soc Trans. 32:733-7.

Wold, W. S. and L. R. Gooding (1991). Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 184(1): 1-8.

Wold, W. S. M., Tollefson, A. E., & Hermiston, T. W. (1995). E3 transcription unit of adenovirus. In The Molecular Repertoire of Adenoviruses I (pp. 237-274). Springer, Berlin, Heidelberg.

Wold W.S., H. M. S. (2007). Adenoviruses. Fields Virology P. M. H. David M. Knipe. Philadelphia, Lippincott Williams & Wilkins. Volume II: 2395-2436.

Wolin, S.L., and P. Walter. (1988). Ribosome pausing and stacking during translation of a eukaryotic mRNA. Embo J. 7:3559-69.

115 Wright, J., Atwan, Z., Morris, S. J., & Leppard, K. N. (2015). The human adenovirus type 5 L4 promoter is negatively regulated by TFII-I and by L4-33K. Journal of virology, JVI- 00683.

Wu, K., Guimet, D., & Hearing, P. (2013). The adenovirus L4-33K protein regulates both late gene expression patterns and viral DNA packaging. Journal of virology, JVI-00652.

Xia, X., and M. Holcik. (2009). Strong eukaryotic IRESs have weak secondary structure. PloS ONE. 4:e4136.

Xi, Q., Cuesta, R., & Schneider, R. J. (2004). Tethering of eIF4G to adenoviral mRNAs by viral 100K protein drives ribosome shunting. Genes & development, 18(16), 1997-2009.

Xi, Q., Cuesta, R., & Schneider, R. J. (2005). Regulation of translation by ribosome shunting through phosphotyrosine-dependent coupling of adenovirus protein 100K to viral mRNAs. Journal of virology, 79(9), 5676-5683.

Yamashita, Y., Kadokura, Y., Sotta, N., Fujiwara, T., Takigawa, I., Satake, A., … & Naito, S. (2014). Ribosomes in a stacked array: elucidation of the step in translation elongation at which they are stalled during S-adenosyl-L-methionine-induced translation arrest of CGS1 mRNA. Journal of Biological Chemistry, jbc-M113.

Yang, Y., Fan, X., Mao, M., Song, X., Wu, P., Zhang, Y., … & Wong, C. C. (2017). Extensive translation of circular RNAs driven by N 6-methyladenosine. Cell research, 27(5), 626.

Yee, S. P. and P. E. Branton (1985). Detection of cellular proteins associated with human adenovirus type 5 early region 1A polypeptides. Virology 147(1): 142-153.

Younis, S., Kamel, W., Falkeborn, T., Wang, H., Yu, D., Daniels, R., ... & Andersson, L. (2018). Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth. Proceedings of the National Academy of Sciences, 115(16), E3808- E3816.

Yueh, A., & Schneider, R. J. (1996). Selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells. Genes & development, 10(12), 1557-1567.

Yueh, A., & Schneider, R. J. (2000). Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA. Genes & development, 14(4), 414-421.

Young, J. A. (2014, October). Preliminary Safety Results and Antiviral Activity from the Open-label Pilot Portion of a Phase 3 Study to Evaluate Brincidofovir (BCV) for the Treatment of Adenovirus (AdV) Infection. In IDWeek 2014.

116 Yusupova, G., L. Jenner, B. Rees, D. Moras, and M. Yusupov. (2006). Structural basis for messenger RNA movement on the ribosome. Nature. 444:391-4.

Zagorska, L., J. Chroboczek, S. Klita, and P. Szafranski. (1982). Effect of secondary structure of messenger ribonucleic acid on the formation of initiation complexes with prokaryotic and eukaryotic ribosomes. Eur J Biochem. 122:265-9.

Zain, S., Sambrook, J., Roberts, R. J., Keller, W., Fried, M., & Dunn, A. R. (1979). Nucleotide sequence analysis of the leader segments in a cloned copy of adenovirus 2 fiber mRNA. Cell, 16(4), 851-861.

Zarai, Y., Ovseevich, A., & Margaliot, M. (2017). Optimal translation along a circular mRNA. Scientific reports, 7(1), 9464.

Zhang, Y., Dolph, P. J., & Schneider, R. J. (1989). Secondary structure analysis of adenovirus tripartite leader. Journal of Biological Chemistry, 264(18), 10679-10684.

Zhang, W., & Imperiale, M. J. (2003). Requirement of the adenovirus IVa2 protein for virus assembly. Journal of virology, 77(6), 3586-3594.

Zhang, P., He, D., Xu, Y., Hou, J., Pan, B. F., Wang, Y., … & Zhou, F. (2017). Genome- wide identification and differential analysis of translational initiation. Nature communications, 8(1), 1749.

Zhao, H., Chen, M., & Pettersson, U. (2014). A new look at adenovirus splicing. Virology, 456, 329-341.

Zheng, Z. M. (2010). Viral oncogenes, noncoding RNAs, and RNA splicing in human tumor viruses. International journal of biological sciences, 6(7), 730.

Zylber, E. A., & Penman, S. (1970). The effect of high ionic strength on monomers, polyribosomes, and puromycin-treated polyribosomes. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 204(1), 221-229.

117 FIGURES

Low Molecular Weight Cellular Extract

Polysome Profile

0.60 Total RNA 1400 1200 0.55 Qubit Total RNA (ng/ul) 1000

40S 800 0.50 60S Polyribosomes 600

Absorbance 280 nm 80S 0.45 400 200 0.40

0 5 10 15

Fraction Number

Figure 15. The polyribosome profile of uninfected HeLa cells shows the separation and detection of ribosomal species Polyribosomes separated by rate zonal centrifugation in sucrose gradients was detected at 280 nm (black curve; Fig. 8). The top of the gradient (left) contained the low MW material. The bottom of the gradient (right) showed a clear, characteristic polysome peak. The proposed 40S, 60S, and 80S peaks are labeled. (red) Total RNA-specific quantification by fluorescence. The most abundant RNA signal corresponded to the polyribosomes.

118

Figure 16. The peaks in the polysome profile, of uninfected cells, are from ribosomes

The digital gel from the bioanalyzer 2100. The first 11 fractions of the polyribosome profile of the uninfected HeLa cell are shown. The bioanalyzer identified and reported the 28S and 18S rRNA (labelled). The system does not report the 5.8S and 5S rRNA. Potential 5.8S and 5S rRNA are labelled.

119 Bioanalyzer 28S/18S rRNA Ratio From Polysome Profile Fractions

●

● 2

28S/18S Ratio ●

●

● 0 ● ● ● ● ●

2 4 6 8 10 12

Fraction Number

Figure 17. The increasing ratio of 28S/18S ribosomal RNA confirms the presence of ribosomes in the polyribosome profile

The first 12 fractions from a polyribosome profile were run on the bioanalyzer using the Agilent RNA 6000 pico kit. The system automatically identified the 18S and 28S peaks. The 28S/18S ratio increases further down the gradient. This confirms the presence of an increasing amount of 80S ribosomes in the polysome peak.

120 Mock Infection with and without RNase 10 2.0 Cell Extract 8 6 4 2 Total RNA (ng/ul) 1.5 0

5 10 15

Fraction Number 1.0 Absorbance 280 nm 80S Ribosome No RNase (Control) 0.5 RNase

Polyribosomes 0.0

0 2 4 6 8 10 12

Fraction Number

Figure 18. Polyribosome and ribosome profile of uninfected HeLa cells shows a decrease in polyribosomes due to RNaseI and a increase in the single ribosome peak

Mock infected HeLa cells were profiled with and without RNaseI. The polyribosome and ribosome peaks are labeled. Inset: Qubit total RNA per fraction with and without RNase. The red (RNase) curve shows a reduction in polyribosomes and a possible 80S peak. The RNase clearly cut the RNA between ribosomes. λ = 280 nm.

121 Mock Infection Profiles +/- RNase

0.3

80S RNase 0.3 units 0.8 units 2.4 units 0 units

0.2 Cellular Extract

Polyribosomes Absorbance 280 nm

0.1

0.0

0 5 10 Fraction Number

Figure 19. Ribosome profile shows a clear single ribosome peak upon RNaseI treatment

Mock infected HeLa Cells were treated with 0, 0.3 U, 0.8 U, and 2.3 Units of RNaseI. As the polyribosome peak decreased the 80S ribosome peak increased in response to RNase treatment. The 80S ribosome peak is clearly identifiable. λ = 280 nm.

122

HAd Infection Profiles +/- RNase

Cell Extract Mock Infection

2.3 Units RNase 1.5 4.6 Units RNase

Infection No RNase 80S Ribosomes

1.0

Polyribosomes Absorbance 280 nm (Scaled)

0.5

0.0

5.0 7.5 10.0 12.5 15.0 Fraction Number

Figure 20. Ribosome profile of adenovirus dl309 infected HeLa cells reveals a distinct peak of ribosome-protected fragments

Red = mock infection no RNase; Green = infected with 2.3 U RNase; Blue = infected with 4.6 U RNase; Purple = infected with no RNase. The blue bar represents the fractions containing RPFs that were collected. λ = 280 nm.

123

Figure 21. The ion torrent summary report of ribosome protected fragment cDNA sequencing

(Top) ISP loading and templating (i.e. live) bead percentages. The system uses test fragments as controls. Templated beads are filtered for polyclonal, low quality signaling, or primer dimers. (Bottom) The RPF sample had a unique barcode for identification. The final number of barcoded RPF reads was 1,633,229.

124

Figure 22. Post-sequencing analysis of ribosome protected fragments shows a mean base-call accuracy of greater than 99.4% and the correct read-length

(top) A box-plot showing the base-call quality as a function of read position. The mean quality score are the red bars. (bottom) The read frequency as a function of sequence read-length.

125 1000 4000 600

750 3000

400 500 2000

200 1000 250

0 0 0 Not Trimmed 0 2000 4000 6000 6000 8000 10000 12000 12000 14000 16000 18000 Trimmed 1250

Counts 1500 Filtered

400 1000

1000 300 750

200 500 500

250 100

0 0 0

18000 20000 22000 24000 24000 26000 28000 30000 32000 34000 HAd Genome Position

Figure 23. Ribosome-protected fragments of varying Phred quality mapped to adenovirus genome

The mapping data of (red) RPFs not quality trimmed or filtered, (green) trimmed to a minimum value of Q20 (99% base-call accuracy), or (blue) filtered for an average quality of Q20 across the entire read.

126 Normalized Mapping of RPFs to rRNA

179 175

150

100

92 Counts

18S 28S 5.8S 5S rRNA

Figure 24. RPF Mapping to rRNA is consistent with 80S ribosomes

The RPF data was first mapped to rRNA and the different rRNA species coverage is identified. The 18S rRNA is associated with the 40S ribosomal subunit. The 5.8S, 5S and 28S rRNA are associated with the 60S ribosomal subunit. Having rRNA from both the 40S and 60S subunits is consistent with RPFs from 80S ribosomes

127

Figure 25. RPF mapping to the L4 region captured the splicing of 33K consistent with 80S ribosomes in translation

The L4 transcription unit has three open-reading frames that partially overlap. The 33K and 22K mRNA share the first 300-nucleotides. The splicing of 33K make a distinct protein. The RPFs captured the splicing of 33K (red arrows). This is consistent with RNA from translating ribosomes.

128

Figure 26. Individual viral mRNA transcripts show a dominant reading frame consistent with translation The mapped RPFs to several late transcripts captured the translation of a dominant reading frame (blue line). This is consistent with RNA from translation ribosomes.

129

Figure 27. The reading frame change in the L4 transcription unit from 100K translation to 33K translation is consistent with translation

The dominant reading frame for the 100K ORF (blue line) is interrupted by the 33K ORF (read line). The RPFs are able to capture the change in reading frames of different mRNA transcripts consistent with 80S ribosomes in translation.

130

Figure 28. Different length MRNA produce a three-nucleotide periodicity in individual transcripts consistent with translation A three-nucleotide periodicity is revealed in late mRNA transcripts depending on the read-length of the RPF. Solid Red Line = 99% Confidence Interval; Dashed Red Line = 95% Confidence Interval.

131

Figure 29. The 5’ position of ribosome protected fragments reveal a 3- nucleotide periodicity consistent with the rigid exit channel of the 80S ribosome in translation The 5’ position, and not the 3’ position, of RPFs shows periodicity. Solid Red Line = 99% Confidence Interval; Dashed Red Line = 95% Confidence Interval.

132

Unique Mapped Reads

Delayed IX ● IVa2 ●

Early DBP ● E1B 19K ● Pol ● E1A ● 10.5 kD protein ● E3 12.5K ● E3 gp19K ● E1B 55K ● E4 ORF6/7 ● E3 RID−alpha ● E4 ORF4 ● E4 ORF3 ● E3 CR1−alpha0 ● E3 14.7K ● U exon ● E4 ORF1 ● E4 ORFB ● E4 34K ● E3 RID−beta ● pTP ●

Late Hexon ● TPL−3 ● 100K ● Penton ● pV ● pVII ● pVI ● TPL−2 ● Fiber ● 33K ● I−leader ● pX ● 52K ● pIIIa ● pVIII ● TPL−1 ● Protease ● 22K ●

0 1000 2000 3000 4000 5000 6000

Read Count

Figure 30. The ribosome protected fragments mapped uniquely to one transcript, most of which from the late phase unit

The mapping of RPFs to the early, delayed early and late phase genes show that most of the reads mapped uniquely to each gene. This increases the confidence that the cDNA sequenced came from a particular transcription unit. The bulk of the reads mapped to the late transcription unit. This is consistent with ribosome profiling late in infection.

133

Figure 31. Adenovirus translation expression normalized for transcript length reveal dramatic ribosome association with the TPL Adenovirus mRNA transcripts are grouped according to the phase of infection. The tripartite leader (TPL) has the highest normalized expression indicating a high association of ribosomes within the TPL (inset). The 5’ UTR of IVa2 also has more association with ribosomes compared to its ORF.

134

Figure 32. Reads mapping to the tripartite leader and coding regions are essentially identical in length

The reads mapping to mRNA coding regions (black) are 28-nucleotides in length. The reads mapping to the TPL (blue) are 28-29 nucleotides in length. Hence the 28-nucleotide length is not exclusive to translating ribosomes.

135

Figure 33. The 80S ribosomes in the tripartite leader show a periodicity of 29-nucleotides The TPL periodicity. The TPL periodicity is 29-nucleotides or equal to the size of one ribosome. This shows that the ribosomes in the TPL are not translating.

136 LEADER 1 6049-6089nt (~67X; n=2,777)

28nt LEADER 2 7111-7182nt (~760X; n=54,729)

31nt

LEADER 3 9644-9733nt (~2400X; n=212,856)

28nt

Figure 34. The ribosomes in the tripartite leader protected the 18S complementary regions The TPL 1-3 regions have 18S complementary regions (Yueh & Schneider, 2000). These regions were found to be important for shunting. The ribosomes in the TPL protected the 18S complementary regions.

137 5’ UTR of IVa2 (7X; n= 283)

26nt

5’ UTR of IVa2 (205X; n=8409)

27nt

Figure 35. The ribosomes in the 5’ untranslated region of IVa2 protected the 18S complementary regions The 5’ UTR of IVa2 has 18S complementary regions (Yueh & Schneider, 2000). These regions were found to be important for shunting. The ribosomes in the 5’ UTR of IVa2 protected the 18S complementary regions.

138

Figure 36. Mapping of protected fragments to individual viral mRNA show ribosome pausing upstream, upon, and downstream of the start codon Tripartite leader RPFs are evenly distributed between late-TPL-mRNAs. The individual mRNA transcripts show ribosome pausing before the start codon (red) in the TPL. Additionally, pausing is found upon the start codon and 100-nucleotides downstream. There were no ribosomes prior to the start codon of the IX transcript.

139

Figure 37. Zoomed in view of mapping to individual MRNA transcripts show pausing near the start codon The RPFs mapped extensively to the 5’ UTR of shunting mRNA exclusively. In addition, many transcripts had ribosome pausing 100- nucleotides downstream of the start codon (red line).

140

Figure 38. The ribosome fragment data correlate with transcriptome data late in infection The transcriptome data of Evans et al., 2012 of adenovirus infected cells at 9 h post- infection. and 24 h post-infection. were correlated with the RPFs. The highest correlation was during late infection.

141 Comparison of Ribosome Protected Fragments and Transcriptome Mapping to the Tripartite Leader

1500000

1000000 Time

RPFs Zero

FPKM Nine Twenty−Four

500000

TPL 1 TPL 2 TPL 3

Figure 39. Comparison of ribosome fragment mapping and transcriptome mapping from Evans et al. shows pronounced translational expression of the tripartite leader Fragment per kilobase million mapped reads (FPKM) normalization was performed on the RPFs and transcriptome data from Evans et al. The reads were mapped to the HAdVC reference used by Evans et al., (i.e. Ref AC_000008.1)

142

Figure 40. Models of late viral mRNA translation by ribosome shunting

(A) The 80S ribosome forms at the 5’ cap of linear late mRNA. The loading is faster than the 80S ribosomes can shunt resulting in stacking in the 5’ UTR. (B) The same as (A) but the 80S ribosomes form 3’ to 5’ on linear mRNA.(C,D) The closed-loop model of late translation. (C) During the initial round of translation, the initiation complex (i.e.40S) binds to the 5’ cap, scans, and shunts. (D) The 80S terminates at the stop codon and then is recycled 3’ to 5’ in the 5’ UTR.

143 APPENDIX I

Adenovirus Infection

The virus family Adenoviridae was first discovered in 1953 as an infectious agent present in human adenoids (Rowe et al., 1953). Today Adenoviridae is divided into 5 genera. The genera include: Atadenovirus that infects mammals, birds and reptiles.

Avianadenoviridae that infects birds. Siadenovirus that infects reptiles and birds.

Ichtadenovirus that infect fish. And Mastadenoviridae that infects mammals including humans (Davison et al., 2003).

Mastadenoviridae is subdivided into 6 species (groups) labelled “A” through “F”.

Each species is subdivided into serotypes. There are some 50-different human viral serotypes, some of which have oncogenic potential (Berk, 2007). The oncogenic potential from HAdVC infection is partly due to the viral induction of dormant cells into proliferation that can lead to uncontrolled cell growth (Bett et al., 1995).

HAdVC is a non-enveloped, icosahedral virion capsid with 240 hexon proteins covering the 20 equilateral triangular faces, with penton base subunits at the icosahedron’s

12 vertices. Projecting from the penton bases are fiber proteins containing a terminal knob.

Other capsid proteins assist in the interactions between hexon and penton (Acheson, N.H.

2007; Berk, 2007).

The terminal knob of the fiber proteins attaches to non-dividing human cells (often epithelial cells) via the cell surface coxsackie and adenovirus receptor (CAR). The binding to CAR receptors triggers fiber dissociation that exposes specific amino acids in the penton base that interact with secondary cell surface integrin receptors (e.g. alphaV/beta3/5). The

144 binding of integrin receptors leads to extracellular relocation of the bound virus: receptor complexes to clathrin-coated pits on the cell membrane that induces endocytosis of the virion into the cytosol (Acheson, N.H. 2007; Berk, 2007).

Early HAdVC capsid containing endosomes have a mildly acidic pH (5.5-6.3) maintained via membrane ATPases. The low pH induces the viral VI protein to lyse the endosome releasing the capsid. To reach the nucleus for viral replication the capsid contains binding domains for the molecular motor Dynein. The motor “walks” the capsid, in a hand-over-hand motion, down microtubular tracks to the microtubule-organizing center near the nucleus. The viral capsid is then disassembled to allow the viral DNA and associated proteins to enter the nuclear pores. Entry into the nucleus is accomplished by about 2 h post infection (h.p.i). Inside the host nucleus the viral genome is replicated, and viral genes are transcribed and eventually translated back in the cytosol (Acheson, N.H.

2007; Berk, 2007; Wold, 2007).

The viral DNA genome is linear, double-stranded, and roughly 36 kb in size. The genome core proteins are histone-like and assist in organizing the genome into coiled domains. A 55-kDa terminal protein is linked to each 5’ end of the genome by serine mediated covalent bonds. Each end of the viral genome has inverted terminal repeat sequences. The repeats allow each single strand to hairpin and serve as replication origins

(Acheson, N.H. 2007; Schaack et al., 1990; Schneider, 1995).

The Early Phase of Infection

Viral gene expression is generally divided into early, delayed early, and late phase relative to DNA replication (Schneider, 1995). There are six early (E1A, E1B, E2, E3, and

145 E4), two delayed-early (IX, IVa2), and one major late transcription unit respectively

(MLTU). All units are transcribed by the host RNA polymerase II. The two virus- associated RNA (VA RNA) are transcribed by host RNA polymerase III. The VA gene products inhibit RNA-dependent protein kinase (PKR) activity to assist in translational control (Akusjarvi et al., 1987; Schneider et al., 1985).

The first viral gene products are from transcription unit E1A at about 2.5 h post- infection E1A products bind to the retinoblastoma (Rb) family of host tumor suppressor proteins. This allows the transcription factor E2F to bind and activate S-phase genes thus inducing dormant cells into the S-phase of cellular replication (Acheson, N.H. 2007;

Whyte, 1988).

When the cell is dormant, the S-phase transcription factor E2F is in complex with

Rb and the histone deacetylase (HDAC) complex. The bound E2F is unavailable to activate transcription. Furthermore, the bound HDACs remove acetyl groups from the amine terminal end of histone proteins. The resultant positive charge on the histones produces tight ionic bonds with negatively charged chromatin. This makes the chromatin inaccessible to any free E2F and therefore transcription is inhibited (Acheson, N.H. 2007;

Berk, 2007; Yee and Branton, 1985).

When a cell is naturally induced to move through the cell cycle, Rb proteins get phosphorylated by cyclin-dependent protein kinases (CDK) releasing E2F and HDACs.

The free E2F binds and activates S-phase genes for cellular replication. When viral E1A gene products accumulate they bind Rb proteins releasing E2F to activate host transcription. E1A gene products also interact with histone acetyl transferases (HACs). The

HACs deposit acetyl groups on histones neutralizing the positive charge. The neutralization

146 results in a more relaxed chromatin structure that is accessible for transcription factors that initiate the S phase of the cell cycle (Acheson, N.H. 2007; Berk, 2007; Whyte et al., 1988).

E1A gene products also bind the host basal transcription TATA-binding protein

(TBP) and bZIP activating transcription factors (ATFs) producing a stable transcription complex (Frisch and Mymryk, 2002). The complex induces the transcription of viral E3 and E4 genes. Finally, E1A, E2A, and E2B direct viral DNA replication. As the infected cell prepares for division, the environment supplies the virus the amino acids and nucleic acids it needs for replication. An unintended result of forcing the cell into replication can be oncogenesis. Hence, E1A is considered an oncogene (Acheson, N.H. 2007; Berk, 2005).

The E1B transcription unit is responsible for preventing cellular apoptosis via E1B-

19K/55K to allow the accumulation of viral progeny (Acheson, N.H. 2007; Berk

2005,2007). The 19K protein is a functional homologue to the cellular apoptosis repressor

BCL-2. The cellular BCL-2 and virus 19K can bind to another family member called Bax preventing the activation of the p53-independent Bax-caspase apoptosis pathway. The 55K protein binds directly to p53 inhibiting the p53-dependent gene expression that would lead to apoptosis. In addition, 55K, with help from the viral E4orf6 protein, can induce p53 ubiquitination and proteasome degradation.

The E2A transcription unit produces the 72K sequence-independent single- stranded DNA binding protein (ssDBP). The E2B transcription unit produces both the viral

DNA polymerase and the 87-kDa pre-terminal protein (pTP). All three gene products are necessary for viral replication (van der Vliet and Levine, 1973; Acheson, N.H. 2007).

The E3 region hinders the immune response of the host. E3 produces proteins that downregulate the expression of the Major Histocompatibility Complex I (MHC).

147 Consequently, this hinders MHC I dependent extracellular viral peptide presentation

(antigen presentation) to cytotoxic T-cells that would ultimately destroy the infected cell.

E3 proteins also inhibit TNFalpha- dependent apoptosis again keeping the cell alive to produce viral progeny (Wold and Gooding, 1991).

The dl309 variant used in this dissertation has all or part of the E3 14.7K, 14.5K, and 10.4K coding regions deleted. Hence dl309 would not survive an infection of a host.

The cells infected in this thesis were cultured and not a part of a human host (Bett et al.,

1995).

The E4 region produces proteins involved in the regulation of 1) transcription and translation, 2) viral DNA replication, 3) host anti-viral response, 4) progeny production, 5) splice-site selection, 6) late viral mRNA stabilization and 7) cell cycle phase growth restriction (Virtanen et al., 1984; Leppard, 1997). The E4orf6 in complex with E1B-55K help suppress host protein synthesis by hindering host mRNA transport from the nucleus to the cytoplasm while preferentially exporting late viral mRNAs for translation (Logan and Shenk, 1984; Schneider, 1985, 1995, 2000, 2003).

Viral DNA Replication

At 6 h post-infection viral DNA replication begins (Ornelles and Shenk, 1991).

Replication is accomplished by using HAd’s E2A-DBP, E2B-pTP, E2B-DNA polymerase, and host machinery (Acheson, 2007; Shenk 2001; Berk, 2007). Since both ends of the genome have identical inverted repeats, replication can begin on either end. The double- stranded inverted repeats contain binding sites for pTP. The viral pTP forms a phosphodiester bond with a deoxycytidine (pTP-C) molecule. The pTP-C-OH residue

148 binds to one end of genome and serves as a primer for viral DNA polymerase to begin replication. The polymerase makes a copy of one viral parent strand while displacing the other parent complementary strand. The double-stranded product consists of a linear parental and daughter strand. The inverted repeats from the displaced parental strand anneal forming a horseshoe-like structure. Another pTP-C-OH binds the short double stranded stem and again serves as a primer for viral DNA polymerase. Hence, both parental DNA strands have been copied. Finally, the 80-kDa pTP on each end of the replicated DNA is cleaved to make the 55-kDa terminal protein (TP) that remains attached to each 5’ end of the genome (reviewed in Acheson, 2007).

The Delayed Early and Late Phase of Infection

Delayed early transcription units (IX and IVa2) are involved in capsid formation and to some extent viral DNA replication (Acheson, 2007; Schneider, 2000). The late phase transcription unit (~12 h post-infection ) produces structural proteins from about 18 different late mRNAs all joined to the major-late tripartite leader promoter (MLP or TPL;

Ramke et al. 2017). The major late unit consists of one large primary transcript that is further processed into five families (L1-L5) of transcripts defined by unique polyadenylation sites (Schneider, 1995). Each family is further processed via alternative splicing into ~ 18 monocistronic mRNAs. Each mRNA is spliced into the same TPL leader sequence resulting in coordinated expression of late structural capsid proteins. The TPL leader is itself a product of splicing together three exons (TPL leader 1-3). After late viral translation, the structural proteins are then transported back to the nucleus where viral genome packaging occurs (Acheson, 2007; Schneider, 1995, 2000). Eventually cell lysis

149 occurs releasing progeny virus associated with the viral E3 11.6-kDa “death protein”

(Acheson, 2007). The “death protein” mechanism is not fully understood nor the interplay between cell death verses cell proliferation and tumor formation. In the adherent HeLa cells used in this thesis, viral release is accomplished at about 42 h post-infection The work in this dissertation was performed with dl309 infection of adherent HeLa cells in the late phase of infection at 15 -16 h post-infection .

150 APPENDIX II

Eukaryotic Translation

Eukaryotic translation involves three stages: Initiation, Elongation and

Termination (Jackson 2010). Initiation requires the eukaryotic initiation factor 4F (eIF4F) and the “ternary complex” made up of eIF2-GTP with the initiating tRNAimet. The eIF4F consists of the eIF4E (24 kDa; 5’ m7GpppN cap-binding protein), the eIF4G scaffolding protein (220 kDa; eIF4G1 or eIF4GIII), and the ATP-dependent RNA helicase eIF4A (45 kDa). The eIF4G scaffolding protein is also bound to the poly-A- binding protein (PABP). The eIF4G is used to associate capped-mRNA with the 43S preinitiation complex (40S ribosomal subunit-eIF2-GTP) via eIF3 (Jackson et al, 2010;

Dever & Green, 2012; Curran, 1993).

The 43S preinitiation complex bind to the 5’ cap of mRNA and in canonical translation, scan until it finds a start codon (usually AUG) in a favorable context (Kozak,

1991,1992; Andreev et al., 2016). When engaged to the start codon other factors help bring in the 60S ribosomal subunit to form the 80S ribosome (Jackson et al, 2010; Dever

& Green, 2012). The formation of the 80S ribosome and initiating methionine anti-codon recognition are accompanied by the hydrolysis of GTP and the release of eIF2-GDP.

Then eIF2-GDP is converted back to eIF2-GTP by the guanine exchange factor eIF2B for the translation initiation cycle to continue (Safer, 1983; Rowlands et al., 1988;

Balachandran et al., 2000).

During translation elongation, the 80S ribosome translates the mRNA Open

Reading Frame (ORF) into protein. The reading frame is a series of read in triplets (i.e.

151 codons) that code for biological information (i.e. amino acids). In general, one reading frame would contain the biological information that would result a functional protein

(Vanderprerre et al., 2013). This is analogous to these three hypothetical reading frames:

Frame 1: HTH-EFA-TCA-TAT-ETH-EBI-GRA-T-... Frame 2: THE-FAT-CAT-ATE-THE-BIG-RAT-... Frame 3: HEF-ATC-ATA-TET-HEB-IGR-AT-... Each word is three letters. Only one reading frame in the string (i.e. starting at the “T” in frame2) would make a sensible sentence. However, in many genes more than one reading frame can be possible (Vanderprerre et al., 2013; Zhang et al., 2017).

The ribosome shifts down the mRNA codon by codon (3-nucleotides) translating each codon of the reading frame into an amino acid. This is like our eyes shifting down each three-letter word in the above sentence translating the word into meaning. At the end of the reading frame or coding sequence the 80S translating ribosome reaches “stop” codons (UAA, UGA, or UAG). The stop codons generally do not code for an amino acid.

When the stop codons enter the A site of the 80S ribosome termination is catalyzed by eukaryotic release factors eRF1 and eRF3 (Jackson et al, 2010; Dever & Green, 2012;

Curran, 1993).

152 APPENDIX III

Translation of Circular-mRNA

Ribosomes, associated with circular mRNA, have been directly observed through electron and atomic force microscopy (Christensen et al., 1987 Wells et al., 1998). High resolution cryo-electron tomography showed circular mRNA coated with polyribosomes

(Afonin et al., 2015).

The removal of either the cap and/or poly(A) tail have shown to decrease translation efficiency (Proweller & Butler, 1996; Vicens et al., 2018). The adding of a cap and/or polyadenylating mRNA have shown a synergistic increase in translation efficiently in vitro

(Keiper, 2019).

Circular mRNAs are found to be more efficiently translated than linear mRNA

(Proudfoot et al., 2002; Jackson et al, 2010; Zarai et al., 2017; Brambilla et al., 2019). The advantages of circular mRNA are at least two-fold: the mRNA half-life is increased by preventing common 3’ nuclease digestion (Wang & Wang., 2015; Zarai et al., 2017) and it is cap-independent due to ribosome recycling. The recycling involves the dissembling of the 80S at the stop codon and then reassembling in the 5’ UTR that is in close proximity.

This reassembling eliminates the rate-limiting step of 5’ cap initiation (Wells et al., 1998;

Malys & McCarthy., 2011). In fact, kinetic studies on the translation of circular mRNA have shown that terminated ribosomes at the 3’ end can reinitiate in the 5’ UTR faster than ribosomes initiating at the cap (see below; Alekhina et al., 2007; Kopeina et al., 2008). In humans, the cap-independent translation of circular mRNAs have been observed during heat shock (Yang et al, 2017).

153 Interestingly, the increase in the translation of circular mRNA is correlated with limited ribosome availability. Michel et al., 2000 reduced the number of available ribosomes via ultracentrifugation in HeLa cells. The 80S ribosome concentration was decreased until it was no longer detectable at 254 nm in sucrose gradients. In addition, they made mRNA that had only a 5’ cap or only a poly(A) tail or both. Translation was performed in vitro. They observed that ribosome depletion reduced the translation of mRNA that did not have both a cap and poly(A) tail. When the mRNA was capped and polyadenylated, translation increased 3-fold increase in translation to 50% that of the non- depleted system. The translation of capped and polyadenylated was restored to normal levels upon introducing a non-depleted extract.

The authors next added rotaviral NSP3, a protein that binds to 4G in the same location of PABP but with 10-fold more affinity. NSP3 disrupts the binding of PABP and the poly(A) tail preventing the circularization of mRNA (Gratia et al., 2015). The treatment of NSP3 had zero effect on the translation on the mRNAs that did not have both the cap and poly(A) tail. However, the translation of mRNAs that were both capped and polyadenylated was reduced by 90%. These results are consistent with the conclusion that capped polyadenylated mRNA, able to be circularized, is translated with high efficiency when ribosome availability is significantly reduced.

154 Curriculum Vitae

Ph.D. Candidate, Biology: Wake Forest University, Expected Graduation: Summer 2019 Advisor: James F. Curran

Thesis: “Ribosome Profiling of Adenovirus Infected Cells Reveals Ribosome Stacking on the 5’ UTR of Late Shunting Messenger RNA

M.S.E., Biology, Chemistry (Honors): State University of NY College at Brockport May 2011

Thesis: “Relevant Chemistry through Inductive Reasoning.” B.S., Biology/Chemistry (Magna Cum Laude): State University of NY College at Brockport May 2004

Research Experience:

• Professional Research 2016-Present: Analytical Lab Coordinator: Forsyth Technical Community College

2010-Present: Developmental Scientist/Consultant: NanoMedica LLC;

2006-2016: Full-Time Research: Wake Forest University Physics Department -Executed analytical analysis on prebiotic products using HPLC and GC-MS (published).

-Constructed and executed novel drug discovery assays on next generation DNA sequencing chips using encoded chemical libraries and/or DNA aptamers (in writing).

-Designed and implemented a novel technique to study the cooperation of multiple kinesin motors in a viscoelastic environment (published).

-Developed and executed a novel technique to generate force-velocity curves for kinesin motors using an opposing magnetic force (published).

-Developed an in vitro centrosome purification and microtubule polymerization assay for electron microscopy imaging of a turbine-like force in rod formation (not published).

• Academic Research 2010- Present Graduate Research: Wake Forest University Biology Department (Part time)

155 -Currently designed and implemented a ribosome profiling strategy for identifying translational control regions in Adenovirus messenger RNA utilizing Next Generation sequencing technology (in preparation).

2006-Analyzed numerous water samples in upstate New York for phosphorus, sodium, calcium, nitrate, sulfate and pesticides in accord with Monroe county standards via HPLC and Gas Chromatography (not publishable).

2004-Undergraduate Research: University of Rochester: - Developed a Green Fluorescent Protein assay for the analysis of G-protein coupled receptors in S.cerevisiae (not published).

Fellowships:

2012-2014 Kenan Fellow (North Carolina Biotechnology Center). “Accelerating Drug Discovery: On-chip selection of DNA-Encoded Chemical Libraries.”

Memberships:

Biophysical Society; North Carolina Science Teachers Association; big brother outreach

Bioinfomatics Skills:

Biolinux;CLC;Tophat;bowtie;trimmomatic;prinseq;samtools;bioconducter;Trinity;Velvet ;Oasis.

Computer Languages:

Python and R

Patents:

Integrated Compound Discovery Systems and Methods (No. 9746466B2)

Publications:

Gagliano J., Walb M., Blaker B., Macosko J.C., and Holzwarth G., Kinesin Velocity Increases with the Number of Motors Pulling against Viscoelastic Drag, European Biophysics Journal 39(5), 801- 813, 2010.

Gagliano, J., Riley, K. R.,Xiao, J., Libby, K., Saito, S., Yu, G., ... & Bonin, K. (2015). Combining capillary electrophoresis and next-generation sequencing for aptamer selection. Analytical and bioanalytical chemistry, 407(6), 1527-1532.

156 Riley, K. R., Saito, S., Gagliano, J., & Colyer, C. L. (2014). Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection. Journal of Chromatography A, 1368, 183-189.

Nagpal, R., Wang, S., Ahmadi, S., Hayes, J., Gagliano, J.,... & Yadav, H. (2018). Human- origin probiotic cocktail increases short-chain fatty acid production via modulation of mice and human gut microbiome. Nature Scientific reports, 8(1), 12649.

Ahmadi, S., Nagpal, R., Wang, S., Gagliano, J., Kitzman, D. W., Soleimanian-Zad, S., ... & Yadav, H. (2019). Prebiotics from acorn and sago prevent high-fat-diet-induced insulin resistance via microbiome–gut–brain axis modulation. The Journal of nutritional biochemistry, 67, 1-13.

Invited Presentations:

2018 Bioscience Industry Fellowship Program. Gagliano J., High Throughput Sequencing and Pharmacogenomics

2017 Bioscience Industry Fellowship Program. Gagliano J., Personalized Medicine via High Throughput Sequencing

2017 National Center for the Biotechnology Workforce. Gagliano J., The Analytical Training Laboratory at the Innovation Quarter

2013 Wake Forest Innovation Initiative. Gagliano J., Commercializing Physics-A Case Study on University-based Entrepreneurship

2013 Wake Forest Biotechnology Center. Gagliano J., Next-Generation Sequencing: Applications and Approaches

2012 Wake Forest Medical Center. Gagliano J., Ribosome Profiling in Adenovirus Infected Cells using the Ion Torrent PGM

2012 Wake Forest Biology Dept. Gagliano J., Next-Generation DNA Sequencing Using the Ion Torrent Personal Genome Machine (PGM)

Spring 2009 Brockport State College (Brockport, NY). Gagliano J., Cooperation of Kinesin motors in a Viscoelastic Medium

Professional Conference Presentations:

Fall 2016 12th International Adenovirus Meeting. Gagliano J1, Ornelles, DA2, Curran, JF1 Ribosome Coverage on Adenovirus Late Messenger RNA Revealed by Ribosome Profiling

157 Fall 2009 North Carolina Science Teachers Association Annual Professional Development Institute (Greensboro, NC). Pecore, J. L., Anderson, A., Dunlap, P., & Gagliano, J. Incorporate laboratory experiments into coupled inquiry activities

Fall 2009 Biophysical Society (Boston, MA). Holzwarth G., Gagliano J., Walb M., Macosko J.C. Kinesin Velocity Increases with the Number of Motors in Gliding Assays against a simple Viscoelastic Load

2008 Carolina Biophysical Symposium (Chapel Hill, NC). Gagliano J., Macosko J.C., Holzwarth G., Kinesin Motility Assays in a Viscous Medium: The Number of Motors Matters

2008 Biophysical Society (Baltimore, MD). Fallesin T., Gagliano J., Macosko J.C., Investigating the Force Generated by Multiple Kinesin Motors in vitro with a Novel Magnetic Technique

Other Awards and Honors:

2015 ‘Combining capillary electrophoresis and next-generation sequencing for aptamer selection“. The top 10 most downloaded articles published 2015 in ‘Analytical and Bioanalytical Chemistry' (ABC).

2010 Biology Graduate Seminar Presentation Award; Wake Forest University

2009 Best Teacher Award; Palmyra NY Middle School

2005 Summer Scholar; University of Rochester

2004 Henry Gould award for Science Academic Achievement; Brockport State

Teaching Experience:

• Professional Teaching 2018 Adjunct Professor, Chemistry 131, Forsyth Tech Community College

Spring 2009 (research break) Living Environment: Pittsford Mendon High School, NY

Spring 2009 (research break) Science 7: Palmyra Middle School, NY

2005 Earth Science: James Madison High School, Rochester, NY,

2005 Science 8: James Madison Middle School, Rochester, NY,

2002-2006 Substitute Teaching: Several School Districts in Monroe County, NY,

158 1998-2000 Paraprofessional: Rochester City School District, Rochester, NY, • Graduate and Undergraduate Teaching 2016 Teaching Assistant, Bio-Physics (Physics 625; Biology 604) Wake Forest University

2014 Teaching Assistant, Bio-Physics (Physics 625; Biology 604) Wake Forest University

2012 Teaching Assistant, Bio-Physics (Physics 625; Biology 604) Wake Forest University

2003-2005 Student Chemistry Workshops: SUNY College at Brockport, NY

159