1. BTY328: Viruses Dr William Stafford wstafford@uwc.ac.za Viral hosts and disease Bacteriophages -viruses that infect bacteria

2. Types of bacteriophages Bacteriophages can have DNA or RNA as genetic material Most can be placed in a few morphological groups: tailless icosahedral phages viruses with contractile tails, viruses with noncontractile and filamentous phages.

3. dsDNA phages e.g. T4 Specific host cell recognition and attachment- T4 Bacteriophage bind to specific host cell receptor sites. The nature of these receptors varies with the phage and variation in receptor properties is partly responsible for phage host preferences. After attachment, conformational changes occur in the proteins of the baseplate and sheath and the sheath reorganizes so that it shortens and the central tube or core is pushed through the bacterial wall. Transfection occurs when the DNA is extruded from the T4 phage head, through the tail tube, and into the host cell….

4. Lifecycle of bacteriophage T4

5. Bacteriophage T4 replication Soon after phage DNA injection the synthesis of host DNA, RNA, and protein is halted:- the cell is forced to make viral constituents. E. coli RNA polymerase starts synthesizing phage mRNA within 2 minutes. This mRNA (early mRNA) direct the synthesis of the protein factors and enzymes required to take over the host cell and manufacture viral nucleic acids. Some virus-specific enzymes degrade host DNA to nucleotides, thereby simultaneously halting host gene expression and providing raw material for virus DNA synthesis. Within 5 minutes, virus DNA synthesis commences…

6. T4 phage DNA replication A Bidirectional DNA replication B. Formation of DNA concatamers Endonuclease cleavage and DNA polymerase fill in to generate mature viral genome DNA ABAB

7. Protection of T4 DNA by modification T4 DNA contains hydroxymethylcytosine (HMC) instead of cytosine. HMC must be synthesized by two phage-encoded enzymes before DNA replication can begin. After T4 DNA has been synthesized, it is glucosylated by the addition of glucose to the HMC residues. Glucosylated HMC residues protect T4 DNA from attack by E. coll called restriction endonucleases, which would otherwise cleave the viral DNA at specific points and destroy it.

8. Assembly of phage particles The Assembly of Phage Particles A complex self-assembly process. Late mRNA ( produced after DNA replication) directs the synthesis of three kinds of proteins: (1) phage structural proteins, (2) proteins that help with phage assembly and (3) proteins involved in cell lysis and phage release.

9. T4 phage release Many phages lyse their host cells at the end of the intracellular phase. The lysis of E. coli by T4 takes place after ~ 22 minutes at 37°C, releasing ~ 300 T4 phage particles per infected bacterial cell. Several T4 proteins are involved in this process: -endolysin that attacks the cell wall peptidoglycan. -holin produces a plasma membrane lesion that stops respiration and allows the endolysin to attack the peptidoglycan…… ……………….forms holes in the membrane and lysis.

10. ssDNA phages: eg  X174  X174 (Microviridae) is a small ssDNA phage and E. coli as its host. The DNA is the same as that of the viral mRNA (except T>U)) and is therefore (+) strand The genome contains overlapping genes  X174 ssDNA must be converted to a double- stranded form before either replication or transcription can occur. Upon infection of the host, it is immediately copied by the bacterial DNA polymerase to form a double-stranded DNA replicative form which in turn directs the synthesis of more DNA and mRNA. The phage is released by host lysis.

11. Genetic map ssDNA is immediately converted to dsDNA replicative form in the host The replicative form directs the synthesis of DNA and mRNA  X174

12. Rolling circle replication Double stranded replicative form  X174

13. Filamentous phages eg. M13 M13 is a filamentous ssDNA phage (Inoviridae). Circular ssDNA is surrounded by a tube made of a proteins organized in a helical arrangement. The virus infects E. coli cells by attaching to the tip of the F’ pilus; and then the DNA enters the host. A replicative form is first synthesized. Replication of the phage DNA uses the rolling-circle method. The filamentous phages do not kill their host cell but establish a symbiotic relationship in which new virions are continually released by a secretory process.

14. Lysogeny Viral genome remains within the host cell and replicates with the bacteria genome to generate a clone of infected cells. Bacteria having the potential to produce phage particles under certain conditions are lysogens, and phages able enter into this relationship are temperate phages. The latent genome of the virus genome that remains within the host but does not destroy it is called the prophage. The prophage usually is integrated into the bacterial genome but sometimes exists independently. When induced and phage reproduction is initiated in a lysogen, the host cells are destroyed with the release of new phages and then enter the lytic cycle..

15. Why lysogeny? When bacteria enter dormancy (eg. due to nutrient starvation) they degrade their own mRNA and protein. However, the phage can only reproduce in actively metabolizing bacteria, and phage reproduction is permanently interrupted by the bacterial mRNA and protein degradation. This predicament can be avoided if the phage becomes dormant (lysogenic) at the same time as its host (nutrient starvation favours lysogeny).

16. Lysogenic conversion Lysogenic conversion involves alterations in bacterial surface characteristics or pathogenic properties. When Salmonella is infected by epsilon phage, the structure of its outer lipopolysaccharide layer is modified. These epsilon-induced changes appear to eliminate surface phage receptors and prevent infection of the lysogen by another epsilon phage.

17. Lysis or Lysogeny? Lambda is a dsDNA phage that reproduce using a normal lytic cycle but also integrate into the host genome in a lysogenic cycle.

18. Lambda repressor coded for by the cI gene (236 amino acids long and folds into a dumbbell shape) One domain is binds DNA, while the other binds with another cI repressor molecule (dimer) In a lysogen (d) the repressor is synthesized continuously and binds to the operators 0L and 0R, thereby blocking RNA polymerase activity.

19. Integration of lambda DNA The int gene codes for the synthesis of an integrase enzyme, that acts on short repeat sequences (att) Induction of the lytic cycle is triggered drop in lambda repressor levels. Occasionally the repressor spontaneously declines, but induction usually is in response to environmental factors such as UV light or chemical mutagens that damage host DNA (the recA protein involved in SOS cleaves the repressor….).

20. Bacteriophages and transposable elements Some temperate phages can integrate into the genome and replicate as transposable elements. e.g. Mu (mutator) phage that infects E.coli K12. The integration of Mu into the host genome is essential for lytic and lysogenic growth Integration is mediated by a transposase and and occurs randomly in the host genome …….it can be used to induce random mutations into the host genome.