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PowerPoint® Lecture Presentations CHAPTER 8 Viruses and their Replication © 2018 Pearson Education, Inc. Virus • Virus: A genetic element that can replicate only inside a living cell (= host cell) • Possess own genome that encodes functions needed to replicate, however… • A virus is not a ce...

PowerPoint® Lecture Presentations CHAPTER 8 Viruses and their Replication © 2018 Pearson Education, Inc. Virus • Virus: A genetic element that can replicate only inside a living cell (= host cell) • Possess own genome that encodes functions needed to replicate, however… • A virus is not a cell, and is not considered a living organism • Rely on host cell for energy, metabolic intermediates, and protein synthesis • Cannot replicate unless it has gained entry into a suitable host cell • = Infection Viral genomes • Diverse genomes: • DNA or RNA • Single-stranded or double-stranded • ssRNA may be + or – sense • Linear or circular • Some highly unusual viruses use both DNA and RNA as genetic material at different stages of the life cycle • Genomes are typically smaller than cellular genomes • Smallest viruses contain < 2000 bp and only two genes! Host infection • Viruses can also be classified on the basis of the hosts they infect • Prokaryotes: Bacterial viruses (bacteriophages) • Archaeal viruses • Protozoan viruses • Fungi viruses (mycoviruses) • Animal viruses • Plant viruses • Types of infection: • Lytic infection: Virus replicates and destroys the host • Redirects host metabolism to support virus replication & assembly of new virions • Lyses host cell to release virions & repeat • Lysogenic infection: Viral genome becomes part of the host genome • Host cell is not destroyed • Genetically alters the host Virus structure • Most viruses are very small! • Most viruses are smaller than prokaryotic cells; range from 0.02 to 0.3 µm • Smallpox (large virus) = ~0.2 µm in size, similar to smallest known bacterial cells • Poliovirus (small virus) = ~28 nm in size, similar to the size of a ribosome • Virion = Extracellular form of the virus • Allows the virus to travel from one host cell to another • Diverse structures that vary widely in size, shape, and chemical composition • Some virions carry enzymes critical to successful infection & replication Virion • A protein shell called a capsid contains the viral genome • Nucleic acid + capsid = nucleocapsid • Proteins in capsid = capsomeres • Arranged in a precise and highly repetitive pattern around the nucleic acid • Most bacterial viruses contain no further layers • = Naked virus • Many animal viruses contain an outer layer of protein plus lipid • = Envelope Virion structure • Nucleocapsids are constructed in highly symmetric ways • Rod-shaped viruses exhibit helical symmetry • Length of virus determined by length of nucleic acid • Width of virus determined by size and packaging of protein subunits • e.g. Tobacco mosaic virus (TMV) • Contains a single type of protein in the capsid • 2130 copies of the protein combine with RNA into a helix Virion structure • Spherical viruses exhibit icosahedral symmetry • 20 triangular faces • Multiple capsomeres arranged in clusters to form each “face” • Simplest = 3 per face for 60 capsomeres per virion • Most have 240 – 360 capsomeres Head and Tail Bacteriophages • Complex structure • Icosahedral head + helical tail • e.g. Bacteriophage T4 • 85 x 110 nm • 170,000 bp genome encoding ~300 proteins • Tail consists of 20 different proteins arranged in helical tube • Joined to head via a “collar” • At other end endplate connects tail to long tail fibres, important for attachment Enveloped viruses • Membrane surrounds nucleocapsid • Generally infect animal cells in which the cytoplasmic membrane is directly exposed to the environment • Typically entire virion enters cell during infection, with envelope assisting by fusing with host membrane • Envelope is primarily host cell membrane that coats virus as it exits cell, with some viral proteins embedded Life cycle of a bacterial virus • A cell that supports the complete replication cycle of a virus is said to be permissive • Five main steps of viral life cycle: • Attachment (adsorption) of the virus to a susceptible host cell • Entry of the virion or its nucleic acid • Synthesis of virus nucleic acid and protein by host cell machinery as redirected by virus • Assembly of capsids and packaging of viral genomes into new virions • Release of mature virions from host cell Virion Protein coat remains outside DNA Viral DNA enters Virions Cell (host) 1. Attachment (adsorption of phage virion) 2. Penetration of viral nucleic acid 3. Synthesis of viral nucleic acid and protein 4. Assembly and packaging of new viruses 5. Cell lysis and release of new virions Attachment • Attachment of virion to host cell is highly specific • Virus has one or more proteins that interact with receptors on susceptible host • In the absence of the receptor, the virus can’t attach & infect cell • If receptor is altered by mutation, host may become resistant to infection • Receptors include proteins, carbohydrates, glycoproteins, lipids, lipoproteins, or cell structures • Carry out normal functions for host cell Entry • The attachment of a virus to its host cell results in changes to both virus and cell surface that facilitate entry • Bacteriophages leave capsid outside the cell and viral genome enters cytoplasm • Viral proteins accompany genome of some viruses (e.g. RNA viruses) to ensure replication Example: Bacteriophage T4 • Infects E. coli • Virions attach to cells via tail fibers that interact with LPS • Tail fibers retract, and tail pins contact E. coli cell wall • Lysozyme-like enzyme forms small pore in peptidoglycan • Tail sheath contracts, and viral DNA passes into cytoplasm through tail tube Transcription & Translation • Within a minute after T4 entry, synthesis of host DNA and RNA ceases and transcription of phage genes begins, followed by translation • Phage DNA replication begins within 4 minutes of infection • T4 genome encodes three sets of proteins: • Early proteins: enzymes needed for DNA replication and proteins that modify host RNA polymerase so that it recognizes only phage promoters • Anti-sigma factor binds to σ70 and prevents it from recognizing host genes • Middle proteins: additional proteins that modify RNA polymerase and direct expression of… • Late proteins: head and tail proteins and enzymes required to liberate mature phage particles T4 nucleases, Phage T4 DNA DNA polymerase, and new sigma factors Infection Phage head Tail, collar, base plate, and tail proteins fiber proteins Phage DNA replication Mature T4 virion T4 lysozyme production Transcription Middle mRNA Early mRNA Late mRNA Self-assembly Translation Early proteins 0 5 Middle proteins 10 Lysis Late proteins Minutes 15 20 25 Packaging of T4 genome • Double-stranded DNA is pumped into a precursor prohead using an ATP- powered motor • After head is filled with DNA, T4 tail, tail fibers, and other components are added • Two very late enzymes compromise host cell membrane & peptidoglycan layer • Cell breaks by osmotic lysis and releases >100 new virions Virus growth curve • “One-step” growth curve • Virion numbers show no increase until cells burst & release new virions • Latent period includes eclipse + maturation • Eclipse: Period following attachment during which infectious virions cannot be detected in the growth medium • Maturation: Newly synthesized viral nucleic acids are packaged inside capsids • Burst size: number of virions released • Varies from a few to a few thousand Culturing, detecting & counting viruses • Viruses replicate only in certain types of host cells • Bacterial viruses are easiest to grow & study • Grow a pure culture of bacteria as a lawn on surface of agar & inoculate with virus • Animal viruses (and some plant viruses) can be cultivated in tissue (cell) culture • Cells of a specific type are isolated & grown in sterile culture media in plastic vessels Plaque Assay • Titer: Number of infectious virions present per volume of fluid • Can be determined by a plaque assay: • Plaques are clear zones that develop on lawns of host cells • Lawn can be bacterial or tissue culture • Each plaque results from infection by a single virus particle • Viral titer usually reported as “plaque-forming units” per mL Bacterial plaque assay Animal cell culture viral plaques Confluent monolayer of tissue culture cells Viral plaques Plating efficiency • The number of plaque-forming units is almost always lower than direct counts by electron microscopy • Efficiency of infection is not 100% • Inactive virions that did not assemble correctly or contain defective genomes • Viral growth conditions are not optimal • Some virions were damaged by handling or storage • Bacteria viruses usually have efficiencies > 50% • Animal viruses may be much lower (0.1 – 1 %) Temperate Bacteriophages & Lysogeny • Some viruses always kill the host following infection • Temperate viruses can alternate between two pathways: • Lytic pathway: Assembly of virions & lysis of host • Lysogenic pathway: Virus genome is replicated in synchrony with host chromosome without killing host • Lysogeny can confer new genetic properties on the bacterial host cell • = Lysogenic conversion • A cell harbouring a temperate virus = a lysogen • Integrated viral DNA = prophage Bacteriophage lambda • Infects E. coli • Linear, dsDNA genome with complementary overhangs at either end • Upon penetration of host cell, DNA circularizes • If entering lysogenic state, DNA integrates into E. coli chromosome using att site • Requires lambda integrase (phage- encoded enzyme) Lambda lytic pathway • When entering into the lytic pathway, long, linear concatemers of viral DNA are synthesized from circular genome by rolling circle replication • Cut into genome-sized lengths at cos sites & packaged into phage heads • Tail added & cell lysis to release virions Lysis or lysogeny? • Whether lysis or lysogeny occurs following lambda infection depends on levels of two key repressor proteins: • cI & Cro • A protein called cIII stabilizes & protect a protein called cII, which activates synthesis of cI • If cI accumulates following infection, it represses Cro • Lambda integrates & becomes prophage = lysogeny • If Cro accumulates, it represses cII expression & thereby decreases cI • Lambda enters lytic pathway Bacterial Virus Diversity • Most common bacteriophages are head-and-tail with double-stranded DNA genomes • ssDNA genomes are converted into a double-stranded replicative form that is transcribed and replicated • MS2 has ss RNA genome that encodes only four proteins • RNA is plus sense – Can be translated directly upon entry into the cell • Encodes RNA replicase: Enzyme to replicate the viral genome • Small genomes may contain overlapping genes An Overview of Animal Viruses • Two key differences: • Entire virion enters the animal cell • Eukaryotic cells contain a nucleus, where many animal viruses replicate • All types of viral genomes exist, and are further classified as enveloped or nonenveloped Consequences of virus infection in animal cells • Virulent infection: Results in lysis of host cell • Most common • Latent infection: Viral DNA does not replicate and host cell is unharmed • Persistent infections: Release of virions from host cell by budding is slow & does not result in cell lysis • Infected cell remains alive and continues to produce virus • Transformation: conversion of a normal cell into tumor cell by a virus The Virosphere and Viral Ecology • Viruses are present in every environment in enormous numbers • 1 mL sea water contains ~ 106 prokaryotes and 107 viruses • Bacteriophages thought to have major impact on evolution of Bacteria • Lysogenic phages can integrate & confer new properties • Agents of transduction • Most of Earth's genetic diversity resides in viruses • Most viral genes have unknown function