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Home , Hepadnaviridae, Reassortment

and cs Lecture 3 Biology W3310/4310 Spring 2013

“...everywhere an interplay between nucleic acids and proteins; a spinning wheel in which the thread makes the spindle and the spindle the thread”

--ERWIN CHARGAFF

1 Virology breakthrough in the 1950’s:

The viral nucleic acid is the genec code

Hershey-Chase experiment with phage T4

Fraenkel-Conrat’s work with TMV

2 Alfred Hershey & Martha Chase, 1952

3 The bigger surprise: thousands of different virions, seemingly infinite complexity of infecons

But a finite number of viral genomes

4 Key fact makes your life easier:

Viral genomes must make mRNA that can be read by host ribosomes

All on the planet follow this rule, no excepon to date

5 David Balmore (Nobel laureate) used this insight to describe a simple way to think about genomes

The original Balmore system missed one genome type: the gapped DNA of the

6 Definions

• mRNA is always the plus (+) strand • DNA of equivalent polarity is also the (+) strand • RNA and DNA complements of (+) strands are negave (-) strands • mRNA is ribosome ready: it can be translated into protein • Not all (+) RNA is mRNA!

7 The elegance of the Balmore system

Knowing only the nature of the viral genome, one can deduce the basic steps that must take place to produce mRNA

8 The seven classes of viral genomes

• dsDNA

• gapped dsDNA

• ssDNA

• dsRNA

• ss (+) RNA

• ss (-) RNA

• ss (+) RNA with DNA intermediate

9 Viral DNA or RNA genomes are structurally diverse

• Linear • Circular • Segmented • Gapped • Single-stranded (+) strand • Single-stranded (-) strand • Single stranded, ambisense • Double-stranded • Some have covalently aached proteins • Some have cross-linked ends of double-stranded DNA • Some have DNA with covalently aached RNA

10 Is there a purpose for such diversity?

• Does it tell us something about virus biology or viral evoluon? • Is one configuraon more advantageous than another? • Do we have to memorize this stuff?

11 Some of these quesons are difficult to answer

What is the purpose of a given structure? - The structure and composion of the genome is a reflecon of the method of replicaon and packaging - The selecve advantage isn’t always clear

12 Some of these quesons are difficult to answer

• Why have DNA or RNA based genomes? - RNA genomes appeared first in evoluon (The RNA World) - Only RNA genomes on planet today are viral - Relics of an ancient RNA world? • When and why was there a switch to DNA for genomes for all life forms except some viruses?

13 Yes, you need to memorize this

Parvovirus

Hepas B virus Adenovirus

Reovirus Poliovirus

Influenza virus Ebola virus

14 Memorize the 7 genome types and the key virus families that use them

• Do not wait unl the first exam! • If you know the genome structure you should be able to deduce - How mRNA is made from the genome - How the genome is copied to make more genomes

15 What informaon is encoded in a viral genome?

Gene products and regulatory signals for: - Replicaon of the viral genome - Assembly and packaging of the genome - Regulaon and ming of the replicaon cycle - Modulaon of host defenses - Spread to other cells and hosts

16 Informaon NOT contained in viral genomes

• No encoding the complete protein synthesis machinery • No genes encoding proteins involved in energy producon or membrane biosynthesis • No classical centromeres or telomeres found in standard host chromosomes • Probably we haven’t found them yet

17 Viral DNA genomes

• The host genec system is based on DNA • In large part, DNA viruses emulate the host • However, almost all viral DNA genomes are NOT like cell chromosomes • Unexpected tricks have evolved

18 dsDNA genomes

• Mammalian viruses: - - Hepadnaviridae - - - -

19 dsDNA genomes

Genomes copied by host DNA polymerase Genomes encode DNA polymerase

Papillomaviridae (8 kbp)

20 Gapped dsDNA genomes

protein RNA

Hepadnaviridae This genome cannot be copied to mRNA Hepas B virus

21 ssDNA genomes

TT virus (ubiquitous human virus) B19 parvovirus (fih disease)

22 RNA genomes

• Cells have no RNA-dependent RNA polymerase (RdRp) • RNA virus genomes encode RdRp • RdRp produce RNA genomes and mRNA from RNA templates • mRNA is readable by ribosomes

23 24 ssRNA: (+) sense

Viruses from eight families infect mammals:

-Picornaviridae (Poliovirus, ) - (gastroenteris) -Astroviridae (gastroenteris) - (SARS) - - (Yellow fever virus, West Nile virus, Hepas C virus) -Retroviridae (HIV) -Togaviridae (, Equine encephalis virus)

25 ssRNA: (+) sense

26 ssRNA (+) sense with DNA intermediate

One viral family: Retroviridae

Two human :

Human immunodeficiency virus (HIV) Human T-lymphotropic virus (HTLV)

27 The remarkable retroviral genome strategy

provirus

28 ssRNA, (-) sense Mammalian viruses:

• (Measles virus, Mumps virus) • (Rabies virus) • • (Ebola virus, Marburg virus) • (Influenza virus) •Arenaviridae (Lassa virus)

29 ssRNA, (-) sense

30 ssRNA, (-) sense

31 – a consequence of segmented genome

32 A curious twist with ss(-) RNA

• Some viral genomes are ‘ambisense’ • Contain both (+) and (-) RNA • Arenaviridae, Bunyaviridae

33 Ambisense RNA genomes

34 Genec methods

Wild-type - Original, oen laboratory-adapted, from which mutants are selected - May not be idencal to a virus isolated from nature - New virus isolates from the natural host = field or clinical isolates

35 Genec methods

DNA-mediated transformaon - The introducon of foreign DNA into cells - Not to be confused with oncogenic transformaon of cells

36 37 Genec methods

Transfecon - Producon of infecous virus aer transformaon of cells by viral DNA, first done with lambda - Transformaon-infecon - Unfortunately the term has come to be used synonymously with DNA-mediated transformaon

38 Genec methods

Mutaon - Change in DNA or RNA comprising base changes and nucleode inserons, deleons, and rearrangements - Include nonsense and missense mutaons - Should not be used to describe changes in protein

39 This method allowed the applicaon of genec methods to animal viruses

40 How were viral mutants isolated?

• RNA viruses: present in stocks. Error-prone RNA polymerases, 1 misincorporaon in 104 - 105 nucleodes polymerized • DNA viruses: 1 misincorporaon in 108 - 109 nucleodes polymerized • Chemical treatments • Screen for desired phenotype, eg ts, plaque size, drug resistance

41 Engineering mutaons into viral genomes - the modern way

• Infecous DNA clone: transfecon • A modern validaon of the Hershey-Chase experiment (1952) • Deleon, inseron, substuon, nonsense, missense

42 43 44 Resurrecng the 1918 influenza virus

45 Virus vectors

• Gene therapy: deliver a gene to paents who lack the gene or carry defecve versions • Infect cells in culture, infuse back into paent • To deliver angens (viral vaccines) • Research uses

46 47 Retrovirus vectors

48 • X-linked adrenoleukodystrophy, defect in ABCD1 transporter • Paents’ marrow derived hematopoiec stem cells infected with len with normal ABCD1 transporter gene • Re-infused into paents • Neurologic status stabilized or improved hp://www.virology.ws/2009/11/25/clinical-benefit-of-lenviral-gene-therapy-in-two-paents-with-a-rare-neurologic-disease/ 49