viruses. what is a virus ? viruses are submicroscopic, obligate intracellular parasites...

Viruses

What is a virus ?

• Viruses are submicroscopic, obligate intracellular parasites

• Morphologically, virus particle is a protein shell, in which the viral genome is enclosed

• Virus particles are produced from the assembly of pre-formed components and do not grow or undergo division

• Viruses lack the genetic information which encodes apparatus necessary for the generation of metabolic energy and for protein synthesis

Origins of Viruses• How did these become independent genetic entities? The only

absolute requirement is an origin of replication in the nucleic acid.

• Regressive theory: viruses are degenerate forms of intracellular parasites. The leprosy bacillus, rickettsiae and chlamydia have all evolved in this direction. Mitochondria and chloroplasts are often suggested to have been derived from intracellular parasites. However viruses do not have their own rRNAs or protein synthesis machinery. Also begs the question of RNA virus evolution ?

• Progressive theory: Cellular RNA and DNA components: Normal cellular nucleic acids that gained the ability to replicate autonomously and therefore to evolve. DNA viruses came from plasmids or transposable elements. They then evolved coat proteins and transmissibility. Retroviruses derived from retrotransposons and RNA virus from mRNA.

• Coevolution theory: Viruses coevolved with life – their evolotion might go all the way back to RNA world!

• All of these could be correct! No compelling reason to think that RNA viruses have evolved in the same way as DNA viruses

The particleCommon things to all

virus particles:

1. It encloses genomic nucleic acid

2. It is a polymer, assembled from one or few different kinds of monomers

The nucleocapsid

• Nucleocapsid is the viral nuleic acid, enclosed in the protein shell

• In case of simple non-enveloped viruses nucleocaspsid and virus particle is the same thing

• In enveloped viruses, lipid bilayer of cellular origin encloses the nucleocapsid

Enveloped and non-enveloped viruses

• Non-enveloped virus • Enveloped virus

DNA or RNA genome

Nucleocapsid

Lipid bilayer

Envelope protein

Matrix protein

Two different kinds of nucleocapsids

• Filamentous • Icosahedral

The helical geometry of filamentous virus TMV (Tobacco mosaic virus)

Pitch of helix 22.8 Å

p=1.4Å (axial rise per subunit)

=16.3 (subunits per helix turn)

The geometry of icosahedral viruses

• Due to geometrical constraints, there are 60 equivalent environments in icosahedron. This means, that icosahedron can be made of 60 equivalent subunits

• Most icosahedral viruses, however are made of more than 60 subunits, making quasi-equvalent contacts.

• The number of subunits is always a certain multiple of 60, called a triangulation (‘T’) number. For example, T=3 virus will have 180 subunits, T=7 virus 7x60=420 subunits. Only certain T values are allowed (1, 3, 4,7, 9, 13, 16,19,21,25...).

T=1 (60 subunits)

T=3 (180 subunits)

T=4 (240 subunits)

The blue triangle represents one face of icosahedron

Structure of T=3 icosahedral bacteriophage MS2

The three subunits A, B and C are in slighly different conformations. A and C subunits are clustered around 3- fold axes, forming hexamers, whereas B subunits gather around 5-fold axes, forming pentamers. Note the differnt conformation of FG loops for A, B and C subunits

FG loops

The genomes• I: Double-stranded DNA. Examples: Adenoviruses, Herpesviruses,

Papillomaviruses, Poxiviruses, T4 bacteriophageSome replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in the cytoplasm

• II: Single-stranded (+)sense DNA. Examples: phage M13, chicken anaemia virus, maize streak virusReplication occurs in the nucleus, involving the formation of a (-)sense strand, which serves as a template for (+)strand RNA and DNA synthesis.

• III: Double-stranded RNA. Examples: Reoviruses, RotaviruesThese viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs.

• IV: Single-stranded (+)sense RNA Examples: Hepatitis A and C, Small RNA phages, common cold viruses, SARSa) Polycistronic mRNA e.g. Picornaviruses; Hepatitis A. Genome RNA = mRNA. Means naked RNA is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins.b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA.

• V: Single-stranded (-)sense RNA. Examples: Influenza viruses, HantavirusesMust have a virion particle, containing RNA directed RNA polymerase.a) Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for genome replication.b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced.

• VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses). Examples: HIV, Avian leukosis virusGenome is (+)sense but unique among viruses in that it is DIPLOID, and does not serve as mRNA, but as a template for reverse transcription.

• VII: Partial double-stranded (gapped) DNA with RNA intermediate (Hepadnaviruses) Example: Hepatitis B This group of viruses also relies on reverse transcription, but unlike the Retroviruses, this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription.

Simple and complex genomes and particles

Phage MS2

Genome: linear +ssRNA 3400 nt, 4 ORFs, 4 proteins (A for receptor binding, C for coat, L for lysis and R for polymerase)

Particle: Genome encapsidated in a single layer coat protein shell

A C R

L

Hepatitis B virusGenome: partial dsDNA 3200 bp, 4ORFs, 6 proteins(S, preS for envelope, C for core, P for polymerase and X for transcription factor)

Particle: Genome with polymerase encapsidated in double-layer protein shell. The outer shell is composed of multiple copies of S, M (S+preS2) and L (S+preS1+preS2) proteins

AdenovirusGenome: linear ds DNA, 35 000 bp, 40 genesParticle: 10 proteins, single layer

Phage T4: complicated genome & particle

Mimivirus: the biggest known genome and particle• Genome: 1,181,404 nt, codes for 1262 proteins• Some proteins are involved in protein synthesis, thus violating one criterium in a definition of “what is a virus”• Infects amoebae

E.coli (4,600,000 bp, 4377 proteins)

Burrelia burgdorferi (1,440,000 bp, 1738 proteins)

Chlamydia trachomatis (1,040,000 bp, 936 proteins)

Mimivirus (1,180,000 bp, 1262 proteins)

T4 phage (173,000 bp, 280 proteins )

Adenovirus (35,000 bp, 40 proteins)

Hepatitis B (3,200 bp, 4 proteins)

Electron micrograph of mimivirus particle and comparison with sizes of other viruses

Smallest viruses

Hepatitis B

Adenovirus

T4 phage head

E.coli

Poxivirus

The viral life cycle•Initation phase:a) Attachment to the host cell receptor (Ig like receptors, cellular adhesion molecules, membrane transport proteins, oligosaccharides, etc)b) Penetration (endocytosis, fusion) c) Uncoating

Most bacteriophages avoid penetration and uncoating stages by injecting the viral nucleic acid into the cellPlant viruses do not use specific receptors and enter the cell either through insect vectors or mechanically damaged parts of plant

Some viruses initiate direct cell fusing. In this process infected cell is fused with uninfected.

Initiation phase

Non-enveloped virus

Enveloped virus

Movie: animal virus penetration by fusion

Movie: DNA injection in E.coli by T4 bacteriophage

The viral life cycle

• Replication phase

a) nucleic acid replication

b) mRNA synthesis

c) protein expression

d) assembly

The viral life cycle

• Release phase

a) exit from cell (lysis, exocytosis, budding)

b) maturation (rearrangement of nucleocapsid, etc)

In enveloped viruses assembly can be coupled to release

Examples of viral life cycles

• Small RNA phages

• HIV

• Influenza

Initiation phase of small RNA phages

Bacterial pili

Transport of genomic RNA into cytoplasm

Attachment

A protein

Replication and release

Translation of viral proteins

Lysis Coat Replicase

Assembly

Release

A

HIV• Retroviridae, Lentivirus (~10 kb diploid

+ssRNA genome)

The life cycle of HIV

HIV maturation

During HIV maturation, structural polyproteins are cleaved in active units. This causes rearangement of virion structure and makes the particle infectious.

Influenza virus• (-)sense segmented

RNA genome• enveloped, a bit

irregular particle• 2 types of spike

proteins (neuraminidase and hemagglutinin)

• Infects birds and mammals

Life cycle of influenza virus

Should we be scared of that avian H5N1 influenza?

• Maybe...• In 1918 influenza of porcine origin killed around 40, 000,

000 people worldwide• H5N1 influenza is not easily transmitted to humans and certainly

not from one human to another• The problem might arise when a chimeric human-avian virus

arises by exchanging genomic RNA segments• That one could be as lethal as avian and as easily transmitted as

human• Most probably H5N1 will not become pandemic, but some day we

will certainly see something similar to 1918...

Virus life cycle and antiviral therapy

• In antiviral therapy, any vital step in viral life cycle can be blocked

• Frequent targets are viral polymerases (HIV, herpes, HCV and others)

• Other targets include viral protease (HIV) amd matrix protein (influenza, blocks uncoating)

Smaller than virus

Satellites: small RNA molecules, absolutely dependent on presence of another virus

Type A: an RNA molecule of more than 700 nt, which encodes its own capsid protein

Type B: an RNA molecule of more than 700 nt, which encodes a non-structural protein

Type C: a linear RNA of less than 700 nt, which does not encode any proteins

Type D: a circular RNA of less than 700 nt, which does not encode any proteins

Several DNA satellites have been described as well

• Satellites often cause different symptoms than the host virus alone

• Most known satellites are associatet with plants (satellite tobaco necrosis virus, satellite panicum mosaic virus, etc)

• Some are dependent on animal viruses – for example dependoviruses, wich are satellites of adenoviruses.

Different symptoms of infection by Tobacco necrosis virus without (left) and with co-

infection of Tobacco sattelite necrosis virus

Viroids

• Viroids are very small (200-400 nt) rod-like RNA molecules with a high degree of secondary structure

• Viroids do not encode any proteins and unlike satellites they are not dependent on the presence of another virus

Structure of viroids

• Conserved central domain is responsible for replication

• Pathogenic or P domain can display similarities with various cellular RNAs sequences (snRNAs, signal recognition particle) and therefore interfer with cellular proceses

• Viroids also have been shown to directly ativate certain protein kinases

Viroid replication

• Viroids utilize cellular RNA polymerases for their replication

• Replication is performed by “rolling circle mechanism”

• The resulting long RNA molecule is cut in pieces and ligated either autocatalytically or by cellular factors (depending on a viroid)

• So in a sense, at least some viroids are ribozymes...

Examples of plants, infected with various viroids

Hepatitis virus – a chimeric molecule, half viroid, half satellite

• Viroid like properties- Rod-like RNA molecule- Central conserved region

similar to plant viroids- Rolling circle replication- Self-cleaving activty

• Satellite like properties- Encodes a protein, which is

necessary both for encapsidation and replication

- Dependent on presence another virus – HBV

- Genome larger than for viroids (1640 nt)

Prions

• Chronic, progressive and always fatal infections of the nervous system

• Infectious agent is protein only, without presence of any nucleic acid

The main known infections

• Animal

-Scrapie

-Transmissible mink encelophathy

-Bovine spongiform encephalophaty

-Feline spongiform enelophaty

• Human- Creutzfeldt-Jakob

disease (CJD)- Gerstmann-Straussler

syndrome- Kuru

Even yeasts have prions!

Kuru• A fatal disease, ”laughing death” found in

New Guinea

• Has been shown to be transmitted through ritual cannibalism

• Certain tribes used to honour their dead close relatives by eating them

Scrapie

The infectous agent in prion deseases• PrPc (PrP cellular) is the normal variant of the protein of

unknown function, expressed in nervous tissue

• PrPSc (PrP Scrapie) is the same protein, which has undergone severe structural rearrangement, forming unsoluble, -sheet rich fibrils, somewhat similar to those, caused by Alzheimers disease

• PrPSc is itself capable to catalytically convert PrPc to another PrPSc molecule

• Knockout mouse lacking PrPc show normal development, indicating that presence of PrPSc fibrils and not absence of PrPc is fatal

• PrPSc form of protein is extermely stable – it can survive temperatures of over 100oC

Mechanism of PrPSc fibril formation

PrPc

PrPSc

Spontaneous structural rearrangement

External PrPSc

PrPSc induced structural rearrangement

PrPScPrPSc

PrPScPrPSc PrPSc PrPSc PrPSc PrPSc

PrPc

PrPc

PrPc

PrPSc polymerization PrPSc fibril

PrPSc

Normal PrPc function ?

Both Alzheimer's and prion diseases are characterized by the deposition of pathological proteins in the brain, often in the form of plaques. The brown color is indicative of immunostained deposits of the Aß peptide and of the PrPSc protein in brains of patients suffering from Alzheimer's disease (A) and Creutzfeldt-Jakob

disease (B), respectively.

PrPc and PrPSc monomers

PrPc (crystal structure) PrPSc (model – not a real structure!)

Different models of fibrous form of prions

Is BSE transmittable to humans?

• As we know from newspapers, there is a clear link from BSE to human CJD

• CJD occurs worldwide with frequency ~1 per million people per year. This makes about 60 cases per year in UK

• Since 1996 in UK there have been identified additional ~20 cases per year of CJD with somewhat different symptoms, called vCJD

• There is no direct evidence that BSE can cause vCJD

BSE and vCJD statistics

3

1010

1915

26

vCJD

BSE