VIROLOGY viruses and nonchromosomal genetic elements VIRAL GENETICS

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 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 t. RNS 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 Ad 12 5’-gala Kpn. I fragments, 589 b. p. From infected cells purified DNA Virion DNA

DNA zonde K DNA zonde S Membrane Treatment - hybridization with a probe S 3 x (+ 273 b. p. no Ad 12 33845 – 34118) 2 x (+ 273 b. p. no Ad 12 33845 – 34118) + 273 b. p. no Ad 12 33845 - 34118 Ad 12 3’-gala Sac. I 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 Cas 9. Science, V 339, 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 (P 1, P 22 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/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 - 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 ( ) 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, mechanism), Q gene transcription {PR}

VIRAL GENETICS THE EARLY STAGE OF INFECTION - A CHOICE http: //biology. bard. edu/ferguson/course/bio 404/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 att. P and att. B 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 to the rolling circle mechanism.

GENETIC SWITCH

GENETIC SWITCH O 1, 2, 3 sequences are similar but not identical; CI has the best affinity to O 1, the weakest – to O 3. Cro - best to the O 3. 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 – phages 21 f 80, 82, 424, 434, crossimmunity; • P 1, the largest lysogene phage, 97 kbp. DNA rarely integrates - more present in plasmid form of Cre protein and lox. P 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; • P 2, 33, 2 kbp. DNA, approx. 10 integration sites in the genome of bacteria, lysis is rare. P 2 encoded capsid proteins can be used for P 4 (11 kpb. DNA) incapsidation, which in P 2 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 (P 2 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. ed u/bio 105 l/EXERCISES/P 1 /masters. pdf