Lesson 4

Molecular Virology

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

1.1. Virus Definition

1. Introduction

1.2. The viral genome

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 genes …)

Introduction

1.3. Virus Classification1. Depending on the host

Prokaryotic viruses: Bacterial viruses = bacteriophages or phages; Archaeal viruses

Eukaryotic viruses: protozoan- , fungi-, plant- , animal-viruses

2. Formal taxonomy:

Taxonomic ranks (taxons): order , family, subfamily, genus and species.

FAMILY: unique virion morphology, genome structure (replication strategies).

e.g. Ortomixoviridae, Hepadnaviridae, Poxviridae

3. Baltimore classification: 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

2. Penetration (injection)

3. Synthesis of nucleic acids and proteins

4. Assembly and packaging

5. Release (lysis)

1. Attachment (adsorption)

Latency?

Introduction

1.5. Attachment or Adsorption

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

Receptors: proteins, carbohydrates, glycoproteins, lipids, 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• Strain (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)

Introduction

2. Prokaryotic Viruses

ssRNA+

dsRNA

ssDNA

dsDNA***

MS2

ØX174

M13

T7

T4

Mu

Lambda

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. Translation of C grants access to the AUG of P

• Protein 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 ribosomes reach the stop of C

2.1. RNA phages

Prokaryotic Viruses

MS2 Coliphage

2.2. ssDNA Phages (Group II)

ssDNA +: replication must precede transcription

Icosahedral: ØX174Filamentous: M13

Prokaryotic Viruses

2.3. dsDNA Phages (Group I)

• T-uneven phages: T7

• T-even phages: T4

• Mu phage

Prokaryotic Viruses

T7 Phage

Strategies to maximize genetic economyStrategies to maximize genetic economyStrategies to maximize genetic economyStrategies to maximize genetic economy

• Gene overlap

• Internal translational reinitiation

• Internal frame-shifts

TEMPORAL EXPRESSION PROFILE:

Injection of the genome / Order of genes

Left end.: Early genes

RM system inhibitor

T7 RNA polymerase

Inh. Cell. RNA pol Inh. Cell Expr

First to penetrate

Use the host RNA polymerase

Middle region: Intermediate genes

Use T7 RNA polymerase

Right end: Late genes

Use T7 RNA polymerase

Genome:

Linear

40kb

Direct terminal repeat of 160 bp

Replication

Bidirectional

One origin (closer to left end)

Prokaryotic Viruses dsDNA Viruses

T4 Phage

Lytic

Linear genome (169 kb)

Complex capsid

• Head

• Neck (with collar)

• Tail (tube + sheath)

• End plate

• Tail fibers

Prokaryotic Viruses dsDNA Viruses

Linear Genome: Ends?

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

- Form concatamers through recombination

- Sequence-independent Endonuclease

- Regularly spaced cuts (> gene dotation)

- Repeated ends

- Permuted end sequences

Terminal repeats (3-6 Kb)

ReplicationReplicationReplicationReplication

Prokaryotic Viruses dsDNA Viruses: T4

The Mu Bacteriophage

Temperate

Linear genome

Replication through transposition

Integration is necessary both for lysis and lysogeny

Lysogeny

C Protein

(repressor)Inhibits Transposase

Non-replicative Transposition

Replicative TranspositionLytic cycle

No repression

Prokaryotic Viruses dsDNA Viruses

50-150 bp Mu=37 kb

1-2 kb

Packaging

Prokaryotic Viruses dsDNA Viruses: Mu

3.1. Positive-strand RNA

Picornaviridae Family

Polioviruses

Rhinoviruses

Hepatitis A virus (HAV)

Eukaryotic Viruses

Picornaviridae

- pico means small

- Naked

- Icosahedral

- ssRNA +

Eukaryotic Viruses Positive strand RNA Viruses

PoliovirusFam. 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)

AAAAAA

Vpg

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

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

Equal amount of every viral protein(anti-economic regulation)

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

Translation of host proteins is blocked

Replication

Requires priming!!: Vpg - UU

Expression

Eukaryotic Viruses Positive strand RNA Viruses: Poliovirus

3.2. Negative-strand RNA viruses

- Ortomyxovirus

- Rhabdovirus

- Ebola virus

Eukaryotic Viruses

Ortomyxovirus Fam. Ortomyxoviridae

Influenza virus

New variants (antigenic shift): Epidemics

mixo = mucus

Nucleocapsid:

N Protein

Transcriptase complex (endonuclease)

Envelope: M Protein

Hemagglutinin

Neuraminidase

Genome: Segmented in 8 RNA fragments

Eukaryotic Viruses Negative strand RNA Viruses

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

Poly-A: Polymerase slippage

5’-GEndonuclease

Host’s mRNA

5’-GPrimer

5’-G

Viral RNA

5’-G AAAAAAA

Eukaryotic Viruses Negative strand RNA Viruses: Ortomyxovirus

3.3. dsRNA: Reoviridae Family

Eukaryotic Viruses

Naked

Double-shell Icosahedral capsid (Transcriptase)

Segmented Genome (10-12 dsRNA)

Antigenic shift

REO: Respiratory Enteric Orphan

Rotavirus: Childhood Diarrhea

Facilitates unwinding (replication)

ProsThe 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 organisms are in a non-division stage

DNA in eukaryotic cells only replicates during the S-phase

3.4. DNA Viruses

ssDNA Parvovirus

dsDNA Papovaviridae (SV40)

Papillomaviridae

Adenoviridae

Herpesviridae

Poxviridae

Eukaryotic Viruses

3.4.1. ssDNA

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

Parvovirus Parvoviridae

3.4.2. dsDNA Viruses

Adenovirus

Herpesvirus

Poxvirus

Eukaryotic Viruses

Adenovirus Adenoviridae

Naked Icosahedral virion

Linear of 36 kb

Inverted terminal repeats(100-1800 pb)

- Replication and assembly in the nucleus

- DNA is associated with histones

TP protein

Adeno : Gland

Genome

Eukaryotic Viruses dsDNA Viruses

- Virus-encoded replication proteins(DNA polymerase)

- Primer: pTP-C

- Initiates at either end:

No Okazaki fragments

Asynchronously

Replication

ssDNA strand

Cyclization, DBPs

Eukaryotic Viruses dsDNA Viruses: Adenovirus

Herpesvirus Herpesviridae

Herpes simplex

Varicella-Zoster

Epstein-Barr virus

Multilayer Virion

Envelope with spikes

Amorphous Tegument

Icosahedral Nucleocapsid

Linear Genome of 150 kb

Latent in neurons of the sensory ganglia

Eukaryotic Viruses dsDNA Viruses

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

TRANSCRIPTION

α Genes (immediate early)

β Genes (delayed early)

γ Genes (late)

+-

-+

Eukaryotic Viruses dsDNA Viruses: Herpesvirus

Poxvirus Poxviridae

- The largest animal viruses (300nm Ø):

Smallpox, Cowpox, Myxomatosis…

- Replication IN THE CYTOPLASM

VIRION

Proteins envelope

Genome:

150-200 kb Direct terminal repeat of 10 kbCovalently closed Replication??

dsDNA Viruses

3.5. Group VI: Retroviridae

Rous sarcoma Virus (RSV)

Human T-cell leukemia Virus (HTLV)

Avian Myeloblastosis Virus (AMV)

AIDS Virus (HIV)

Genome

2 copies of ssRNA + of 7-10 kb

Direct terminal repeats

Cap and Poly-A

Not translated!!

Envelope

Glycoproteins

Icosahedral Virion

2 copies of the genome (ssRNA +)

NucleocapsidRetrotranscriptase

Integrase

tRNA from the previous host

Eukaryotic Viruses

ssRNA +

Retrotranscription

dsDNA in cytoplasm

Integration in the genome

Transcription:

ssRNA + (genomes, mRNA)

THE REVERSE TRANSCRIPTASE

4 activities :

- RNA-dependent DNA polymerase

- DNA-dependent DNA polymerase

- RNase H, degrades RNA in DNA-RNA hydrids

- Endonuclease

Eukaryotic Viruses Retrovirus

LTR LTRgag pol env

Retrovirus

cccDNA

Host DNA

Viral

Integrase

ssRNA +

(mRNA, Genomes)

Eukaryotic Viruses Retrovirus

GENE ORGANIZATION

Three regions:

gag (capsid proteins + protease)

pol ( Retrotranscriptase and Integrase)

env ( Envelope glycoproteins)

Expression (1 RNA = Genome)

Polyprotein

Eukaryotic Viruses Retrovirus