Lesson 4 Molecular 1. Introduction

1.1. Definition

Genetic elements made of living matter molecules, that are capable of growth and multiplication only in living cells

DIFFERENCE WITH RESPECT TO OTHER GENETIC ELEMENTS:

“They can exist in an extracellular phase that allows their spread”

The extracellular form of the virus (i.e. virion) is metabolically inactive Introduction 1.2. The viral

ssDNA dsDNA ssRNA dsRNA

Genome variation (Phase)

Circular or Linear / A single molecule or Segmented

Size: 3-200kb (Phage G, 670 kb)

Gene compaction (overlapping …) Introduction 1.3.

1. Depending on the Prokaryotic : Bacterial viruses = or phages; Archaeal viruses Eukaryotic viruses: protozoan- , fungi-, plant- , animal-viruses

2. Formal taxonomy: Taxonomic ranks (taxons): order , family, subfamily, and . FAMILY: unique virion morphology, genome structure (replication strategies). e.g. Ortomixoviridae, ,

3. : Type of genome and its method of replicaton Group I: dsDNA (except Group VII) Group II: ssDNA Group III: dsRNA Group IV: ssRNA , positive-sense Group V: ssRNA, negative -sense Group VI: Positive-sense ssRNA viruses that replicate through a DNA intermediate Group VII: dsDNA viruses that replicate through a ssRNA intermediate Introduction 1.4. The replication cycle of viruses

1. Attachment (adsorption)

2. Penetration (injection)

Latency?

3. Synthesis of nucleic acids and

4. Assembly and packaging

5. Release () Introduction 1.5. Attachment or Adsorption

Interaction between proteins on the outside of the virus and cell receptors

Receptors : proteins, carbohydrates, glycoproteins, , lipoproteins or complexes

Carry out normal functions in the cell. e.g., Lambda-MalT

Related viruses may not share receptors

The presence of receptors determine which cells are susceptible:

• Species • (phage typing) • Cell type • Tissue

Examples: M13 / E. coli F+

Influenza virus/ Mucous membrane, red blood cells (Sialic acid)

Measles / Any human cell, except red blood cells (CD46) 2. Prokaryotic Viruses

ssRNA+ MS2 dsRNA

ssDNA ØX174 M13

dsDNA***

T7 T4 Mu Lambda Prokaryotic Viruses 2.1. RNA phages

MS2 Coliphage

Expression (Transcriptional regulation is not possible )

Temporal programme based on accessibility to AUG codons (RNA structure):

• The more accessible AUG is that of C (translated along the infection cycle)

• Access to the AUG of P is limited. of C grants access to the AUG of P

• C binds to AUG of P and inhibits its translation

• The AUG of A is accessible only in nascent RNA+ molecules

• The AUG of L is partially accessible only when reach the stop of C Prokaryotic Viruses 2.2. ssDNA Phages (Group II)

ssDNA +: replication must precede

Filamentous: M13 Icosahedral: ØX174 Prokaryotic Viruses 2.3. dsDNA Phages (Group I)

• T-uneven phages: T7 • T-even phages: T4 • Mu phage Prokaryotic Viruses dsDNA Viruses T7 Phage

Strategies to maximize genetic economy

• overlap • Internal translational reinitiation • Internal frame-shifts

Genome : TEMPORAL EXPRESSION PROFILE: Injection of the genome / Order of genes Linear Left end .: Early genes 40kb RM system inhibitor T7 RNA polymerase Direct terminal repeat of 160 bp Inh. Cell. RNA Inh. Cell Expr First to penetrate Use the host RNA polymerase Replication Middle region : Intermediate genes Bidirectional Use T7 RNA polymerase

One origin (closer to left end) Right end : Late genes Use T7 RNA polymerase Prokaryotic Viruses dsDNA Viruses

T4 Phage

Lytic Linear genome (169 kb) Complex • Head • Neck (with collar) • Tail (tube + sheath) • End plate • Tail fibers Prokaryotic Viruses dsDNA Viruses: T4

TEMPORAL EXPRESSION PROFILE

Sequential modification of the host RNA polymerase specificity:

1. Early genes: RNApol-σ 70 2. Intermediate genes: Modification of RNApol α subunit + phage proteins binding 3. Late genes: RNApol-σ Phage

Replication Linear Genome: Ends? - Form concatamers through recombination Terminal repeats (3-6 Kb) - Sequence-independent Endonuclease

- Regularly spaced cuts (> gene dotation)

- Repeated ends

- Permuted end sequences Prokaryotic Viruses dsDNA Viruses The Mu

Temperate Linear genome Replication through transposition

Integration is necessary both for lysis and lysogeny

Lysogeny Non-replicative Transposition C Protein Inhibits Transposase (repressor)

Lytic cycle Replicative Transposition

No repression Prokaryotic Viruses dsDNA Viruses: Mu

Packaging

50-150 bp Mu=37 kb

1-2 kb

Eukaryotic Viruses 3.1. Positive-strand RNA

Picornaviridae Family

Polioviruses

Rhinoviruses

Hepatitis A virus (HAV) Eukaryotic Viruses Positive strand RNA Viruses

Picornaviridae

- pico means small

- Naked

- Icosahedral

- ssRNA + Eukaryotic Viruses Positive strand RNA Viruses Fam. Picornaviridae

- Capsid: 4 proteins, (VP1- 4) x 60

- Intracellular cycle in the cytoplasm

- Genome:

3’ –terminus poly-A tail (copied during replication)

Absence of 5’ cap (Vpg protein)

Vpg AAAAAA

7.5 kb - Polymerase is not present in the virion

- Translation of viral proteins must take place before replication Eukaryotic Viruses Positive strand RNA Viruses: Poliovirus

Degradation of EIF-4B (binding of the to the methylguanosine cap)

Translation of host proteins is blocked

Replication

Requires priming!! : Vpg - UU

Expression 1 (6620 nt) 1 Polyprotein (proteolytic activity)

Equal amount of every (anti-economic regulation) Eukaryotic Viruses 3.2. Negative-strand RNA viruses

- Ortomyxovirus - Rhabdovirus - virus Eukaryotic Viruses Negative strand RNA Viruses

Ortomyxovirus Fam. Ortomyxoviridae virus mixo = mucus

New variants (): Epidemics

Nucleocapsid: N Protein Transcriptase complex (endonuclease)

Envelope: M Protein Hemagglutinin

Genome: Segmented in 8 RNA fragments Eukaryotic Viruses Negative strand RNA Viruses: Ortomyxovirus

Transcriptase requires 5’ priming to initiate transcription: “stolen” caps

Poly-A: Polymerase slippage

Host’s mRNA Viral RNA Endonuclease 5’-G 5’-G

5’-G Primer 5’-G AAAAAAA Eukaryotic Viruses 3.3. dsRNA: Family

REO: Respiratory Enteric Orphan

Rotavirus: Childhood Diarrhea

Naked Double-shell Icosahedral capsid (Transcriptase)

Segmented Genome (10-12 dsRNA)

Facilitates unwinding (replication)

Antigenic shift Eukaryotic Viruses 3.4. DNA Viruses

Pros The virus can utilize the cellular transcription machinery

The virus con utilize part or all the replication machinery of the cell

Cons

Most cells in an are in a non-division stage

DNA in eukaryotic cells only replicates during the S-phase

ssDNA Parvovirus

dsDNA Papovaviridae (SV40) Poxviridae Eukaryotic Viruses

3.4.1. ssDNA

Parvovirus

Genome: 4,5- 5 Kb (2 genes)

3’ Terminal hairpin Replication primer

Depends on the replication machinery of the host cell

Infect tumor cells or those infected with other viruses (e.g., Adenovirus) Eukaryotic Viruses

3.4.2. dsDNA Viruses

Adenovirus Herpesvirus Poxvirus Eukaryotic Viruses dsDNA Viruses Adenovirus Adenoviridae

Adeno : Gland

Genome

TP protein Linear of 36 kb

Inverted terminal repeats (100-1800 pb)

Naked Icosahedral virion - Replication and assembly in the nucleus

- DNA is associated with histones Eukaryotic Viruses dsDNA Viruses: Adenovirus

Replication

- Virus-encoded replication proteins (DNA polymerase)

- Primer : pTP-C

- Initiates at either end:

No Okazaki fragments

Asynchronously

ssDNA strand

Cyclization, DBPs Eukaryotic Viruses dsDNA Viruses Herpesvirus Herpesviridae

Herpes simplex Varicella-Zoster Epstein-Barr virus

Latent in neurons of the sensory ganglia

Multilayer Virion

Linear Genome of 150 kb Envelope with spikes

Amorphous Tegument

Icosahedral Nucleocapsid Eukaryotic Viruses dsDNA Viruses: Herpesvirus

TRANSCRIPTION

α Genes (immediate early) - β Genes (delayed early) + γ Genes (late) + -

REPLICATION

In the nucleus

Circularizes

Rolling circle replication

Concatamers

ASSEMBLY

In the nucleus

Envelope via budding of the nucleus inner membrane

Release through the endoplasmic reticulum dsDNA Viruses

Poxvirus Poxviridae

- The largest animal viruses (300nm Ø):

Smallpox, , …

- Replication IN THE CYTOPLASM

VIRION Proteins envelope

Genome : 150-200 kb Direct terminal repeat of 10 kb Covalently closed Replication?? Eukaryotic Viruses 3.5. Group VI: Retroviridae

Rous sarcoma Virus (RSV) Envelope Human T-cell leukemia Virus (HTLV) Avian Myeloblastosis Virus (AMV) AIDS Virus (HIV) Glycoproteins

Icosahedral Virion

2 copies of the genome (ssRNA +) Genome

2 copies of ssRNA + of 7-10 kb

Nucleocapsid Direct terminal repeats Retrotranscriptase Cap and Poly-A tRNA from the previous host Not translated!! Eukaryotic Viruses

ssRNA +

Retrotranscription

dsDNA in cytoplasm

Integration in the genome

Transcription:

ssRNA + (, mRNA)

THE 4 activities : - RNA-dependent DNA polymerase - DNA-dependent DNA polymerase - RNase H, degrades RNA in DNA-RNA hydrids - Endonuclease Eukaryotic Viruses Retrovirus Retrovirus cccDNA

Viral Integrase Host DNA

LTR gag pol LTR

ssRNA + (mRNA, Genomes) Eukaryotic Viruses Retrovirus

Expression (1 RNA = Genome)

GENE ORGANIZATION

Three regions:

gag (capsid proteins + protease) Polyprotein pol ( Retrotranscriptase and Integrase)

env ( Envelope glycoproteins)