VIROLOGY

(viruses and non-chromosomal genetic elements)

VIRAL GENETICS

Mutation types :Biochemical characterization phenotypic expression

MUTATION FREQUENCIES OF VIRUSES Interaction between viruses and

between viruses and cellsphenotypic mixing

ReasortimentsHelper virusesInterference

restriction-modificationCRISP/Cas system

The lytic and lysogenic development cycle, immunityTransduction

VIRAL GENETICS

TYPES OF MUTATION:

single nucleotide replacement : transition or transversion misssense, nonsense or silent

insertion /deletion of nucleotidesrecombination genomic mutations:

translocations inversions deletions duplications

VIRAL GENETICS

Zero (silent) mutations:inactivating of the gene (nonsense, missense)

nonsense suppression

Temperature sensitivity (ts) mutation: conditionally lethal (missense) Host range mutations Plaque morphology, enzyme resistance mutations; “hot" mutants, attenuated mutants

E.coli sup D, E, F, P tRNS

amber UAG ser, glu, tyr, leuochre UAA (UCG) (CAA) (UAU) (UUG)opal UGA

J.W. Drake, B. Charlesworth, D. Charlesworth, J. F. CrowRates of Spontaneous MutationGenetics, Vol. 148, 1667-1686, 1998

MUTATION RATES

G – size of genome (bp); Ge – size of encoding genome;b – mutation rate per bp in a replication cycleg – mutation rate per genome in a replication cycleeg – mutation rate per genome equivalent encoding replication in a replication cycle

MUTATION RATES

MUTATION RATES

R.Sanjua, et al. (2004)The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus (VSV) PNAS, 101, 8396–8401

MUTATION OUTCOMES

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

REASSORTMENTof viruses with segmented genome

Opportunities for the development of vaccines using the reassortment of influenza virus genome

PHENOTYPIC MIXING

VIRAL GENETICS

VIRAL GENETICS

PHENOTYPIC MIXING

VIRAL GENETICS

PHENOTYPIC MIXING

PHENOTYPIC MIXING

VIRAL GENETICS

Helper viruses

VIRAL GENETICS

CHIMERIC VIRUS-LIKE PARTICLES

Interference

The defective particles compete for the coat proteins and inhibit the replication

VIRAL GENETICS

DNA–DNA hybridization

(Southern blotting)

From infected cells purified DNA Virion DNA

DNA zonde K DNA zonde S

Membrane Treatment - hybridization with a probe K

Ad12 5’-gala KpnI fragments, 589 b.p.

Virion DNA From infected cells purified DNA

DNA zonde K DNA zonde S

Membrane Treatment - hybridization with a probe S

+ 273 b.p. no Ad12 33845 - 34118

2x (+ 273 b.p. no Ad12 33845 – 34118)

3x (+ 273 b.p. no Ad12 33845 – 34118)

Ad12 3’-gala SacI fragments, 615 b.p.

What makes up the Ad 12 genome 3'-end "excess" sequence?

Restriction - modification

VIRAL GENETICS

CRISPR (clustered regularly interspaced short palindromic repeat)

Cas (CRISPR-associated) genes, CRISPR-based adaptive immune systems Terns and Terns, 2011

Bacterial defence against viral infections

CRISP-Cas

Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824, 2013

Novel approaches to genome modification

CRISP-Cas

Transfection

Protein unprotected viral delivery of genetic material in the cell (electroporation, liposomes, hydroxyapatite)

Transduction

Gene transfer with the help of virus

Specialized ( phage, gal, bio operons)

Non-specific (P1,P22 phage, 40-50 kbp. genomic fragments)

VIRAL GENETICS

Lysis / Lysogeny

Strategy Choice of the –phage replication

VIRAL GENETICS

Lysis / Lysogeny

VIRAL GENETICS

Genetic map of the lambda () phage

VIRAL GENETICS

http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%209.htm

Virulence / LysogenyVIRAL GENETICS

Lysis / Lysogeny

Early stages of the 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 - first, then rolling circle mechanism, specific cleavage in cos sequences, the separation of the sticky ends, assembling of phage

5. Lysis of bacterial cell

VIRAL GENETICS

cos site nucleotide sequence of the phage

teta () mechanism of DNA replication

Lambda () phage replication

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, mechanism), Q gene transcription {PR}

VIRAL GENETICS

THE EARLY STAGE OF INFECTION - A CHOICE

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

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.

Vīrusu ģenētika

Choice - INTEGRATION

Choice - INTEGRATIONVIRAL GENETICS

Choice - INTEGRATIONVIRAL GENETICS

att site nucleotide sequence of the phage

Choice - INTEGRATION

VIRAL GENETICS

Choice - INTEGRATIONVIRAL GENETICS

Choice - INTEGRATIONVIRAL GENETICS

Lysogenic cells:

•Contain phage genome integrated in the chromosome, the inactive state

•Immune to infection with the closely related phages

•Prophages can be activated by a variety of factors (UV, mutagenic, adverse environmental conditions)

PROPHAGES

Choice - INTEGRATIONVIRAL GENETICS

Gene expression in prophageVIRAL GENETICS

INDUCTION

VIRAL GENETICS

VIRAL GENETICS Choice – LYTIC CYCLE

DNA replication, rolling circle mechanism

Lambda () phage replication

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 to the rolling circle mechanism.

VIRAL GENETICS

Choice – LYTIC CYCLE

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

GENETIC SWITCH

OTHER E. coli LYSOGENE PHAGES

• phage-like – phages 21 f80, 82, 424, 434, crossimmunity;

• 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

TRANSDUCTION

Gene transfer with the help of LYSOGENE virus

Specialized ( phage, gal, bio operons)

Non-specific (P2 phage, 40-50 KBP. genomic fragments)

VIRAL GENETICS

SPECIFIC TRANSDUCTION

SPECIFIC TRANSDUCTION

NON-SPECIFIC (GENERAL) TRANSDUCTION

NON-SPECIFIC (GENERAL) TRANSDUCTION

NON-SPECIFIC (GENERAL) TRANSDUCTION

http://bio.classes.ucsc.edu/bio105l/EXERCISES/P1/masters.pdf

NON-SPECIFIC (GENERAL)

TRANSDUCTION