Genomes and Genetics

Lecture 3
Biology 3310/4310
Virology
Spring 2017

“...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
 Virology breakthrough in the 1950’s:
The viral nucleic acid genome is the genetic code

Hershey-Chase
experiment with
phage T4

Fraenkel-Conrat’s work with TMV

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 Alfred Hershey & Martha Chase, 1952

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 The bigger surprise: thousands of different virions,
seemingly infinite complexity of infections

But a finite number of viral genomes

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Key fact makes your life easier:

Viral genomes must make mRNA

that can be read by host ribosomes

All viruses on the planet follow this rule, no

exception to date

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

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

The original Baltimore

system missed one
genome type: the
VII gapped DNA of the
Hepadnaviridae

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 Definitions

• mRNA (ribosome ready) is always the plus (+) strand

• DNA of equivalent polarity is also the (+) strand
• RNA and DNA complements of (+) strands are negative (-) strands
• Not all (+) RNA is mRNA!

VII

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 The elegance of the Baltimore system

Knowing only the nature of the viral genome,

one can deduce the basic steps that must take place
to produce mRNA

VII

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 The seven classes of viral genomes
• dsDNA

• gapped dsDNA

• ssDNA VII

• dsRNA

• ss (+) RNA

• ss (-) RNA

• ss (+) RNA with DNA

intermediate

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
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Why is mRNA placed at the center of the Baltimore scheme?

A. Because all virus particles contain mRNA

B. There is no specific reason
C. Because all viral genomes are mRNAs
D. Because mRNA must be made from all viral genomes
E. Because Baltimore studied mRNA

1
 Viral DNA or RNA genomes are structurally diverse

• Linear

• Circular

• Segmented

• Gapped

• Single-stranded (+) strand

• Single-stranded (-) strand

• Single stranded, ambisense

• Double-stranded

• Covalently attached proteins

• Cross-linked ends of double-stranded DNA

• DNA with covalently attached RNA

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 What is the function of genome diversity?

• DNA and RNA based

- RNA genomes appeared first in evolution (RNA World)
- Switch to DNA genomes
- Only RNA genomes on planet today are viral
- Viroids: Relics of RNA world?
• Linear, circular, segmented, ds, ss, (+), (-)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Memorize 7 genome types and key virus families
Parvovirus

Hepatitis B virus
VII
Retrovirus
Adenovirus
Herpes simplex virus

Poliovirus Reovirus

Influenza virus

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

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University
 What information is encoded in a viral genome?

Gene products and regulatory signals for:

- Replication of the viral genome
- Assembly and packaging of the genome
- Regulation and timing of the replication cycle
- Modulation of host defenses
- Spread to other cells and hosts

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Information NOT contained in viral genomes

• No genes encoding the complete protein synthesis machinery (AARS, eIFs, tRNAs)
• No genes encoding proteins involved in energy production or membrane
biosynthesis
• No classical centromeres or telomeres found in standard host chromosomes
• Probably we haven’t found them yet - 90% of giant virus genes are novel

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Largest known viral genomes
Virus Length Protein

Pandoravirus salinus 2,473,870 2,541

Pandoravirus dulcis 1,908,524 1,487

Megavirus chilensis 1,259,197 1,120

Mamavirus 1,191,693 1,023

Mimivirus 1,181,549 979

Moumouvirus 1,021,348 894

Mimivirus M4 981,813 620

C. roenbergensis virus 617,453 544

Mollivirus sibericum 651,000 523

Pithovirus sibericum 610,033 467

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University
 Smallest known viral genomes
Virus Length Protein

Viroid 120 none

Satellite 220 none

Hepatitis delta satellite 1,700 1

Circovirus 1,759 2

Anellovirus 2,170 4

Geminivirus 2,500 4

Hepatitis B virus 3,200 7

Levivirus 3,400 4

Partitivirus 3,700 2

Barnavirus 4,000 7
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What information may be encoded in a viral genome?

A. Gene products that catalyze membrane biosynthesis

B. Gene products that catalyze energy production
C. Complete protein synthesis systems
D. Centromeres or telomeres
E. Enzymes to replicate the viral genome

2
 Viral DNA genomes

• The host genetic system is based on DNA

• Many DNA viruses emulate the host
• However, almost all viral DNA genomes are NOT like cell chromosomes
• Unexpected tricks have evolved

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 dsDNA genomes

Adenoviridae


Herpesviridae


Papillomaviridae


Polyomaviridae


Poxviridae

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 dsDNA genomes

Genomes copied by host DNA polymerase Genomes encode DNA polymerase

Papillomaviridae (8 kbp)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 Gapped dsDNA genomes

reverse
protein RNA transcriptase

This genome cannot be copied to mRNA

Hepadnaviridae
Hepatitis B virus

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 ssDNA genomes

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

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
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Which DNA genome, on entry into the cell, can be

immediately copied into mRNA?

A. dsDNA
B. gapped dsDNA
C. circular ssDNA
D. linear ssDNA
E. All of the above

3
 RNA genomes

• Cells have no RNA-dependent RNA polymerase (RdRp)

• RNA virus genomes encode RdRp
• RdRp produce RNA genomes and mRNA from RNA templates

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 dsRNA genome

Rotavirus (human gastroenteritis)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 5' c

4
P1234
3 5' c
5' c
nsP1 nsP4 5' c

ssRNA: (+) sense

nsP2 nsP3

Picornaviridae (Poliovirus, Rhinovirus)





Caliciviridae (gastroenteritis)




Coronaviridae (SARS)



10
Flaviviridae (Yellow fever virus, West Nile virus, Hepatitis C virus, Zika virus)

11




12
Togaviridae (Rubella virus, Equine encephalitis virus)
13

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 ssRNA: (+) sense

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 CA NC
NC PR
PR RT
IN

ssRNA(+) sense with DNA intermediate

One viral family: Retroviridae

15
Two human pathogens: 16

Human immunodeficiency virus (HIV)

Human T-lymphotropic virus (HTLV)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 The remarkable retroviral genome strategy
A ss (+) RNA with DNA intermediate: Retroviridae

+ RNA

– DNA

provirus
DNA

+ RNA

B Retroviridae (7–10 kb)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
U5 U3
5' c AnAOH3’
 c M2, NEP 6

6
7

5' c 3' 7
ssRNA,
c (-)
c sense
HA
Nucleus NA
9
ER
M2
NEP/NS2 M1 NS1
15 5' c
Paramyxoviridae (Measles virus, Mumps virus)

3'

 10b
Golgi
17
Rhabdoviridae (Rabies virus)


 18
16

Filoviridae (Ebolavirus, Marburg virus)





Orthomyxoviridae (Influenza virus)
 19


 9

Arenaviridae (Lassa virus)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press

os
 ssRNA, (-) sense
A ss (–) RNA: Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae

– RNA

+ RNA – RNA

B Segmented genomes: Orthomyxoviridae C Ambisense (–) strand RNA

(10–15 kb in 6–8 RNAs)
Arenaviridae (11 kb in 2 RNAs)
(–) strand RNA segments Bunyaviridae (12–23 kb in 3 RNAs)
1 2 3 4
3' 5' 3' 5' 3' 5' 3' 5'
L RNA 5’ c
5 6 7 8
3' 5' 3' 5' 3' 5' 3' 5'

Nonsegmented genomes: Paramyxoviridae (15–16 kb) S RNA 5’ c 3’

3' 5'

Rhabdoviridae (13–16 kb)

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
3' 5'
 Reassortment: Consequence of segmented genome
A B
L M
L R3 M
1 1
2 2
3 3

4 4
5 5

6
6

L M R3
7
7

8 8

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 Nucleus
Ambisense RNA genomes

NP
3’

c 5’

4
NP
9
5’
Arenaviridae
RNA pol in virion

6
Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press

ER
 A
Linear (+) strand RNA genome of a picornavirus

5' VPg AnAOH3’

UTR UTR

5’

3’
4252

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
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Which statement about viral RNA genomes is correct?

A. (+)ssRNA genomes may be translated to make viral protein  

B. dsRNA genomes can be directly translated to make viral protein 
C. (+)ssRNA virus replication cycles do not require a (-) strand intermediate
D. RNA genomes can be copied by host cell RNA-dependent RNA polymerases
E. All of the above 

4
 This method allowed the application of genetic
methods to animal viruses

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Engineering mutations into viral
genomes - the modern way

• Infectious DNA clone: transfection

• A modern validation of the Hershey-Chase experiment (1952)
• Deletion, insertion, substitution, nonsense, missense

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 Genetic methods

Transfection
- Production of infectious virus after transformation of cells by viral
DNA, first done with bacteriophage lambda
- Transformation-infection

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University

 A Poliovirus C

Infection
Cultured cells
(+) Viral RNA
Transfection Vaccinia vir
5' 3' encoding T
RNA polym
cDNA synthesis
and cloning Infection

Transfection Transfection

Poliovirus 5'
DNA 3'
In vitro RNA (+) strand RNA
synthesis transcript

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press

B Viral protein PB1

Translation
Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
 Resurrecting the 1918 influenza virus

• Influenza virus was not identified until 1933

• In 2005, influenza RNA was isolated from formalin-fixed, paraffin-embedded lung tissue sample from
autopsy of victim of influenza in 1918

• Influenza RNA also isolated from frozen sample obtained by in situ biopsy of the lung of a victim buried in
permafrost since 1918

• Complete nucleotide sequence of all 8 RNA segments determined

• Virus was recovered by transfection of cells with 8 plasmids containing genome sequences
Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University http://www.sciencemag.org/content/310/5745/77.long
 Synthetic Virology and Biosecurity
NSABB: National Science Advisory Board for Biosecurity

• Federal advisory committee to provide advice, guidance, and leadership

regarding biosecurity oversight of dual use research to all Federal
departments and agencies with an interest in life sciences research
• Advises on and recommends specific strategies for the efficient and effective
oversight of federally conducted or supported dual use biological research,
taking into consideration national security concerns and the needs of the
research community.

Virology Lectures 2017 • Prof. Vincent Racaniello • Columbia University http://osp.od.nih.gov/office-biotechnology-activities/biosecurity/nsabb