Research and collections:Research and collections:

JAVAD RAMEZANI AVVAL REIABIJAVAD RAMEZANI AVVAL REIABI

MS in Plant PathologyMS in Plant PathologyAgricultural Management Ghaen cityAgricultural Management Ghaen city

VirusesViruses

What is a virus ?What is a virus ?

• Viruses are submicroscopic, obligate intracellular Viruses are submicroscopic, obligate intracellular parasitesparasites

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

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

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

Origins of VirusesOrigins of Viruses• How did these become independent genetic entities? The only absolute requirement is How did these become independent genetic entities? The only absolute requirement is

an origin of replication in the nucleic acid.an origin of replication in the nucleic acid.

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

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

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

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

The particleThe particleCommon things to all Common things to all

virus particles:virus particles:

1.1. It encloses genomic It encloses genomic nucleic acidnucleic acid

2.2. It is a polymer, It is a polymer, assembled from one or assembled from one or few different kinds of few different kinds of monomersmonomers

The nucleocapsidThe nucleocapsid

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

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

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

Enveloped and non-enveloped virusesEnveloped and non-enveloped viruses

• Non-enveloped virus • Enveloped virus

DNA or RNA genome

Nucleocapsid

Lipid bilayer

Envelope protein

Matrix protein

Two different kinds of Two different kinds of nucleocapsidsnucleocapsids

• Filamentous • Icosahedral

The helical geometry of filamentous virus The helical geometry of filamentous virus TMV (Tobacco mosaic 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 virusesThe geometry of icosahedral viruses

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

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

• The number of subunits is always a certain multiple The number of subunits is always a certain multiple of 60, called a triangulation (‘T’) number. For of 60, called a triangulation (‘T’) number. For example, T=3 virus will have 180 subunits, T=7 example, T=3 virus will have 180 subunits, T=7 virus 7x60=420 subunits. Only certain T values are virus 7x60=420 subunits. Only certain T values are allowed (1, 3, 4,7, 9, 13, 16,19,21,25...). 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 genomesThe genomes• I: Double-stranded DNAI: Double-stranded DNA.. Examples: Examples: AdenovirusesAdenoviruses,, Herpesviruses Herpesviruses, Papillomaviruses, , Papillomaviruses,

Poxiviruses,Poxiviruses, T4 bacteriophageT4 bacteriophageSome replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in Some replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in the cytoplasmthe cytoplasm

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

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

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

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

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

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

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

Phage T4Phage T4: complicated genome & particle: complicated genome & particle

                                                                             

Mimivirus: the biggest known genome and particleMimivirus: the biggest known genome and particle• Genome: Genome: 1,181,404 nt1,181,404 nt, codes for 1262 proteins, codes for 1262 proteins• Some proteins are involved in protein synthesis, thus violating one criterium in a definition of “what is a virus”Some proteins are involved in protein synthesis, thus violating one criterium in a definition of “what is a virus”• Infects amoebaeInfects 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 cycleThe viral life cycle•Initation phase:Initation phase:a)a) Attachment to the host cell receptorAttachment to the host cell receptor ( (Ig like receptorsIg like receptors,, cellular cellular adhesion molecules, membrane transport proteins,adhesion molecules, membrane transport proteins, oligos oligosaaccharidesccharides, , etcetc))b)b) PenetrationPenetration (endocytosis, (endocytosis, fusion fusion) ) c)c) UncoatingUncoating

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

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

Initiation phaseInitiation phase

Non-enveloped virus

Enveloped virus

Movie: animal virus penetration by fusion

Movie: DNA injection in E.coli by T4 bacteriophage

The viral life cycleThe viral life cycle

• Replication phaseReplication phase

a)a) nucleic acid replicationnucleic acid replication

b)b) mRNA synthesismRNA synthesis

c)c) protein expressionprotein expression

d)d) assembly assembly

The viral life cycleThe viral life cycle

• Release phaseRelease phase

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

b)b) maturation (rearrangement of maturation (rearrangement of nucleocapsid, etc)nucleocapsid, etc)

In enveloped viruses assembly can be coupled to release

Examples of viral life cyclesExamples of viral life cycles

• Small RNA phagesSmall RNA phages

• HIVHIV

• InfluenzaInfluenza

Initiation phase of small RNA Initiation phase of small RNA phagesphages

Bacterial pili

Transport of genomic RNA into cytoplasm

Attachment

A protein

Replication and release

Translation of viral proteins

Lysis Coat Replicase

Assembly

Release

A

HIVHIV• Retroviridae, Lentivirus (~10 kb diploid

+ssRNA genome)

The life cycle of HIVThe life cycle of HIV

HIV maturationHIV maturation

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

Influenza virusInfluenza 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 virusLife cycle of influenza virus

Should we be scared of that Should we be scared of that avian H5N1 influenza?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 Virus life cycle and antiviral therapytherapy

• 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 virusSmaller than virus

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

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

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

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

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

Several DNA satellites have been described as wellSeveral DNA satellites have been described as well

• Satellites Satellites often often cause different symptoms cause different symptoms than the host virus alonethan the host virus alone

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

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

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

infection of Tobacco sattelite necrosis virusinfection of Tobacco sattelite necrosis virus

ViroidsViroids

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

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

Structure of viroidsStructure of viroids

                                                                                 

• Conserved central domain is responsible for replicationConserved central domain is responsible for replication

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

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

Viroid replicationViroid replication

• Viroids utilize cellular RNA polymerases for their Viroids utilize cellular RNA polymerases for their replicationreplication

• Replication is performed by “rolling circle Replication is performed by “rolling circle mechanism”mechanism”

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

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

Examples of plants, infected with various viroids

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

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

similar to plant viroidssimilar to plant viroids- Rolling circle replicationRolling circle replication- Self-cleaving activtySelf-cleaving activty

• Satellite like propertiesSatellite like properties- Encodes a protein, which is Encodes a protein, which is

necessary both for necessary both for encapsidation and encapsidation and replicationreplication

- Dependent on presence Dependent on presence another virus – HBV another virus – HBV

- Genome larger than for Genome larger than for viroids (1640 nt)viroids (1640 nt)

PrionsPrions

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

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

The main known infectionsThe main known infections

• AnimalAnimal

-Scrapie-Scrapie

-Transmissible mink -Transmissible mink encelophathyencelophathy

-Bovine spongiform -Bovine spongiform encephalophatyencephalophaty

-Feline spongiform -Feline spongiform enelophatyenelophaty

• HumanHuman- Creutzfeldt-Jakob Creutzfeldt-Jakob

disease (CJD)disease (CJD)- Gerstmann-Straussler Gerstmann-Straussler

syndromesyndrome- KuruKuru

Even yeasts have prions!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 deseasesThe infectous agent in prion deseases• PrPPrPc c (PrP (PrP ccellular) is the normal variant of the protein of ellular) is the normal variant of the protein of

unknown function, expressed in nervous tissueunknown function, expressed in nervous tissue

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

• PrPPrPScSc is itself capable to catalytically convert PrP is itself capable to catalytically convert PrPcc to to another PrPanother PrPSc Sc moleculemolecule

• Knockout mouse lacking PrPKnockout mouse lacking PrPcc show normal development, show normal development, indicating that presence of PrPindicating that presence of PrPScSc fibrils and not absence of fibrils and not absence of PrPPrPcc is fatal is fatal

• PrPPrPScSc form of protein is extermely stable – it can survive form of protein is extermely stable – it can survive temperatures of over 100temperatures of over 100ooC C

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.

PrPPrPcc and PrP and PrPScSc monomers monomers

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

Different models of fibrous form of prionsDifferent models of fibrous form of prions

Is BSE transmittable to humans?Is BSE transmittable to humans?

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

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

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

• There is no direct evidence that BSE can cause vCJDThere is no direct evidence that BSE can cause vCJD

BSE and vCJD statisticsBSE and vCJD statistics

3

1010

1915

26

vCJD

BSE