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10/28/13 Chapter 13-Viruses. Viroids, and Prions Viruses: Obligate Intracellular Parasites Viruses simply genetic information: DNA or RNA contained within protective coat • Inert particles: no metabolism, replication, motility • Genome hijacks host cell’s replication machinery • Inert outside cells; inside, direct activities of cell • Infectious agents, but not alive • Can classify generally based on type of cell they infect: eukaryotic or prokaryotic • Bacteriophages (phages) infect prokaryotes • May provide alternative to antibiotics History began with the Tobacco Mosaic Virus (TMV) • 1886 Aldolf Mayer showed that a virus was transmissable between plants • 1892 Iwanowski tried to isolate it by filtering with porcelain filter 1 10/28/13 General Characteristics of all viruses • Contain a single type of nucleic acid • Contain a protein coat • Obligate intracellular parasites General Characteristics of Viruses Most viruses notable for small size • Smallest: ~10 nm ~10 genes • Largest: ~500 nm Virion (viral particle) is nucleic acid with a protein coat Contain either RNA or DNA 2 10/28/13 2,288 species 348 genera 6 orders Common Shapes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Icosahedral Three shapes: Icosahedral Helical Complex Protein coat (capsid) Nucleic acid Adenovirus 75 nm (a) Helical Nucleic acid Protein coat (capsid) Tobacco mosaic virus (TMV) 100 nm (b) Complex Protein coat (capsid) Nucleocapsid Head with nucleic acid (DNA) Tail Base plate Tail spike Tail fibers T4 Bacteriophage 100 nm (c) Two different types of Viruses Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Naked viruses lack envelope; more resistant to disinfectants • Enveloped viruses have lipid bilayer envelope Protein capsid: protects nucleic acids Made of identical subunits -capsomers Capsomere subunits Nucleic acid Nucleocapsid Capsid (entire protein coat) Spikes (a) Naked virus Spikes Matrix protein Nucleic acid Nucleocapsid Capsid (entire protein coat) Envelope (b) Enveloped virus • Capsid + nucleic acids = nucleocapsid DNA or RNA Genome may be linear or circular Double- or single-stranded Viruses have components for attachment • Phages have tail fibers • Many animal viruses have spikes • attach to specific receptor sites 3 10/28/13 A complex virus showing attachment fibers Relationship of virus with host cell Bacterial viruses • • Known as bacteriophages or phages Two different life cycles 1. Lytic cycle (lytic or virulent phage)-results in lysis of the cell 2. Lysogenic cycle (temperate or lysogenic phage)-may result in lysis of the cell or becomes a permanent part of the chromosome by integrating 4 10/28/13 T4 phage replication 1. Attachment Phage uses bacterial receptors 2. Genome entry T4 lysozyme degrades cell wall, Tail contracts, injects genome 3. Synthesis of proteins and genome 4. Assembly--Some components spontaneously assemble, others require protein scaffolds 5. Release Lysozyme produced late in infection; digests cell wall--Cell lyses, releases phage Burst size of T4 is ~200 Lambda Phage replication • Lytic infection or incorporation of DNA into host cell genome • Lysogenic infection • Infected cell is lysogen • Lambda (λ) phage as model 5 10/28/13 Lambda integrates into the chromosome • Site specific recombination • Integrated phage DNA termed prophage phage-encoded enzymes • A repressor prevents, maintains lysogenic state Temperate Phage Infections (continued...) • Lambda (λ) phage: DNA excised from chromosome only about once per 10,000 divisions of host bacterium • If DNA damaged (e.g., UV light exposure), SOS repair system turns on, activates a protease • Protease destroys repressor, allows prophage to be excised, enter lytic cycle • Called phage induction; allows phage to escape damaged host Properties conferred by prophage 6 10/28/13 Some phage are filamentous 13.2. Bacteriophages Filamentous Phages • Single-stranded DNA phages • Used to produce only single- stranded recombinant DNA • Look like long fibers • Cause productive infections Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Filamentous phage F pilus Phage DNA • Host cells not killed, but grow more slowly • M13 phage as model • Attaches to protein on F pilus of E. coli • Single stranded DNA genome enters cytoplasm Phage attaches to the F pilus of a bacterial cell and injects its single-stranded DNA. Phage DNA replicates; phage capsomeres are synthesized and embedded in the host cell membrane. Outside environment Carrier cell Carrier cell Phage nucleic acid gains its capsid as it extrudes through the membrane. The bacteria do not lyse. Phage DNA Capsomeres Replication of filamentous phage • DNA polymerase synthesizes complementary strand Replicative form (RF)—one strand for mRNA synthesis, the other for genome • M13 phage coat protein molecules inserted into cytoplasmic membrane • Other proteins form pores • As phage DNA excreted through pores, coat proteins coat the DNA, form nucleocapsids 7 10/28/13 M13 is ssDNA…how does it replicate the ssDNA? Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ssDNA (+) strand Host enzyme synthesizes complementary strand. dsDNA (±) strand (RF) Replication (–) strand DNA transcribed Into mRNA (+) strand DNA functions as phage genome mRNA translated into phage coat protein • DNA polymerase synthesizes complementary strand • Replicative form (RF); one strand used as template for synthesis of mRNA, copies of genome • M13 phage coat protein molecules inserted into cytoplasmic membrane • Other proteins form pores • As phage DNA excreted through pores, coat proteins coat the DNA, form nucleocapsids Virion How do bacteria protect themselves against phage? • Prevent phage attachment • Attacking foreign DNA with restriction enzymes, protecting native DNA with methylation • CRISPR system degrades incoming viral nucleic acid CRISPR defense system against phage 8 10/28/13 Methods to study bacteriophage • Plaque Assay used to quantitate phage How do animal viruses differ from bacterial viruses? • Attachment or entry into the cell • Replication of viral nucleic acid (remember eukaryotic cells have a nucleus) • Uncoating step is required by animal viruses • Exit the host cell by budding or shedding Effects of animal virus on cells 9 10/28/13 Entry of animal virus Replication strategies • Watch the type of nucleic acid • What enzymes are needed for the process? Release of enveloped viruses 10 10/28/13 Acute viral infections • Usually short in duration • Host develops long lasting immunity • Infection of the virus results in a productive infection…host cells die as a result of infection General Steps of Acute Viral infection • • • • • • • Attachment Entry into host cell Targeting where it will reproduce Uncoating of the capsid Synthesis of proteins, replication of nucleic acid Maturation Cell lysis Can you identify some examples of viruses that produce an acute viral infection? 11 10/28/13 Persistent infections • Virus is continually present in the body, released by budding • Three categories – Latent infections – Chronic infections – Slow infections Persistent: Latent Infections • Persistent infection with symptomless period followed by reactivation of virus and symptoms • Example of latent viruses are found in the family Herpesviridae – Herpes simplex virus -1 – Herpes simplex virus -2 Latent Viral infections • All of these viruses are in the Herpesviridae family 12 10/28/13 Herpesviridae Family • Double stranded DNA (dsDNA), enveloped viruses -herpes simplex virus type 1(cold sores) -herpes simplex virus type 2 (genital herpes) -Varicella-zoster virus (chicken pox, shingles) -Epstein-Barr (infectious mono and Burkitt s lymphoma) Herpes Simplex virus-1 HSV-1 reactivation 13 10/28/13 Herpes simplex-1 • HSV-1 causes fever blisters, HSV-2 genital herpes • Symptoms: fluid filled skin lesions • Treatment: Acyclovir Varicella (chickenpox) and Herpes Zoster (Shingles) • HSV-3 causes chicken pox and latent activation known as shingles • Acquired by respiratory route, 2 weeks later see vesicles on skin • Vaccine established in 1995 for chickenpox Epstein Barr • Causes infectious mononucleosis • Acquire by saliva, incubation period is 4-7 weeks • Identify by -lobed lymphocytes -heterophile antibodies -fluorescent antibody tests 14 10/28/13 Chronic infections • Infectious virus present at all times • Disease may be present or absent • Examples are Hepatitis Type B and Type C viruses Type Hepadnaviridae family: Hepatitis B • dsDNA virus, enveloped • Hepatitis B -passes through intermediate stage (RNA) for replication -three particles found in blood sample 1. Dane 2. filamentous 3. sphericle -exposure through blood/ body fluids Hepatitis Type B • Incubation period is ~12 weeks • 10% of cases become chronic, mortality rate is less than 1% • About 40% of the chronic cases die of liver cirrhosis 15 10/28/13 Flaviviridae Family: Hepatitis Type C • Hepatitis C virus – (+) ssRNA virus, enveloped – Obtain from blood/body fluids – Incubation period averages 6 weeks – Hard to screen blood for the virus – 85% of all cases become chronic What other types of Hepatitis viruses are known to infect humans? • Hepatitis Type A – Found in the Picornaviridae family (+) ssRNA -obtain through fecal-oral route, enters GI tract and multiplies -incubation period is ~4 weeks -symptoms include: anorexia, malaise, nausea, diarrhea, abdominal discomfort, fever, and chills lasting 2-21 days Slow Infections • Infectious agent increases in amount over a long time during which there are no symptoms • Examples are HIV found in the Retroviridae family • Retroviruses use reverse transcriptase to replicate ssRNA 16 10/28/13 Retroviridae-multiple strands of (-)RNA • HIV -infects Helper T cells -requires the enzyme reverse transcriptase -integrates as a provirus -is released by budding, or lyses the cell HIV replication Viruses associated with cancers 17 10/28/13 Viruses and tumors • dsDNA viruses are most common to cause viral-induced tumors • Cancer is result of integration of viral genes into the host chromosome • Transforming genes are called oncogenes • Examples: papillomavirus, herpesvirus Orthomyxoviridae-multiple strands of (-)RNA • Influenza virus – Consists of 8 segments of RNA – Envelope has H spikes (hemagglutinin) and N spikes (neuraminidase) – Incubation is 1-3 days – Symptoms include: chills, fever, headache, muscle aches, may lead to cold-like symptoms Influenza virus 18 10/28/13 If multiple forms infect one cell…reassortment can occur Antigenic shift vs antigenic drift Ways to study viruses • Since viruses grow in living cells….need a live cell to culture them – Cell culture/tissue culture – Embryonated chicken eggs 19 10/28/13 Cell Culture Proteinaceous infectious particles: PRIONS • 1982 Stanley Prusiner proposed that there were infectious proteins • Caused the disease scrapie in sheep • Caused the mad-cow disease in 1987 • Human forms suggest a genetic component Prions • Contain no nucleic acid • Abnormal protein promotes conformational change to normal protein • Results in damage to neurons… transmissible spongiform encephalopahthies 20 10/28/13 Brain with spongiform encephalopathy Infections caused by prions Mechanism of prion replication 21