2
Structural Virology
Fall 2024
Pavel Plevka
3
Class rules
• Turn off anything that beeps or rings.
• Reading any material that is not related to the class,
texting, or checking the internet during the class is
rude and will not be tolerated.
• Please refrain from eating during class. Having
something to drink is fine.
• Ask questions - it will help to clarify the issue not only
for you but for your peers as well!
• In class discussions, be respectful of other students'
opinions.
4
• learn elementary molecular virology
• appreciate role of structural information in
understanding of biological processes
• learn to read and evaluate scientific papers
• learn history of some fundamental discoveries
in biology
• be able to evaluate what is an important
research question
Aims of the course
5
Elementary molecular virology
1. Attachment
2. Entry
3. Transcription
4. Translation
5. Genome replication
6. Assembly
7. Exit
HIV replication cycle
6
Structural biology in understanding virus
infection
Virus binding to receptor Binding of antibodies
to virus particle
7
Read and evaluate scientific papers
CHALLENGES IN IRREPRODUCIBLE RESEARCH:
- more than 50% of drug-related research
articles in high profile journals contain
irreproducible data
Reasons for irreproducibility:
- Under supervised students and post-docs
- “Publish or Perish” threat
- wrong design of experiments – “know your statistics”
8
History of fundamental discoveries
WILHELM CONRAD RÖNTGEN (1845-
1923)
• 1901 Nobel Laureate in Physics
discovery of the remarkable rays
subsequently named after him
9
What is important research question?
1. It asks about something other people care about
2. It builds on what you and others already know
3. It allows you to learn something you don't already know
Interesting X Important
What are evolutionary relationships of anaerobic
protists?
What is the probability that current Ebola epidemics is
going to spread outside of Africa?
10
What is asked of you:
• Read assigned readings before the day for
which they are assigned
• Bring five-sentence essay describing the
importance of a scientific paper to each
lecture
• Participate in discussions
• Submit two mini-assignments
• I am here to help, learning is up to you!
11
Course textbook:
14 copies are available in campus library
12
Viruses and their importance
13
Virosphere!
14
• Phage typing of bacteria
• Sources of enzymes (polymerases, T4 ligase,
reverse transcriptase)
• Pesticides (baculoviruses – lepidoptera,
hymenoptera, myxoma virus - rabits) SPECIFICITY!
• Anti-bacterial agents (S. aureus phage phi812)
• Anti-cancer agents (adeno-associated viruses)
• Gene vectors (capsids used as vehicles for gene
delivery)
• Mass protein production
Useful viruses
15
• Phage T2 and E. coli were used to provide evidence that genes are
composed of DNA.
• The first enhancers to be characterized were in genes of simian
virus 40 (SV40).
• The first transcription factor to be characterized was the
transplantation (T) antigen of SV40.
• The first nuclear localization signal of a protein was identified in the
T antigen of SV40.
• Introns were discovered during studies of adenovirus transcription.
• The role of the cap structure at the 5’ end of eukaryotic messenger
RNA was discovered during studies with vaccinia virus and a
reovirus.
• The first internal ribosomal entry site to be discov- ered was found
in the RNA of poliovirus.
• The first RNA pseudoknot to be discovered was that in the genome
of turnip yellow mosaic virus.
Fundamental discoveries in biology
Most viruses are 25-400nm in size
Units of length
20
Using TMV to increase surface of electrodes
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
21
Methods to study virus structures
Wavelength and diffraction
Wavelength comparison of X-rays and visible light
38l
Cellular organisms Viruses
dsDNA
ssRNA
Protein
Replication
Transcription
Translation
DNA
RNA Protein
25
Viruses are intracellular parasites
HIV replication cycle
“Virion” – virus
particle
Two forms of virus existence
Cell infection
Are viruses alive?
Pro-life Anti-life
Have genes
Do not have cellular
structure
They evolve No metabolism
Subject to natural
selection
Require host cell for pro-
creation
Procreate
New viruses assemble
from parts
28
Virus definition
A virus is a very small, non-cellular
parasite of cells. Its genome, which
is composed of either DNA or RNA,
is enclosed in a protein coat.
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
29
• discuss reasons for studying viruses
• explain how viruses differ from other
organisms
• define the term ‘virus’
Learning outcomes
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
30
Methods used in virology
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
31
Virus production in eggs
32
Virus production honeybee pupae
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
33
Virus production in vitro in tissue culture
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
34
Sterile work with tissue cultures
BSL levels
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
36
Plaque assay for animal viruses
HeLa 300 tis bb./ml, 10.2.14
Fixed after 72h
BEST OF CMC
HRV16 from stock 28.11.13,
PFU 5.103
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
38
Phage plaque assay
39
Differential centrifugation
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
40
Density gradient centrifugation
41
SDS-PAGE
Israeli acute bee paralysis virus
tartrate
gradient
CsCl
gradient CsCl gradient – middle fraction
1.F tartrate A260=133.083, A280 = 160.379
2.F tartrate A260=36.575, A280 = 28.762
3.F tartrate A260=86.795, A280 = 70.081
4.F tartrate A260=10.029, A280 = 6.267
1.F CsCl A260=111.613, A280 = 63.564
1.F CsCl A260=2.555, A280 = 1.556
1.F CsCl A260=6.836, A280 = 3.918
SDS-PAGE of IAPV
1. Virus purification
3. cryo-EM 4. Reconstruction
2. Grid preparation
45
Cryo-EM of phage phi812
1. Virus purification 2. Crystallization
3. Diffraction
data
4. Solve structure
Crystals of IAPV
0.1 M Cadmium Chloride
0.1 M Na acetate pH 4.5
15 % PEG 400
0.2 M Na/K Phosphate
0.1 M BisTris Propane pH 7.5
20 % PEG (w/v)3350
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
48
Hybridization detection of nucleic acids
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
49
Microarrays
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
50
rtPCR assay
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
51
TCID50 assay
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
52
Sequencing of virus genomes
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
53
• outline methods for
◦ cultivation of viruses;
◦ purification of viruses;
◦ detection of viruses and their
components;
◦ assay of virus infectivity;
◦ investigation of virus gene function;
• assess the value of virus genome sequencing.
Learning outcomes
Helical Icosahedral
Complex
Irregular
Virus structures
55
Virus structures
Picornavirus virion
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
62
Icosahedron
63
64
65
66
67
E18 Fab E19 Fab
69
70
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
71
72
73
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
74
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
75
76
77
78
79
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
80
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
81
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
82
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
83
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
84
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
85
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
86
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
87
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
88
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
89
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
90
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
91
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
92
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
93
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
94
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
95
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
96
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
97
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
98
© 2012 John Wiley & Sons Ltd.
www.wiley.com/college/carter
99