VIROLOGY
(viruses and non-chromosomal
genetic elements)
VIRAL GENETICS
VIRAL GENETICS
Mutation types :
Biochemical characterization
phenotypic expression
MUTATION FREQUENCIES OF VIRUSES
Interaction between viruses and
between viruses and cells
phenotypic mixing
Reasortiments
Helper viruses
Interference
restriction-modification
CRISP/Cas system
The lytic and lysogenic development
cycle, immunity
Transduction
TYPES OF MUTATION:
single nucleotide replacement :
transition or transversion
misssense, nonsense or silent
insertion /deletion of nucleotides
recombination
genomic mutations:
translocations
inversions
deletions
duplications
VIRAL GENETICS
Zero (silent) mutations:
inactivating of the gene (nonsense, missense)
nonsense suppression
E.coli
sup
amber
ochre
opal
UAG ser, glu, tyr, leu
UAA (UCG) (CAA) (UAU) (UUG)
UGA
D,
E,
F,
P
tRNS
Temperature sensitivity (ts) mutation: conditionally
lethal (missense)
Host range mutations
Plaque morphology,
enzyme resistance mutations; “hot" mutants,
attenuated mutants
MUTATION RATES
G – size of genome (bp);
Ge – size of encoding genome;
mb – mutation rate per bp in a replication cycle
mg – mutation rate per genome in a replication cycle
meg – mutation rate per genome equivalent encoding replication in
a replication cycle
J.W. Drake, B. Charlesworth, D. Charlesworth, J. F. Crow
Rates of Spontaneous Mutation
Genetics, Vol. 148, 1667-1686, 1998
MUTATION RATES
MUTATION RATES
MUTATION OUTCOMES
R.Sanjua, et al. (2004)The distribution of fitness effects caused by singlenucleotide substitutions in an RNA virus (VSV) PNAS, 101, 8396–8401
HOMOLOGOUS RECOMBINATION
The mechanism of copy
choice in the replication of
viruses
The mechanism of strand exchange in
replication of eucariot cells
Mapping genomes, Marker rescue, Inclusion of host cell genome
fragments into virus
REASSORTMENT
of viruses with segmented genome
Opportunities for the
development of
vaccines using the
reassortment of
influenza virus genome
VIRAL GENETICS
PHENOTYPIC MIXING
VIRAL GENETICS
PHENOTYPIC MIXING
VIRAL GENETICS
PHENOTYPIC MIXING
VIRAL GENETICS
PHENOTYPIC MIXING
VIRAL GENETICS
Helper viruses
CHIMERIC VIRUS-LIKE PARTICLES
VIRAL GENETICS
Interference
The defective particles compete for the coat proteins
and inhibit the replication
DNA–DNA hybridization
(Southern blotting)
DNA zonde K
DNA zonde S
Membrane Treatment - hybridization with a probe K
Ad12 5’-gala KpnI
fragments, 589 b.p.
From infected cells purified DNA
Virion DNA
DNA zonde K
DNA zonde S
Membrane Treatment - hybridization with a probe S
3x (+ 273 b.p. no
Ad12 33845 – 34118)
2x (+ 273 b.p. no Ad12
33845 – 34118)
+ 273 b.p. no Ad12
33845 - 34118
Ad12 3’-gala SacI
fragments, 615 b.p.
Virion DNA
From infected cells purified DNA
What makes up the Ad 12 genome 3'-end "excess"
sequence?
VIRAL GENETICS
Restriction - modification
Bacterial defence against viral infections
CRISP-Cas
CRISPR (clustered regularly interspaced short palindromic repeat)
Cas (CRISPR-associated) genes,
CRISPR-based adaptive immune systems Terns and Terns, 2011
Novel approaches to genome modification
CRISP-Cas
Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824,
2013
VIRAL GENETICS
Transfection
Protein unprotected viral delivery of genetic
material in the cell (electroporation, liposomes,
hydroxyapatite)
Transduction
Gene transfer with the help of virus
Specialized (l phage, gal, bio operons)
Non-specific (P1,P22 phage, 40-50 kbp. genomic
fragments)
VIRAL GENETICS
Lysis / Lysogeny
Strategy Choice of the l–phage replication
VIRAL GENETICS
Lysis / Lysogeny
VIRAL GENETICS
Genetic map of the lambda (l) phage
http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%209.htm
VIRAL GENETICS
Virulence / Lysogeny
VIRAL GENETICS
Lysis / Lysogeny
Early stages of the l infection:
1. Adsorption to the cell receptor (maltose
transport protein)
2. DNA injection, cos sequence – the union of
the sticky ends and ligase
3. Transcription - immediate early, delayed
early, late genes
4. Replication - Q first, then rolling circle
mechanism, specific cleavage in cos
sequences, the separation of the sticky
ends, assembling of phage
5. Lysis of bacterial cell
cos site nucleotide sequence of the l phage
Lambda (l) phage replication
teta (Q) mechanism of DNA replication
VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
1. Weak transcription from PL and PR.
Antitermination protein N that interacts with RNA
polymerase and promotes transcription in both
directions is formed. Cro regulatory protein that
promotes transkription of PR is formed.
2. N promotes CIII (CII stabilizer) {PL}; as well
as CII (CI stimulator) O, P, (DNA synthesis, Q
mechanism), Q gene transcription {PR}
VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
http://biology.bard.edu/ferguson/course/bio404/Lecture_08.pdf
VIRAL GENETICS
THE EARLY STAGE OF INFECTION - A CHOICE
Vīrusu ģenētika
Choice - INTEGRATION
LYSOGENY. CII activates the PRE (CI
synthesis starts) and PI (integrase). Formed
CI, which extorts Cro from PL and PR,
activates PRM
Int promotes attP and attB interaction and a
fusion of DNA of phage with the DNA of
bacteria.
VIRAL GENETICS
Choice - INTEGRATION
VIRAL GENETICS
Choice - INTEGRATION
VIRAL GENETICS
Choice - INTEGRATION
att site nucleotide sequence of the l phage
VIRAL GENETICS
Choice - INTEGRATION
VIRAL GENETICS
Choice - INTEGRATION
VIRAL GENETICS
Choice - INTEGRATION
Lysogenic cells:
• Contain l phage genome integrated in the
chromosome, the inactive state
• Immune to infection with the closely related
phages
PROPHAGES
• Prophages can be activated by a variety of
factors (UV, mutagenic, adverse environmental
conditions)
VIRAL GENETICS
Gene expression in prophage
VIRAL GENETICS
INDUCTION
VIRAL GENETICS
Choice – LYTIC CYCLE
Lambda (l) phage replication
DNA replication, rolling circle mechanism
VIRAL GENETICS
Choice – LYTIC CYCLE
LYSE. If there is enough Cro, CI synthesis is
blocked (first), but later the PL and PR in
general. Decisive role is played by PR’ in
context with Q antitermination, that runs a
phage capcid protein and lysis protein
synthesis.
DNA synthesis moves from Q to the rolling
circle mechanism.
GENETIC SWITCH
GENETIC
SWITCH
O1, 2, 3 sequences are
similar but not identical;
CI has the best affinity to
O1, the weakest – to O3.
Cro - best to the O3.
In average, CI binds to
the operator sites approx.
5 times more efficient
than the Cro
GENETIC SWITCH
OTHER E. coli LYSOGENE PHAGES
• l phage-like
crossimmunity;
–
phages
21
f80,
82,
424,
434,
• P1, the largest lysogene phage, 97 kbp. DNA rarely
integrates - more present in plasmid form of Cre protein and
loxP recombination site, 40% of the DNA filling required for
aggregation, non-specific transduction;
• Mu, 42 kbp. DNA, at the ends of phage genome – bacteria
sequence, effective transposon, mutation induction;
• P2, 33,2 kbp. DNA, approx. 10 integration sites in the
genome of bacteria, lysis is rare. P2 encoded capsid proteins
can be used for P4 (11 kpb. DNA) incapsidation, which in P2
free cells are in multicopy plasmid form
VIRAL GENETICS
TRANSDUCTION
Gene transfer with the help of LYSOGENE virus
Specialized (l phage, gal, bio operons)
Non-specific (P2 phage, 40-50 KBP. genomic
fragments)
SPECIFIC TRANSDUCTION
SPECIFIC TRANSDUCTION
NON-SPECIFIC (GENERAL) TRANSDUCTION
NON-SPECIFIC (GENERAL) TRANSDUCTION
NON-SPECIFIC (GENERAL) TRANSDUCTION
NON-SPECIFIC
(GENERAL)
TRANSDUCTION
http://bio.classes.ucsc.e
du/bio105l/EXERCISES/
P1/masters.pdf