University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Student Research Projects, Dissertations, and Theses - Chemistry Department Chemistry, Department of 3-30-2006 A COMPARATIVE GENOMIC ANALYSIS OF CHLORELLA NC64A VIRUS NY-2A AND CHLORELLA Pbi VIRUS MT325 FROM THE FAMILY PHYCODNAVIRIDAE Lisa A. Fitzgerald University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/chemistrydiss Part of the Chemistry Commons Fitzgerald, Lisa A., "A COMPARATIVE GENOMIC ANALYSIS OF CHLORELLA NC64A VIRUS NY-2A AND CHLORELLA Pbi VIRUS MT325 FROM THE FAMILY PHYCODNAVIRIDAE" (2006). Student Research Projects, Dissertations, and Theses - Chemistry Department. 1. https://digitalcommons.unl.edu/chemistrydiss/1 This Article is brought to you for free and open access by the Chemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Student Research Projects, Dissertations, and Theses - Chemistry Department by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. A COMPARATIVE GENOMIC ANALYSIS OF CHLORELLA NC64A VIRUS NY-2A AND CHLORELLA Pbi VIRUS MT325 FROM THE FAMILY PHYCODNAVIRIDAE by Lisa A. Fitzgerald A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Chemistry Under the Supervision of Professor James L. Van Etten Lincoln, Nebraska May 2006 A COMPARATIVE GENOMIC ANALYSIS OF CHLORELLA NC64A VIRUS NY-2A AND CHLORELLA Pbi VIRUS MT325 FROM THE FAMILY PHYCODNAVIRIDAE Lisa A. Fitzgerald, Ph.D. University of Nebraska, 2006 Adviser: James L. Van Etten The family Phycodnaviridae consists of a morphologically similar but genetically diverse group of large dsDNA viruses which infect both fresh and marine water eukaryotic algae. Two viruses, the 370 kb Chlorella NC64A virus NY-2A and the 313 kb Chlorella Pbi virus MT325, from the family Phycodnaviridae, genus Chlorovirus, were sequenced, analyzed, and compared to the prototype chlorella virus PBCV-1. The NY-2A genome, the largest chlorella viral genome sequenced to date, contains 886 open reading frames (ORFs) of 65 codons or larger and encodes 404 putative proteins and 7 tRNAs. The MT325 genome, the smallest chlorella viral genome sequenced to date, contains 845 ORFs and encodes 331 putative proteins and 10 tRNAs. The protein- encoding genes are evenly distributed on both strands, and the intergenic space is minimal. Approximately 50% of the viral gene products have been identified, including some which are the first of their type to be detected in a virus. Domain scatter plots revealed that NY-2A and MT325 are mosaics of both prokaryotic- and eukaryotic-like proteins. A comparison of the three chlorella viruses sequenced, NY-2A, MT325, and the previously sequenced PBCV-1, revealed that ~75% of the viral encoded proteins are homolgous. However, 64% of these homologs are classified as unknowns based on a lack of sequence similarity to proteins in public databases. In addition to the genomic annotations, I conducted an extensive comparison of the chloroviruses to other phycodnavirus members as well as other large dsDNA viruses. Surprisingly, only 10 (~3%) of the chlorovirus genes are encoded by all six of the sequenced phycodnaviruses. iv TABLE OF CONTENTS TITLE PAGE……………………………………………………………………. i ABSTRACT…………………………………………………………………………. ii TABLE OF CONTENTS…………………………………………………………… iv LIST OF FIGURES AND TABLES…………………………………………………viii LIST OF ABBREVIATIONS……………………………………………………… xi ACKNOWLEDGMENTS……………………………………………………………xiii DEDICATION…………………………………………………………………………xiv CHAPTER 1 INTRODUCTION AND BACKGROUND: PHYCODNAVIRIDAE- LARGE DNA ALGAL VIRUSES 1.1 INTRODUCTION………………………………..……………………………… 1 1.2 DISCOVERY OF ALGAL VIRUSES……………………………...…………… 2 1.3 GENERAL CHARACTERISTICS AND TAXONOMY…………...…………… 4 1.4 CHLORELLA VIRUSES……………………………………...………………… 7 1.5 NATURAL HISTORY OF THE CHLORELLA VIRUSES………………..…… 8 1.6 PARAMECIUM BURSARIA CHLORELLA VIRUS TYPE-1……………….……15 1.6.1 The Life Cycle……………………………………………………….……15 1.6.2 PBCV-1 Genome……………………………………………..………… 17 1.6.3 PBCV-1-encoded Proteins……………………………..…………………18 1.7 DIVERSITY OF CHLOROVIRUS GENOMES…………………………...…… 20 1.8 PHYCODNAVIRUS GENES ARE LIKELY VERY OLD……………………… 22 1.9 CONCLUSION……………………………………………….……………………24 1.10 REFERENCES…………………………………………..……………………… 26 v CHAPTER 2 GENOMIC ANALYSES OF CHLORELLA NC64A VIRUS NY-2A AND CHLORELLA Pbi VIRUS MT325 2.1 INTRODUCTION…………………………………………………………….. 37 2.2 EXPERIMENTAL METHODS………………………………………………. 38 2.2.1 Viral DNA Isolation and Sequencing…………………………………… 38 2.2.2 Genomic Sequence Analysis…………………………………………… 39 2.2.2.1 Initial Analysis of Viral Genomes…………………………………… 39 2.2.2.2 Identification of Putative tRNAs…………………………………… 39 2.2.2.3 Identification and Naming of Open Reading Frames…………………39 2.2.2.4 Identification of Major versus Minor Open Reading Frames……… 40 2.2.2.5 Characterization of Open Reading Frames………………………… 42 2.2.2.6 Analysis with the Non-redundant Database at NCBI…………………43 2.2.2.7 Analysis with the Protein Families Database…………………………43 2.2.2.8 Identification of Clusters of Orthologous Groups…………………… 43 2.2.2.9 Identification of Inteins and Introns………………………………… 43 2.3 RESULTS………………………………………………………………………… 45 2.3.1 Analysis and Annotation of the NY-2A Genome……………………… 45 2.3.1.1 Identification of Putative tRNAs…………………………………… 47 2.3.1.2 Identification of Major and Minor Open Reading Frames……………47 2.3.1.3 Characterization of Open Reading Frames………………………… 53 2.3.1.4 Annotation of NY-2A Genome……………………………………… 59 2.3.1.5 Identification of Inteins and Introns………………………………… 61 2.3.1.6 Identification of Gene Families……………………………………… 62 2.3.2 Analysis and Annotation of the MT325 Genome……………………… 62 2.3.2.1 Identification of Putative tRNAs…………………………………… 64 2.3.2.2 Identification of Major and Minor Open Reading Frames……………65 2.3.2.3 Characterization of Open Reading Frames………………………… 70 2.3.2.4 Annotation of MT325 Genome……………………………………… 76 2.3.2.5 Identification of Inteins and Introns………………………………… 78 2.3.2.6 Identification of Gene Families……………………………………… 78 2.4 DISCUSSION………………………………………………………………… 79 2.4.1 DNA Replication and Repair-Associated Proteins……………………… 82 2.4.2 Transcription-Associated Proteins……………………………………… 83 2.4.3 Protein Synthesis, Modification, and Degradation……………………… 84 vi 2.4.4 Nucleotide Metabolism-Associated Proteins…………………………… 85 2.4.5 Protein Kinases, Phosphatases, and Channel Proteins……………………86 2.4.6 Sugar- and Lipid-Manipulating Proteins………………………………… 87 2.4.7 Cell Wall-Degrading Enzymes………………………………………… 88 2.4.8 Restriction-Modification Enzymes……………………………………… 88 2.4.9 Integration and Transposition Enzymes………………………………… 88 2.4.10 Miscellaneous Proteins………………………………………………… 89 2.5 REFERENCES…………………………………………………………………… 91 2.6 ADDENDUM………………………………………………………………….. 96 2.6.1 Termini of the PBCV-1, NY-2A, and MT325 Genomes…………………96 2.6.1.1 Termini of the PBCV-1 Genome…………………………………… 96 2.6.1.2 Termini of the NY-2A Genome………………………………………99 2.6.1.3 Termini of the MT325 Genome………………………………………102 2.6.2 NY-2A Genome…………………………………………………………105 2.6.2.1 General Characteristics of Open Reading Frames……………………105 2.6.2.2 Analysis of Open Reading Frames……………………………………128 2.6.2.3 Results from Analysis with the Protein Families (Pfam) Database…162 2.6.2.4 Results of Analysis with Clusters of Orthologous Groups (COGs)…166 2.6.3 MT325 Genome…………………………………………………………169 2.6.3.1 General Characteristics of Open Reading Frames……………………169 2.6.3.2 Analysis of Open Reading Frames……………………………………190 2.6.3.3 Results from Analysis with the Protein Families (Pfam) Database…222 2.6.3.4 Results of Analysis with Clusters of Orthologous Groups (COGs)…225 CHAPTER 3 GENOMIC COMPARISONS OF THREE CHLORELLA VIRUSES, PBCV-1, NY- 2A, AND MT325 3.1 INTRODUCTION…………………………………………………………….. 228 3.2 EXPERIMENTAL METHODS………………………………………………. 229 3.2.1 Comparing the Orientations of the Chlorella Viral Genomes……………229 3.2.2 Genomic Comparisons……………………………………………………229 3.2.3 Domain and Kingdom Scatter Plots………………………………………230 3.3 RESULTS……………………………………………………………………… 230 3.3.1 Comparison of the Orientations of the Chlorella Viral Genomes……… 230 vii 3.3.2 Comparison of the Chlorella Virus Open Reading Frames………………234 3.3.3 Domain and Kingdom Scatter Plots………………………………………238 3.4 DISCUSSION…………………………………………………………………… 239 3.5 REFERENCES……………………………………………………………………248 3.6 ADDENDUM…………………………………………………………………… 250 3.6.1 Homologous ORFs in Chlorella Viruses…………………………………250 3.6.1.1 PBCV-1 Homologous ORFs…………………………………………250 3.6.1.2 NY-2A Homologous ORFs………………………………………… 258 3.6.1.3 MT325 Homologous ORFs………………………………………… 268 3.6.2 Domain and Kingdom Scatter Plots………………………………………277 3.6.2.1 PBCV-1 Domain and Kingdom Scatter Plots……………………… 277 3.6.2.2 NY-2A Domain and Kingdom Scatter Plots…………………………281 3.6.2.3 MT325 Domain and Kingdom Scatter Plots…………………………285 CHAPTER 4 COMPARATIVE ANALYSES OF CHLOROVIRUSES, PHYCODNAVIRIDAES, AND NUCLEO-CYTOPLASMIC LARGE DNA VIRUSES 4.1 INTRODUCTION…………………………………………………………………289 4.2 BACKGROUND………………………………………………………………. 290 4.2.1 Characteristics and Taxonomy of Sequenced Phycodnaviruses…………290 4.2.1.1 Coccolithoviruses……………………………………………………290 4.2.1.2 Phaeoviruses…………………………………………………………291 4.2.2 Nucleo-Cytoplasmic Large DNA Viruses……………………………… 292 4.3 EXPERIMENTAL METHODS……………………………………………………294 4.3.1 Comparisons of Phycodnavirus Metabolic Domains……………………294 4.3.2 Analysis of NCLDVs Core Genes……………………………………… 294