Viral Genomics Wednesday, October 27, 2010 Genomics 260.605.01 J. Pevsner [email protected]

Viral Genomics

Wednesday, October 27, 2010

Genomics260.605.01J. [email protected]

Many of the images in this powerpoint presentationare from Bioinformatics and Functional Genomics (2nd edition) by J Pevsner (ISBN 0-471-21004-8). Copyright 2009 by Wiley.

These images and materials may not be usedwithout permission from the publisher.

Visit http://www.bioinfbook.org Copyright notice

Outline of todays lectureIntroductionClassification of VirusesDiversity and Evolution of VirusesMetagenomics and Virus DiversityBioinformatics Approaches to Problems in VirologyInfluenza VirusHerpesvirus: From Phylogeny to Gene ExpressionHuman Immunodeficiency VirusBioinformatic Approaches to HIV-1Measles Virus

Learning objectives for todays 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)

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 virusesPage 567

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 568Introduction to viruses

Fig. 14.1Page 569

Fig. 14.2Page 570The International Committee on Taxonomy of Viruses(ICTV) offers a website, accessible via NCBIs Entrez sitehttp://www.ncbi.nlm.nih.gov/ICTVdb/

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 microbePage 569

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 microbePage 569

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 metagenomicsPage 573

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)

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.

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

Categories of phage proteins with significant matches in the uncultured human fecal viral libraryMya Breitbart M. et al. (2003) Metagenomic Analyses of an Uncultured Viral Community from Human Feces. J Bacteriol. 185: 62206223.

Vaccine-preventable viral diseases include:

Hepatitis A Hepatitis BInfluenzaMeaslesMumpsPoliomyelitisRubellaSmallpoxPage 571Human disease relevance of virusesSource: Centers for Disease Control website

DiseaseVirus

Hepatitis A Hepatitis A virusHepatitis BHepatitis B virusInfluenzaInfluenza type A or BMeaslesMeasles virusMumpsRubulavirusPoliomyelitisPoliovirus (three serotypes)RotavirusRotavirusRubellaGenus RubivirusSmallpoxVariola virusVaricellaVaricella-zoster virusPage 571Source: Centers for Disease Control websiteHuman disease relevance of viruses

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 virusesPage 574

The unique nature of viruses presents special challengesto 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 virusesDiversity and evolution of virusesPage 574

archaeabacteriaeukaryotavirusesinfluenzaSARSviruses

Overview of viral complete genomesPASC

Overview of viral complete genomes

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 virusInfluenza AInfluenza virus leads to 200,000 hospitalizations and ~36,000 deaths in the U.S. each year.Page 574

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 virusPage 575

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 virusPage 575

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

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

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 analysisHolmes 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

Evolutionary Relationships of Concatenated Major Coding Regions of Influenza A Viruses Sampled in New York State during 19992004. 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.

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.

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

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.

207 H3N2 isolatesamino acid positions in influenza proteins

Herpesviruses are double-stranded DNA viruses thatinclude herpes simplex, cytomegalovirus, and Epstein-Barr.

The genomic DNA is packed inside an icosahedral capsid; with a lipid bilayer the diameter is ~200 nanometers.HerpesvirusPage 578

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 578Herpesvirus

Fig. 14.6Page 578Millions of years before presentHerpesvirus: three main groups

McGeoch et al. (Virus Res. 117:90-104, 2006) describe a new herpesvirus taxonomy.

Family HerpesviridaeSubfamilies Alpha-, Beta-, Gammaherpesvirinae

New family Alloherpesviridae (piscine, amphibian herpesviruses)Herpesvirus taxonomyPage 578

AlphaherpesvirinaeGammaherpesvirinaeBetaherpesvirinaeAlloherpesviridae(piscine, amphibian)Malacoherpesviridae(invertebrate HV)protein-coding regionsBlocks of core genes (IVII)putative ATPase subunit of the terminaseMcGeoch DJ et al. (Virus Res. 117:90-104, 2006)

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

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 herpesvirusPage 579

clusters NCBI virus site includes tools (e.g. Protein clusters) to analyze herpesviruses

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

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 genesPage 579

Fig. 14.11Page 581

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 herpesvirusPage 582

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

Paulose-Murphy et al. (2001)

Human Immunodeficiency Virus (HIV) is the cause ofAIDS. 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 asa 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 HIVPage 583

Fig. 14.13Page 584HIV phylogeny based on pol suggests five cladesHahn et al., 20001. Simian immunodeficiency virus from the chimpanzee Pan troglodytes (SIVcpz) with HIV-1

HIV phylogeny based on pol suggests five cladesHahn et al., 20002. SIV from the sooty mangabeys Cerecocebus atys (SIVsm), with HIV-2 and SIV from the macaque (genus Macaca; SIVmac)Fig. 14.13Page 584

HIV phylogeny based on pol suggests five cladesHahn et al., 20003. SIV from African green monkeys (genus Chlorocebus)(SIVagm)Fig. 14.13Page 584

HIV phylogeny based on pol suggests five cladesHahn et al., 20004. SIV from Sykes monkeys, Cercopithecus albogularis (SIVsyk)Fig. 14.13Page 584

HIV phylogeny based on pol suggests five cladesHahn et al., 20005. SIV from lHoest monkeys (Cercopithecus lhoesti); from suntailed monkeys (Cercopithecus solatus); and from mandrill (Mandrillus sphinx)

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

Example of genotyping tool from NCBI retrovirus resourcereference sequence with the highest score

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 toolBioinformatic approaches to HIV: LANLPage 586

LANL offers many HIV tools including analysis algorithms

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

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

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Viral Genomics Wednesday, October 27, 2010 Genomics 260.605.01 J. Pevsner [email protected]