1. Viral Genomics Wednesday, October 27, 2010 Genomics 260.605.01 J. Pevsner pevsner@jhmi.edu

2. Many of the images in this powerpoint presentation are from Bioinformatics and Functional Genomics (2 nd edition) by J Pevsner (ISBN 0-471-21004-8). Copyright © 2009 by Wiley. These images and materials may not be used without permission from the publisher. Visit http://www.bioinfbook.org Copyright notice

3. Outline of today’s lecture Introduction Classification of Viruses Diversity and Evolution of Viruses Metagenomics and Virus Diversity Bioinformatics Approaches to Problems in Virology Influenza Virus Herpesvirus: From Phylogeny to Gene Expression Human Immunodeficiency Virus Bioinformatic Approaches to HIV-1 Measles Virus

4. Learning objectives for today’s lecture Describe how viruses are classified Explain bioinformatics approaches to virology Describe the influenza virus genome including the new H1N1 virus Provide a descriptio of the Herpesviruses Use NCBI and LANL resources to identify the function and evolution of Human Immunodeficiency Virus (HIV-1)

6. Viruses are small, infectious, obligate intracellular parasites. They depend on host cells to replicate. Because they lack the resources for independent existence, they exist on the borderline of the definition of life. The virion (virus particle) consists of a nucleic acid genome surrounded by coat proteins (capsid) that may be enveloped in a host-derived lipid bilayer. Viral genomes consist of either RNA or DNA. They may be single-, double, or partially double stranded. The genomes may be circular, linear, or segmented. Introduction to viruses Page 567

7. Viruses have been classified by several criteria: -- based on morphology (e.g. by electron microscopy) -- by type of nucleic acid in the genome -- by size (rubella is about 2 kb; HIV-1 about 9 kb; poxviruses are several hundred kb). Mimivirus (for Mimicking microbe) has a double-stranded circular genome of 1.2 megabases (Mb). -- based on human disease Page 568 Introduction to viruses

8. Fig. 14.1 Page 569

9. Fig. 14.2 Page 570 The International Committee on Taxonomy of Viruses (ICTV) offers a website, accessible via NCBI’s Entrez site http://www.ncbi.nlm.nih.gov/ICTVdb/

10. Mimivirus is the sole member of the Mimiviridae family of nucleocytoplasmic large DNA viruses (NCLDVs). It was isolated from amoebae growing in England. The mature particle has a diameter of ~400 nanometers, comparable to a small bacterium (e.g. a mycoplasma). Thus, mimivirus is by far the largest virus identified to date. Mimivirus: mimicking microbe Page 569

11. The mimivirus genome is 1.2 Mb (1,181,404 base pairs). It is a double-stranded DNA virus. ► Two inverted repeats of 900 base pairs at the ends (thus it may circularize) ► 72% AT content (~28% GC content) ► 1262 putative open-reading frames (ORFs) of length >100 amino acids. 911 of these are predicted to be protein-coding genes ► Unique features include genes predicted to encode proteins that function in protein translation. The inability to perform protein synthesis has been considered a prime feature of viruses, in contrast to most life forms. See Raoult D et al. (2004) Science 306:1344. Mimivirus: mimicking microbe Page 569

12. Viral metagenomics refers to the sampling of representative viral genomes from the environment. A typical viral genome is ~50 kilobases (in comparison, a typical microbial genome is ~2.5 megabases). A sample is collected (e.g. seawater, fecal material, or soil). Cellular material is excluded. Viral DNA is extracted, cloned, and sequenced. Viral metagenomics Page 573

13. Comparison of viral metagenomic libraries to the GenBank non-redundant database. Viral metagenomic sequences from human faeces, a marine sediment sample and two seawater samples were compared to the GenBank non-redundant database at the date of publication and in December 2004. The percentage of each library that could be classified as Eukarya, Bacteria, Archaea, viruses or showed no similarities (E-value >0.001) is shown. Edwards RA, Rohwer F. Nature Reviews Microbiology 3, 504-510 (2005)

14. Edwards RA, Rohwer F. Nature Reviews Microbiology 3, 504-510 (2005) “The Phage Proteomic Tree is a whole-genome-based taxonomy system that can be used to identify similarities between complete phage genomes and metagenomic sequences. This new version of the tree contains 167 phage genomes. Phages in black cannot be classified into any clade. In the key, each phage is defined in a clockwise direction.”

15. Genomic overview of the uncultured viral community from human feces based on TBLASTX sequence similarities. (A) Numbers of sequences with significant matches (E values of <0.001) in GenBank. (B) Distribution of significant matches among major classes of biological entities. (C) Types of mobile elements recognized in the library. (D) Families of phages identified in the fecal library. Mya Breitbart M. et al. (2003) Metagenomic Analyses of an Uncultured Viral Community from Human Feces. J Bacteriol. 185: 6220–6223.

16. Categories of phage proteins with significant matches in the uncultured human fecal viral library Mya Breitbart M. et al. (2003) Metagenomic Analyses of an Uncultured Viral Community from Human Feces. J Bacteriol. 185: 6220–6223.

17. Vaccine-preventable viral diseases include: Hepatitis A Hepatitis B Influenza Measles Mumps Poliomyelitis Rubella Smallpox Page 571 Human disease relevance of viruses Source: Centers for Disease Control website

18. DiseaseVirus Hepatitis A Hepatitis A virus Hepatitis BHepatitis B virus InfluenzaInfluenza type A or B MeaslesMeasles virus MumpsRubulavirus PoliomyelitisPoliovirus (three serotypes)Rotavirus RubellaGenus Rubivirus SmallpoxVariola virus VaricellaVaricella-zoster virus Page 571 Source: Centers for Disease Control website Human disease relevance of viruses

19. Outline of today’s lecture Introduction Classification of Viruses Diversity and Evolution of Viruses Metagenomics and Virus Diversity Bioinformatics Approaches to Problems in Virology Influenza Virus Herpesvirus: From Phylogeny to Gene Expression Human Immunodeficiency Virus Bioinformatic Approaches to HIV-1 Measles Virus

20. Some of the outstanding problems in virology include: -- Why does a virus such as HIV-1 infect one species (human) selectively? -- Why do some viruses change their natural host? In 1997 a chicken influenza virus killed six people. -- Why are some viral strains particularly deadly? -- What are the mechanisms of viral evasion of the host immune system? -- Where did viruses originate? Bioinformatic approaches to viruses Page 574

21. The unique nature of viruses presents special challenges to studies of their evolution. viruses tend not to survive in historical samples viral polymerases of RNA genomes typically lack proofreading activity viruses undergo an extremely high rate of replication many viral genomes are segmented; shuffling may occur viruses may be subjected to intense selective pressures (host immune respones, antiviral therapy) viruses invade diverse species the diversity of viral genomes precludes us from making comprehensive phylogenetic trees of viruses Diversity and evolution of viruses Page 574

22. archaea bacteria eukaryota viruses influenza SARS viruses

23. Overview of viral complete genomes PASC ►

24. Overview of viral complete genomes

25. Outline of today’s lecture Introduction Classification of Viruses Diversity and Evolution of Viruses Metagenomics and Virus Diversity Bioinformatics Approaches to Problems in Virology Influenza Virus Herpesvirus: From Phylogeny to Gene Expression Human Immunodeficiency Virus Bioinformatic Approaches to HIV-1 Measles Virus

26. Influenza viruses belong to the family Orthomyxoviridae. The viral particles are about 80- 120 nm in diameter and can be spherical or pleiomorphic. They have a lipid membrane envelope that contains the two glycoproteins: hemagglutinin (H) and neuraminidase (N). These two proteins determine the subtypes of Influenza A virus. Influenza virus Influenza A Influenza virus leads to 200,000 hospitalizations and ~36,000 deaths in the U.S. each year. Page 574

27. Since 1976, the H5N1 avian influenza virus has infected at least 232 people (mostly in Asia), of whom 134 have died. A major concern is that a human influenza virus and the H5N1 avian influenza strain were to combine, a new lethal virus could emerge causing a human pandemic. In a pandemic, 20% to 40% of the population is infected per year. ►The 1918 Spanish influenza virus killed tens of millions of people (H1N1 subtype). ►1957 (H2N2) ► 1968 (H3N2) ► Asia 2003-2005 (H5N1) ► Current, 2009 (H1N1, “swine flu”) Influenza virus Page 575

28. There are three types: A, B, C ► A and B cause flu epidemics ► Influenza A: 20 subtypes; occurs in humans, other animals. For example, in birds there are nine subtypes based on the type of neuraminidase expressed (group 1: N1, N4, N5, N8; group 2: N2, N3, N6, N7, N9). The structure of H5N1 avian influenza neuraminidase has been reported (Russell RJ et al., Nature 443:45, 2006). ► Influenza A genome consists of eight, single negative- strand RNAs (from 890 to 2340 nucleotides). Each RNA segment encodes one to two proteins. Influenza virus Page 575

29. Page 576

30. NCBI offers an Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html)

31. Growth of Influenza Virus Sequences in GenBank 10/10 http://www.ncbi.nlm.nih.gov/genomes/FLU/growth.html

32. Holmes et al. (2005) performed phylogenetic analyses of 156 complete genomes of human H3N2 influenza A viruses collected over time (1999-2004) in one location (New York State). Phylogenetic analysis revealed multiple reassortment events. One clade of H3N2 virus, present since 2002, is the source for the HA gene in all subsequently sampled viruses. Large-scale influenza virus genome analysis Holmes EC, et al. Whole-genome analysis of human influenza A virus reveals multiple persistent lineages and reassortment among recent H3N2 viruses. PLoS Biol. 2005 Sep;3(9):e300. Page 576

33. Evolutionary Relationships of Concatenated Major Coding Regions of Influenza A Viruses Sampled in New York State during 1999– 2004. The maximum likelihood phylogenetic tree is mid-point rooted for purposes of clarity, and all horizontal branch lengths are drawn to scale. Bootstrap values are shown for key nodes. Isolates assigned to clade A (light blue), clade B (yellow), and clade C (red) are indicated, as are those isolates involved in other reassortment events: A/New York/11/2003 (orange), A/New York/182/2000 (dark blue), and A/New York/137/1999 and A/New York/138/1999 (green). Holmes EC, et al. Whole-genome analysis of human influenza A virus reveals multiple persistent lineages and reassortment among recent H3N2 viruses. PLoS Biol. 2005 Sep;3(9):e300.

35. Ghedin et al. (2005) sequenced 209 complete genomes of human influenza A virus (sequencing 2,821,103 nucleotides). See Nature 437:1162. Large-scale influenza virus genome analysis

36. Each row represents a single amino acid position in one protein. Amino acids (single-letter abbreviations are used) are colour-coded as shown in the key, so that mutations can be seen as changes in colour when scanning from left to right along a row. For simplicity, only amino acids that showed changes in at least three isolates are shown. Each column represents a single isolate, and columns are only a few pixels wide in order to display all 207 H3N2 isolates in this figure. Isolates are ordered along the columns chronologically according to the date of collection; boundaries between influenza seasons are indicated by gaps between columns. A more detailed version of this figure, showing positions that experienced any amino acid change and showing identifiers for the isolates in each column, is available as Supplementary Fig. 1. Ghedin E, et al. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature. 2005 Oct 20;437(7062):1162-6.

37. 207 H3N2 isolates amino acid positions in influenza proteins

39. Outline of today’s lecture Introduction Classification of Viruses Diversity and Evolution of Viruses Metagenomics and Virus Diversity Bioinformatics Approaches to Problems in Virology Influenza Virus Herpesvirus: From Phylogeny to Gene Expression Human Immunodeficiency Virus Bioinformatic Approaches to HIV-1 Measles Virus

40. Herpesviruses are double-stranded DNA viruses that include herpes simplex, cytomegalovirus, and Epstein-Barr. The genomic DNA is packed inside an icosahedral capsid; with a lipid bilayer the diameter is ~200 nanometers. Herpesvirus Page 578

41. Phylogenetic analysis suggests three major groups that originated about 180-220 MYA. Mammalian herpesviruses are in all three subfamilies. Avian and reptilian herpesviruses are all in the Alphaherpesvirinae. Page 578 Herpesvirus

42. Fig. 14.6 Page 578 Millions of years before present Herpesvirus: three main groups

43. McGeoch et al. (Virus Res. 117:90-104, 2006) describe a new herpesvirus taxonomy. Family Herpesviridae Subfamilies Alpha-, Beta-, Gammaherpesvirinae New family Alloherpesviridae (piscine, amphibian herpesviruses) Herpesvirus taxonomy Page 578

44. Alphaherpesvirinae Gammaherpesvirinae Betaherpesvirinae Alloherpesviridae (piscine, amphibian) Malacoherpesviridae (invertebrate HV) protein-coding regions Blocks of core genes (I–VII) putative ATPase subunit of the terminase McGeoch DJ et al. (Virus Res. 117:90-104, 2006)

45. Genome sizes range from 124 kb (simian varicella virus from Alphaherpesvirinae) to 241 kb (chimpanzee cytomegalovirus from Betaherpesvirinae). ► GC content ranges from 32% to 75%. ► Protein-coding regions occur at a density of one gene per 1.5 to 2 kb of herpesvirus DNA. ► There are immediate-early genes, early genes (nucleotide metabolism, DNA replication), and late genes (encoding proteins comprising the virion). ► Introns occur in some herpesvirus genes. ► Noncoding RNAs have been described (e.g. latency- associated transcripts in HSV-1). Herpesvirus taxonomy

46. Consider human herpesvirus 8 (HHV-8)(family Herpesviridae; subfamily Gammaherpesvirinae). Its genome is ~140,000 base pairs and encodes ~80 proteins. Its RefSeq accession number is NC_003409. We can explore this virus at the NCBI website. Try NCBI  Entrez  Genomes  viruses (this is on the right sidebar)  dsDNA Bioinformatic approaches to herpesvirus Page 579

47. clusters► NCBI virus site includes tools (e.g. “Protein clusters”) to analyze herpesviruses

48. Fig. 14.7 Page 579 NCBI virus site includes tools (e.g. “Protein clusters”) to analyze herpesviruses

49. HHV-8 proteins include structural and metabolic proteins. There are also viral homologs of human host proteins such as the apoptosis inhibitor Bcl-2, an interleukin receptor, and a neural cell adhesion-related adhesin. Mechanisms by which viruses may acquire host proteins include recombination, transposition, splicing. A blastp search using HHV-8 interleukin IL-8 receptor as a query reveals several other viral IL-8 receptor molecules. Viruses can acquire host genes Page 579

50. Fig. 14.11 Page 581

51. Functional genomics approaches have been applied to human herpesvirus 8 (HHV-8). For example, microarrays have been used to define changes in viral gene expression at different stages of infection (Paulose-Murphy et al., 2001). Conversely, gene expression changes have been measured in human cells following viral infection. Bioinformatic approaches to herpesvirus Page 582

52. Fig. 14.12 Page 582 Paulose-Murphy et al. (2001) described HHV-8 viral genes that are expressed at different times post infection

53. Paulose-Murphy et al. (2001)

54. Outline of today’s lecture Introduction Classification of Viruses Diversity and Evolution of Viruses Metagenomics and Virus Diversity Bioinformatics Approaches to Problems in Virology Influenza Virus Herpesvirus: From Phylogeny to Gene Expression Human Immunodeficiency Virus Bioinformatic Approaches to HIV-1 Measles Virus

55. Human Immunodeficiency Virus (HIV) is the cause of AIDS. Some have estimated that 33 million people were infected with HIV (2006). HIV-1 and HIV-2 are primate lentiviruses. The HIV-1 genome is 9181 bases in length. Note that there are >300,000 Entrez nucleotide records for this genome (but only one RefSeq entry). Phylogenetic analyses suggest that HIV-2 appeared as a cross-species contamination from a simian virus, SIVsm (sooty mangebey). Similarly, HIV-1 appeared from simian immunodeficiency virus of the chimpanzee (SIVcpz). Bioinformatic approaches to HIV Page 583

56. Fig. 14.13 Page 584 HIV phylogeny based on pol suggests five clades Hahn et al., 2000 1. Simian immunodeficiency virus from the chimpanzee Pan troglodytes (SIVcpz) with HIV-1

57. HIV phylogeny based on pol suggests five clades Hahn et al., 2000 2. SIV from the sooty mangabeys Cerecocebus atys (SIVsm), with HIV-2 and SIV from the macaque (genus Macaca; SIVmac) Fig. 14.13 Page 584

58. HIV phylogeny based on pol suggests five clades Hahn et al., 2000 3. SIV from African green monkeys (genus Chlorocebus)(SIVagm) Fig. 14.13 Page 584

59. HIV phylogeny based on pol suggests five clades Hahn et al., 2000 4. SIV from Sykes’ monkeys, Cercopithecus albogularis (SIVsyk) Fig. 14.13 Page 584

60. HIV phylogeny based on pol suggests five clades Hahn et al., 2000 5. SIV from l’Hoest monkeys (Cercopithecus lhoesti); from suntailed monkeys (Cercopithecus solatus); and from mandrill (Mandrillus sphinx)

61. NCBI offers a retrovirus resource with reference genomes and protein sets, and several tools (alignment, genotyping). Bioinformatic approaches to HIV: NCBI Page 585

62. 10/08

63. Example of genotyping tool from NCBI retrovirus resource reference sequence with the highest score

64. Los Alamos National Laboratory (LANL) databases provide a major HIV resource. See http://hiv-web.lanl.gov/ LANL offers -- an HIV BLAST server -- Synonymous/non-synonymous analysis program -- a multiple alignment program -- a PCA-like tool -- a geography tool Bioinformatic approaches to HIV: LANL Page 586

66. LANL offers many HIV tools including analysis algorithms

67. Fig. 14.17 Page 588 http://resdb.lanl.gov/Resist_DB/protease_mutation_map.htm

68. On Friday Egbert Hoiczyk will give a lecture on bacteria. Monday we will cover chapter 14 (bacteria and archaea). Then on Wednesday we will have computer lab on bacteria. Check the www.bioinfbook.org/chapter14 site to find a word document describing the exercises, as well as web links. After that we move on to the eukaryotes. Next…