Viral Growth Dr Paul Brown firstname.lastname@example.org BC10M: Introductory Biochemistry Lecture 7
Characteristics of Viruses • Non-living agents that infect all life forms (Bacteriophages, plant viruses, and animal viruses) • One virus particle = “virion” (size: 10 – 500 nm) • Virus architecture • Nucleic acid (DNA or RNA – not both!) • Protein coat = capsid of various shapes (isometric, helical, complex) • Naked virions vs. enveloped viruses • Genome: ss-/ds-RNA, ss-/dsDNA; linear or circular
Methods of Study • Much more expensive and difficult to study animal viruses than bacteriophages • Cultivation in host cells • Living animal • Embryonated chicken eggs • Cell or tissue culture (= in vitro)
Methods of Study cont: Quantitation • Plaque assay (useful for infective and lytic viruses) • Virion counting with EM • Quantal assay (ID50 or LD50) • Haemagglutination (e.g.influenza virus)
Culturing viruses requires an appropriate host cell. In this example a bacteriophage is grown using bacterial cells as host cells. Animal or plant viruses can be grown in tissue culture.
Plaques in a lawn of bacterial cells caused by viruses lysing bacterial cells.
Replication Cycle - Overview • Obligate intracellular parasites using host cell machinery • Very limited number of genes encode proteins for • Capsid formation • Viral nucleic acid replication • Movement of virus into and out of cell • Kill or live in harmony within the host cell – Outside the cell, viruses are inert Fig 13.3 Size comparison(see also Table 13.1)
Bacteriophage (Phage) • Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery • Significance • Models for animal cell viruses • Gene transfer in bacteria (transduction) • Medical applications • Identification of bacteria - phage typing • Treatment and prophylaxis??? • Examples: T4 and Lambda ()
Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail
Infection of Host Cells • Irreversible attachment • Adsorption • LPS for T4 • Sheath Contraction • Nucleic acid injection
Lytic Bacteriophage • Lytic or virulent phage: Phage that can only multiply within bacteria and kill the cell by lysis. (e.g., T4)
Total Phage Extracellular Phage Number of Infectious Particles Lysis Intracellular accumulation phase Eclipse Time after Infection Lytic Phage Multiplication Cycle • Eclipse • Early genes • Phage DNA synthesis • Late genes • Intracellular accumulation • Lysis and Release
Time course of events occurring during one-step growth curve of bacteriophage T4.
Lysogenic Bacteriophage • Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell. (e.g.,) • Expression of most phage genes repressed • Prophage • Lysogen
Lysogenic Phage • DNA integrates into host chromosome • Phage DNA = Prophage • Infected bacterial cell = lysogenic cell or lysogen • Prophage state can be indefinite • Lysogenic conversion confers new properties onto host cells (e.g.: toxin production of S. pyogenes – scarlet fever) • Phage induction converts lysogenic to lytic state
Cohesive Ends Ligase Linear Double Stranded Closed Circle Opened Circle Events Leading to Lysogeny • Circularization of the phage chromosome • Cohesive ends
bio gal gal bio bio gal Events Leading to Lysogeny • Site-specific recombination • Phage coded enzyme • Repression of the phage genome • Repressor protein • Specific • Immunity to superinfection
gal bio bio bio gal gal bio gal Termination of Lysogeny • Induction • Adverse conditions • Role of proteases • recA protein • Destruction of repressor • Gene expression • Excision • Lytic growth
Lytic vs Lysogenic Cycle? • Role of repressor • Role of cro gene product • Role of proteases
Significance of Lysogeny • Model for animal virus transformation • Lysogenic or phage conversion • Definition: A change in the phenotype of a bacterial cell as a consequence of lysogeny • Modification of Salmonella O antigen • Toxin production by Corynebacterium diphtheriae
Host Range of Phages Phage host cell interaction usually very specific Limiting factors for host range • Phage has to bind to bacterial surface receptors • Bacterial surface receptors mutate resistant cell • Lysogenic conversion changes surface receptors and protects host • Restriction modification system of host cell
Medical Applications of Phage • “I strongly believe phage could become an effective antibacterial tool” - Carl Merril, Chief of the Laboratory of Biochemical Genetics, National Institute of Mental Health, NIH. • “It might be another string on the bow, such that when (conventional antibiotics) fail, here’s something that has a chance of working. But it’s not going to be a panacea” - Joshua Lederberg, Sackler Foundation Scholar at The Rockefeller University Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic Uses American Society for Microbiology News 64:620-623, 1998
Medical Applications of Phage • Exponential Biotherapies (Rockville, MD) • Vancomycin resistant Enterococcus faecium and Streptococcus pneumoniae • Phage Therapeutics (Bothell, WA) • Staphylococcus aureus and Staphylococcus epidermidis • Intralytix, Inc. (Baltimore, MD) • Salmonella in meat and poultry • Biopharm Ltd. (Tblisi, Georgia) • Infections associated with burns • University of Idaho • Escherichia coli O157:H7 in cattle Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic Uses. American Society for Microbiology News 64:620-623, 1998. Phages eyed as agents to protect against harmful E. coli. American Society for Microbiology News 65:666-667, 1999. The End