Research and collections: JAVAD RAMEZANI AVVAL REIABI MS in Plant Pathology

1. Research and collections: JAVAD RAMEZANI AVVAL REIABI MS in Plant Pathology Agricultural Management Ghaen city

2. Viruses

3. 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

4. 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

5. The particle • Common things to all virus particles: • It encloses genomic nucleic acid • It is a polymer, assembled from one or few different kinds of monomers

6. 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

7. Non-enveloped virus Enveloped virus Enveloped and non-enveloped viruses DNA or RNA genome Matrix protein Lipid bilayer Nucleocapsid Envelope protein

8. Filamentous Icosahedral Two different kinds of nucleocapsids

9. The helical geometry of filamentous virus TMV (Tobacco mosaic virus) Pitch of helix 22.8 Å m=16.3 (subunits per helix turn) p=1.4Å (axial rise per subunit)

10. 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...).

11. T=4 (240 subunits) T=1 (60 subunits) T=3 (180 subunits) The blue triangle represents one face of icosahedron

12. Structure of T=3 icosahedral bacteriophage MS2 FG loops 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

13. 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.

14. 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.

15. 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) A C R L Particle: Genome encapsidated in a single layer coat protein shell

16. 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

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

18. Phage T4: complicated genome & particle

19. Hepatitis B (3,200 bp, 4 proteins) Adenovirus (35,000 bp, 40 proteins) T4 phage (173,000 bp, 280 proteins ) Mimivirus (1,180,000 bp, 1262 proteins) Chlamydia trachomatis (1,040,000 bp, 936 proteins) Burrelia burgdorferi (1,440,000 bp, 1738 proteins) E.coli (4,600,000 bp, 4377 proteins) 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

20. Electron micrograph of mimivirus particle and comparison with sizes of other viruses E.coli Poxivirus T4 phage head Adenovirus Hepatitis B Smallest viruses

21. The viral life cycle • Initation phase: • Attachment to the host cell receptor (Ig like receptors, cellular adhesion molecules, membrane transport proteins,oligosaccharides, etc) • Penetration (endocytosis, fusion) • Uncoating Most bacteriophages avoid penetration and uncoating stages by injecting the viral nucleic acid into the cell Plant 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.

22. Initiation phase Enveloped virus Non-enveloped virus

23. Movie: animal virus penetration by fusion

24. Movie: DNA injection in E.coli by T4 bacteriophage

25. The viral life cycle • Replication phase • nucleic acid replication • mRNA synthesis • protein expression • assembly

26. The viral life cycle • Release phase • exit from cell (lysis, exocytosis, budding) • maturation (rearrangement of nucleocapsid, etc)

27. In enveloped viruses assembly can be coupled to release

28. Examples of viral life cycles • Small RNA phages • HIV • Influenza

29. Initiation phase of small RNA phages A protein Attachment Bacterial pili Transport of genomic RNA into cytoplasm

30. Replication and release Translation of viral proteins Lysis Coat Replicase A Assembly Release

31. HIV • Retroviridae, Lentivirus (~10 kb diploid +ssRNA genome)

32. The life cycle of HIV

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

36. Influenza virus • (-)sense segmented RNA genome • enveloped, a bit irregular particle • 2 types of spike proteins (neuraminidase and hemagglutinin) • Infects birds and mammals

37. Life cycle of influenza virus

38. 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...

39. 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)

40. Smaller than virus

41. 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

42. 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.

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

44. 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

45. Structure of viroids

47. 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

48. 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...

49. Examples of plants, infected with various viroids

50. 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) Hepatitis d virus – a chimeric molecule, half viroid, half satellite