Virus and bacteriophage

for under graduate students

1. VIRUSES AND BACTRIOPHAGES HMB 5111 2 (1+1)

2. Viruses Infectious pathogens. Too small to be seen with a electron microscope. The simplest viruses are composed of 1. A small piece of nucleic acid 2. Surrounded by a protein coat Obligate parasites that depend on the cellular machinery of their hosts. Not active outside of their hosts. Organisms including animals, plants, fungi, and bacteria are hosts for viruses, but most viruses infect only one type of host.

3. History of virus discovery In the late 1800s, botanists had been trying to find the cause of tobacco mosaic disease. In 1892, D. IWANOWSKI tried to filter the sap of infected tobacco plants (Filter capable of removing particles the size of all known bacteria).

4. Experiment by Iwanoski

5. The filtrate was Fully capable of producing the ORIGINAL DISEASE in new hosts. Nothing could be seen in the using the most powerful microscopes, nor could anything be cultivated from the filtrates. Iwanowski concluded that the bacteria was so small / or they made a filterable toxin. A Dutch botanist named Martinus Beijerink ruled out the filterable toxin conclusion because the filtered sap are capable of causing undiluted infection. The agent cannot be cultivated on nutrient media (need a host). In 1935, Stanley discovered this agent after crystallization.

6. General properties Obligate intracellular parasites. Do not have the molecular machinery to replicate without a host. Pathogenic to higher plants. Plant viruses infect plants. A virus particle, also known as a virion is an extremely small infectious agent. Essentially a nucleic acid (DNA or RNA) enclosed in a protein coat called a capsid.

7. Cont.. Viral genetic material can be 1. Double-stranded DNA 2. Double-stranded RNA 3. Single-stranded DNA or 4. Single-stranded RNA Most plant viruses are classified as single-stranded RNA or double-stranded RNA virus particles. Cause various types of plant diseases. These diseases do not typically result in plant death.

8. Plant diseases produce symptoms such as Ring spots Mosaic pattern development Leaf yellowing and distortion Deformed growth Some plant viruses are not limited to one particular plant host. May infect different varieties of plants. Plants including tomatoes, peppers, cucumbers, and tobacco may all be infected by the tobacco mosaic virus

9. Composition & Architecture of plant viruses

10. Morphology of Viruses About 50 % of all known plant viruses are elongate (flexuous threads or rigid rods). About 50 % of all known plant viruses are spherical (isometric or polyhedral). A few viruses are cylindrical bacillus-like rods.

11. Chemical composition of plant viruses Protein (Capsid) Capsomere Nucleic acids RNA + ve strand RNA - ve strand RNA ssRNA dsRNA DNA ssDNA dsDNA

12. Cont.. Proteins 60-95% of the virion. Repeating subunits, identical for each virus type but varies from virus to virus and even from strain to strain . subunits - 158 amino acids with a mass of 17,600 Daltons (17.6 kDa , kd or K) TYMV – 20,600 Dalton protein Nucleic acid is 5-40% of the virion Spherical viruses: 20-40% Helical viruses: 5-6%

13. Nucleic acid (5-40%) represents the genetic material, indispensable for replication . Nucleic acid alone is sufficient for virus replication – Fraenkel-Conrat , Schramm. Protein (60-95%) protects virus genome from : degradation facilitates movement through the host and transmission from one host to another

14. Percentage of protein & n/a in some viruses

15. Terminology for virus components Capsid is the protein shell that encloses the nucleic acid. Capsomers are the morphological units seen on the surface of particles and represent clusters of structure units. Capsid and enclosed nucleic acid is called the nucleocapsid. The virion is the complete infectious virus particle

16. Rod shaped particles Helix (rod) e.g., TMV TMV rod is 18 nanometers (nm) X 300 nm

17. TMV Tobacco mosaic virus is typical, well-studied example . Each particle contains only a single molecule of RNA (6395 nt ) and 2130 copies of the coat protein subunit (158 aa; 17.3 kDa ) 3 nt /subunit 16.33 subunits/turn 49 subunits/3 turns TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion also occurs in the absence of RNA

18. Plant viruses are diverse, but not as diverse as animal viruses – probably because of size constraints imposed by requirement to move cell-to-cell through plasmodesmata of host plants

19. Icosahedral arrangement is typical in virus structure An icosahedron has 20 triangular (equilateral) faces (facets), 12 vertices (corner), 30 edge. Icosahedron (sphere) e.g., BMV

20. The Bacteriophages Bacteriophages are obligate intracellular parasite on bacteria that uses bacterial machinery system for its own multiplication and development. These are commonly referred as “phage”. Bacteriophages were jointly discovered by Frederick Twort (1915) in England and by Felix d'Herelle (1917) at the Pasteur Institute in France. “Bacteriophage” term was coined by Felix d'Herelle. Examples of phages: T-even phages such as T2, T4 and T6 that infect E. coli Temperate phages such as lambda and mu Spherical phages with single stranded DNA such as PhiX174 Filamentous phages with single stranded DNA such as M13 RNA phages such as Q beta

21. Fig. : Basic structure of bacteriophage

22. Composition Depending upon the phage, the nucleic acid can be either DNA or RNA but not both. The nucleic acids of phages often contain unusual or modified bases, which protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection. Simple phages may have only 3-5 genes while complex phages may have over 100 genes. Certain phages are known have single stranded DNA as their nucleic acid.

23. Morphology Most phages range in size from 24-200 nm in length. T4 is among the largest phages; it is approximately 200 nm long and 80-100 nm wide. All phages contain a head structure, which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous. The head encloses nucleic acid and acts as the protective covering. Some phages have tails attached to the phage head. The tail is a hollow tube through which the nucleic acid passes during infection.

24. T4 tail is surrounded by a contractile sheath, which contracts during infection of the bacterium. At the end of the tail, phages like T4 have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the bacterial cell. Not all phages have base plates and tail fibers.

25. STEPS IN INFECTION

26. Bacteriophage replication cycle Adsorption: The first step in the infection process is the adsorption of the phage to the bacterial cell. This step is mediated by the tail fibers or by some analogous structure on those phages that lack tail fibers. Phages attach to specific receptors on the bacterial cell such as proteins on the outer surface of the bacterium, LPS, pili , and lipoprotein. This process is reversible. One or more of the components of the base plate mediates irreversible binding of phage to a bacterium.

27. Penetration The irreversible binding of the phage to the bacterium results in the contraction of the sheath (for those phages which have a sheath) and the hollow tail fiber is pushed through the bacterial envelope. Some phages have enzymes that digest various components of the bacterial envelope. Nucleic acid from the head passes through the hollow tail and enters the bacterial cell. The remainder of the phage remains on the outside of the bacterium as “ghost”. Even a non-susceptible bacterium can be artificially infected by injecting phage DNA by a process known as transfection .

28. Life cycle

29. TYPES OF LIFE CYCLE Depending on the life cycle, phages can either by lytic (virulent) or lysogenic (temperate). While lytic phages kill the cells they infect, temperate phages establish a persistent infection of the cell without killing it. In lytic cycle the subsequent steps are synthesis of phage components, assembly, maturation and release.

30. Lytic cycle There are five steps in a typical bacteriophage lytic reproduction, Attachment- A virus will attach to a suitable host cell. Penetration- The whole virus or only the genetic material (nucleic acid) will penetrate the cell’s cytoplasm. A bacteriophage capsid remains on the outside of the bacterial cell whereas many viruses that infect animal cell enter a host cell intact.

31. III. Replication and synthesis - The viral DNA/RNA directs the host cell to produce many copies of viral nucleic acids and proteins necessary for its replication. IV. Assembly - The viral nucleic acids and proteins are assembled together to form new infectious particles. V. Release - Newly generated viral particles are released from the host cell.

32. lysogenic cycle The infection will enter a latent period. The host cell is not killed in this process, but the viral nucleic acid will undergo genetic recombination with the host cell’s chromosome. This integrated structure is called a prophage. When the bacterial DNA replicates, the prophage also replicates. Certain external condition such as UV light and x-rays cause viruses to revert to a lytic cycle and then destroy their hosts.