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Microbiology and Immunology
MICR 221
Eric B Carstens

MICR 221: Virology Section Dr. Eric Carstens Learn:  Material about viruses discussed in lectures. o Material in text book related to what is discussed in class.  Classification of Viruses 7 ed. (on Moodle under the Taxonomy of Viruses)  Characteristics of major families of viruses discussed during lectures -ViralZone -Virus Database On-line Should be able to:  Identify major virus families and understand their structure and replication strategies.  Know how information about viruses is derived.  Recognize that role viruses play in life sciences in addition to their potential as pathogens. Background to the Discovery of Viruses Pasteur, Kock and Lister recognized diseases and were trying to discover what caused them. Their discoveries lead to a new experimental approach for medical science, defining whether an organism was the causative agent of disease. Koch’s Postulates If you can identify these four components you know what has caused a disease. The pathogen: 1. Must be present in every case of the disease. 2. Must be isolated from the diseased host and grown in pure culture. 3. From the pure culture must cause the disease when inoculated into a healthy, susceptible laboratory animal. 4. Must be isolated from the inoculated animal and must be shown to be the original organism. The Germ Theory as proposed by Pasteur and articulated by Kock (and his postulates) did not recognize the existence of viruses. -In many cases viruses cause different diseases – cause different infections or don’t cause and infection at all. -Couldn’t at the time grow viruses in any culture. -Needed a different way to look at viruses – a way to answer: what is a virus? What is a Virus? At first: Agent of Disease or Symptoms Some of the first work was done on tobacco - looking to find what caused white and dark spots on tobacco leaves.  Adolf Mayer was a German working in Holland  “Tobacco mosaic disease” – light and dark spots on leaves  Juice from infected leaves inoculated into healthy plants caused disease transmission.  Agent was unknown, speculated it was caused by an unknown infections form. Filterable Infections Agent  Dimitri Ivanofsky  Commissioned by Russian Department of Agriculture – tobacco disease.  Repeated Mayer’s experiments, with additional step:  Passed infected sap through a filter with pores small enough to block bacteria.  1892 – “the sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filer candles” Provided operational definition of viruses. Chamberland Filter Made of a porous porcelain tube through which water is forced under pressure. The residue left on the outside of tubes can easily be removed, and tubes themselves periodically sterilized by boiling. Used in municipal water-works for filtration. Obligate Intracellular Parasite  Martinus Beijerinck collaborated with Mayer.  Filtered sap could be diluted and then regain its strength (virulence) after transfer through living, growing plant tissue o Contagium vivum fluidum (soluble living germ)  Agent remained infective through several transfers so was not a toxin o Explained failure to culture the pathogen outside its host o 1998 centenary celebration of virology (Tobacco mosaic virus)  New concept for the turn of the 20 century o A filterable agent too small to observe in the light microscope but able to cause disease by multiplying in living cells.  Loeffler and Frosch (1898) isolated first filterable agent from animals (foot and mouth disease virus – family Picornaviridae)  Reed et. Al (1900) recognized first human filterable agent (Yellow fever virus – family Flaviviridae) and also showed mosquito transmission of agent. Chemical makeup of filterable agents  Methods to purify proteins (precipitation) also purified Tobacco mosaic virus o How did they detect the agent? Assayed by infectivity (in host plants!)  TMV, like proteins, migrated in an electric field.  Rabbit antibodies directed against TMV neutralized infectivity.  Led to the question – Are viruses proteins? Viruses contain both protein and nucleic acid  By 1929, concluded that viruses consisted of protein  But in 1934, Max Schlesinger in Germany showed that bacteriophages contained both protein and deoxyribonucleic acid (DNA) o First suggestion that viruses were composed of nucleoprotein (nucleic acid plus protein) Viruses contain only one kind of nucleic acid  In 1935, Wendell Stanly crystallized TMV o These infections crystals contained protein and 5% RNA  In 1940, Hoagland at the Rockefeller Institute showed that vaccinia virus contained DNA but no RNA.  Concept: virus genetic material is DNA or RNA but not both! What do viruses look like?  In 1939, the first electron micrographs showed TMV was rod shaped while x- ray diffraction suggested TMV was built up from repeating subunits.  Concept: most simple viruses consist of one or a few identical subunits. What is a virus?  Infectious, obligate intracellular parasite.  Genetic material of a virus enters a host cell and directs the production of the building blocks of new virus particles (called virions)  New virions are made in the host cell by assembly of these building blocks  The new virions produced in a host cell then transport the viral genetic material to another host cell or organism to carry out another round of infection.  Differ from bacteria in size, their inability to grow in “lifeless” media and that they contain only one kind of nucleic acid, either DNA or RNA.  Filterable, disease-causing agent, completely dependent upon living cell (intracellular parasite) o Self replicating in host cell o Inert (dormant) outside a living cell  Contrasts with dynamic growth of bacteria Viruses are everywhere! Why aren’t we always sick?  Host defenses: physical and immunological (innate and adaptive immunity).  Specificity: proportionally, few microbes are capable of infecting us.  Inapparent infections: often we are infected but with no overt signs.  Viruses are the most abundant biological entity in the oceans and the second largest component of biomass after prokaryotes. Affect climate. All of the small plants and animals living in the ocean are going through their normal life cycles. If a virus infection kills a large proportion of the microorganism that alters the carbon make up which helps sequester carbon out of the atmosphere. Carbon sequestration involves a lot of sinking of dead mass. If you lyse these materials the carbon can be released. Remember:  There is more biological diversity within viruses than in all the rest of bacterial, plant and animal kingdoms put together!  Virus/host interactions are molecular negotiations of fundamental and practical significance that must be understood if we are to defend ourselves against viruses.  The very things that make viruses insidious and lethal can be exploited for practical use through the development of viruses as productive tools and therapeutic agents. Lecture 2 Largest Virus  Amoeba-infecting Mimivirus (mimicking microbe virus) o DNA genome encodes roughly 1000 genes (some viruses have only 1)  Megavirus chilensis: 77kb larger that Mimivirus o Possess DNA genome in excess of a “megabase” o Isolated from water near Las Cruces, Chile o Very large, can be seen by light microscopy Identification of Viruses – How are they characterized? th  Once the concept of filterability took hold (late 19 century), this experimental procedure was applied to many diseased organisms/tissues.  A wide variety of viruses were discovered based on physical properties: o Size: using filters with different pore sizes o Resistance to chemical or physical agents: e.g. alcohol, ether, temp. o Pathological effects: effect on host (plants and animals) Investigating the Invisible  Early characterizations caused confusion, leading to debates whether filterable viruses were animals or plants (at the time, this was a big deal!)  1917 – Felix d’Herelle, a Canadian bacteriologist at the Pasteur Institue, in Paris, working with Shigella dysentery, noticed clear circular spots on his Petri dishes where no bacteria grew. o Called them taches vierges or plaques o Plaque turned out to be very important in characterizing agents.  d’Herelle – credited with development of principles of modern virology. Important techniques in virology developed by d’Herelle  Realized that plaques were caused by viruses that he called bacteriophage (bacteria eater).  Used limiting (serial) dilution and plaque assay to measure virus titer (quantify infectious virus – plaque forming units PFU)  Reasoned that appearance of plaques meant that virus was a physical entity (particulate or corpuscular)  Demonstrated first step in virus infection was attachment (adsorption) to host -Co-sedimentation of virus and host after mixing and centriguging (retained virus in pellent). Loss of virus from supernatant fluid.  Host range specificity can occur at adsorption step -Attachment only occurred when bacteria sensitive to specific virus were mixed.  Described cell lysis as the release of infectious virus -Recognized that passage of time was required for bacteriophage maturation and concluded that phage must be “reproducing”. Serial Dilution Concept o Prepare serial dilutions using sterile technique. Add 0.5ml of each dilution to monolayer of susceptible cells. Adsorb virus for 1 hr, then remove, wash and add agarose overlay. Start out with sample virus. Make 10-fold dilutions (0.1mL + 0.9mL diluents). Then you add this material to a monolayer of cells – in the lab you use bacteria. Add constant volume to plate containing susceptible host cells, then add an agarose overlay to prevent bacteria from moving around (fixes them in place). Then you incubate at a specific temperature and wait for plaques to appear.  Note the technical differences between what you did in lab with bacteriophage and this protocol used with eukaryotic viruses. IT’S DIFFERENT. Viruses Require Living Cells for Reproduction  Plant viruses – required plants  Insect viruses – required insects  Animal viruses – required animals  Bacteriophage- required bacteria o But could easily be studied in Petri dishes and test tubes so became model. The Phage Group  Established by Max Duelbruck (physicist) and Salvador Luria (MD) in 1941  Viewed bacteriophage/bacteria as model system for understanding cancer viruses, fertilization and developmental biology.  Phage were seen as probes to learn how genetic information could determine biological structure and function. Beginnings of Virology/Molecular Biology  1950 – 1975: highly productive period of virus research using phage o Delbruck standardized experimental protocols so results would be comparable from lab to lab.  Combined genetics and biochemistry to elegantly describe, for example: o Lytic infection of E. coli with T-even phage o Nature of lysogeny, using lambda phage o Replication and properties of both RNA and DNA phage  Search to apply same techniques to animal viruses Conversion of Animal Virology to Laboratory Science Simplification of the experimental system under study (1948-1955) Study animals in wildlab animals (hen’s egg) culture of tissues cells in culture Animal Cell culture for Eukaryotic Viruses  Required development of methods for production of single cells, medium (‘food for cells’) and characterization of primary and continuous cell lines. o Primary cell culture – finite life span in culture. o Continuous cell culture – abnormal, often transformed, grow ‘forever’  Cells produced by trypsinization (serine protease) from: o Embryonic tissue (primary cell line) o Carcinoma tissue (continuous cell line) e.g. HeLa cells  1949: replication of poliovirus in non-neuronal human embryonic explants Prep. of Primary Cell Cultures: Break tissue down to single cells mechanically and with protease (trypsin) and place cells: feed with synthetic liquid media). Cell Culture and Virology  Led to development of the polio vaccine, “killed” virus o 1955 first vaccine produced in cell culture (monkey kidney cells)  All other then in use (against smallpox, rabies, yellow fever, influenza) produced in animals or embryonated hen’s eggs.  Exploitation of cell culture began modern era of molecular virology o E.g. first plaque assay for an animal virus was with poliovirus, lead to analysis as detailed and important as contemporary work with phage. Primary to Continuous Cell Lines Transformations where cells actually become immortalized because of accumulation of mutations – these become a continuous cell line and can be grown forever in the laboratory (the rest die). The important point is that we keep diluting these cells so that they are not touching each other. Different Viruses Produce Different Particle-to-PFU Ratios  High ratio of shows that not all virions are successful in replicating.  High ratio sometimes caused by presence of noninfectious particles with genomes that harbor lethal mutations or that have been damaged during growth or purification.  Although all viruses in a preparation are capable of initiating infection, not all succeed because of the complexity of the infectious cycle. Failure at any one stop in the cycle prevents completion.  High ration does not indicate that most articles are defective, but rather, they have failed to complete the infection. Metagenomics and Virus Identification  Nucleotide sequences from environmental samples o No isolation of virus or host  Massive sequencing approach of unculturable microorganisms.  Data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. o Computational analysis of viral metagenomic data is particularly challenging.  E.g. Most viral genes have no annotated homolog in sequence databases. Summary  Viruses require living cells to replicate o Characterization of viruses usually in cell cultures o Not all viruses can be propagated in cell cultures  Viruses can be quantified by physical or biological methods: o Counting physical particles by EM (electron microscopy) or epifluorescence microscopy. o Genome copy number by quantitative PCR o Plaque assay (quantifies infectious particles – PFU/ml) o Hemagglutination assay (quantifies particles approximately)  Recent application of metagenomics to identify hundreds of newly recognized virus-like “genomes”. Lecture 3 – Virus Structure Virions  A virus particle (virion) is a structure evolved to transfer nucleic acid from one cell (and/or host) to another.  Nucleic acid may be either RNA or DNA  Virions of varying complexity carrying either type (RNA or DNA) are found.  Virion structure is related to replication strategies.  Evolved to transfer nucleic acid from one cell (and/or host) to another.  Nucleic acid may be either RNA or DNA.  Virions of varying complexity carrying either type are found.  Virion structure is related to replication strategies. How small are viruses? Why do viruses need to build a particle to protect their genomes? Why doe viruses need to build a particle to protect their genomes?  It’s dangerous out there in the ‘real world’ both inside and outside hosts: o Proteolytic and nucleolytic enzymes (degrade DNA/RNA of viruses) o Extremes of pH o Extremes of temperature (e.g. in nose and throat) o Various forms of natural radiation (UV) o Shearing by mechanical forces (large DNA and RNA viruses)  Part of packaging DNA or RNA is to prevent shearing that would nick strands of nucleic acid & kill the virus. Virus Ultra-Structure 1956: Crick and Watson proposed principles of virus structure:  Nucleic acid in small virions insufficient coding capacity for many proteins. o Genetic economy  More efficient to use the same protein redundantly: identical protein subunits.  Identical protein subunits used over and over again as building blocks to make virus particle. o Expected subunits packaged to provide each a “quasi-identical” environment: symmetrically.  Subunits in an identical environment must be packed together to fit some form of cubic symmetry. Nomenclature of Virus Structure: Definitions  Protein subunit o Single folded polypeptide chain (gene product) o E.g. VP1 of poliovirus (Virus Protein 1)  Protomer (structural unit) o Collections of one or more (non-identical) protein subunits that together form the chemical building block of a larger assembly. o E.g. VP1, VP2, VP3 (and VP4) together in poliovirus. o VP1 in Simian virus 40  single polypeptide makes up polymer.  Assembly unit (usually symmetrical) o A set of subunits or structural units (protomer) that is an important intermediate or subassembly in the formation of a larger structure.  Polio virus VP1-VP2-VP3 pentamer.  SV40 VP1 pentamer: made up pf 5 VP1 protomers. 5 polio virus protomers (structural units) assemble to make a pentamer (assembly unit), introducing curvature to the pentamer. 12 pentamers make up the virion capsid. Definitions cont.  Capsomere: Apparent lumps or clusters seen on the surface of a particle by electron microscopy (may not correspond to chemically unique proteins). Morphological assembly unit of icosahedral capsid.  Capsid: Coat of protein directly surrounding and protecting viral nucleic acid (also called coat or shell).  Core: Internal part of virion, consisting of nucleic acid nad closely associated proteins.  Nucleocapsid: protein-nucleic acid complex (core) that is the packaged form of the viral genome in a virus particle. [Capsid + nucleic acid]  Envelope (when present): Lipid bilayer (membrane) and associated glycoproteins (spikes) surrounding many types of virus particle.  Virion: The mature infections virus particle. Variety of Capsomeres  Adenoviridae o Fibre (12) o Penton base (12) o Hexon (240) o Ninemer (9 hexons) o Peripentonal hexons  Polyomaviridae o Pentamer on face o Pentamer at vertex Capsid Protects Fragile Genome  Capsid can help prevent: o Physical damage – shearing by mechanical forces. o Chemical damage – UV irradiation (from sunlight) leading to chemical modification. o Enzymatic damage – nucleases derived from dead or leaky cells or deliberately secreted by vertebrates as defense against infection.  The capsid must be stable enough to protect the nucleic acid when in the extracellular environment BUT o Virus particles also mediate the attachment of the virus to an appropriate host cell and deliver the genome to the interior of that cell, where the particle is at least partially disassembled. Virion Capsid  Capsid is a “molecular machine” for selective genome packaging and delivery. o E.g. DNA packaging into Mimivrius capsid. Capsid Assembly  Non-covalent bonds between capsid subunits are the same sort that stabilize folded protein domains o Interface between two subunits looks much like interior of a single domain. o Arms of one subunit extend under or over neighbouring subunit domains.  Contacts between individual subunits determine overall structure. Protein Structure 3D structure of a protein is stabilized by non-covalent interactions between its amino acid residues  Viral structural proteins contain one or more regions forming globular domains separated by extended and flexible regions (arms and hinges) o Domains are independently folded regions, 100 — 200 amino acids in size. o Arms or hinges form defined structures only when interacting with other chains in an assembled virion. Virus particles can form spontaneously  If subunits are identical, repeated contacts occur, resulting in symmetry.  Free energy minimum state – high stability  Some simple virus capsids can form spontaneously from dissociated subunits (important concept) o Self assembly via hydrophobic and electrostatic interactions (non- covalent) o Protein-protein, protein-nucleic acid and protein-lipid o Empty “virus-like particles” (VLPs)  No nucleic acid Influenza Virus-Like Particles o Infections influenza virus with surface antigens, lipid membrane, internal proteins and genetic material. o Virus like particle (VLP) is a non-infectious and a more efficient way of presenting antigens to the immune system. Virion Symmetry  Three major mechanisms used to package genome: o Rod-like structures have helical symmetry  Hollow cylinder, rigid or flexible  E.g. filamentous nucleocapsid of influenza virus, tobacco mosaic virus, baculovirus.  o Spherical viruses have icosahedral symmetry  20 triangular faces and 12 vertices  E.g. adenovirus, herpesvirus, poliovirus  o Complex viruses may have mixed elements  E.g. many bacteriophage, poxvirus  Examples of Esoteric Viruses o Ampullaviridae: Acidianus bottle-shaped virus o Bicaudaviridae: Acidianus two-tailed virus Virus Envelopes  Many viruses are enveloped o Lipids and carbohydrates usually derived from host cell nuclear or cytoplasmic membrane components. o Envelope proteins are coded by virus genes, may include spikes or peplomers.  Function during virus cell entry and replication o Associated with receptor binding, membrane fusion (hemagglutinin spike), secondary uncoating or transcriptase activation, receptor destruction (enzymatic activity – neuraminidase spike), virus assembly, immune evasion. Why do some viruses have envelopes? Most enveloped viruses acquire their envelope by budding through a membrane of the host cell into some extra-cytoplasmic compartment. By contrast, non-enveloped viruses generally escape by lysis of the infected cell. Cell lysis is terminal. Thus enveloped viruses do not necessarily have to kill the cell in the course of their replication. The presence of an envelope also provides viruses with an opportunity to insert virally encoded glycoproteins (spikes) into the envelope. These glycoproteins can play roles in multiple facets of the virus lifecycle including: o Entry and host range determination o Assembly and egress o Evasion from the vertebrate immune system Week 4 Lecture 1 Viral Genomes are Diverse  DNA (deoxyribonucleic aicd) viruses: o Linear OR circular. o Single stranded OR double stranded. o Single stranded can be either (+) or (-) polarity. o OR partially double stranded and circular.  RNA (ribonucleic acid) viruses: o Linear OR circular (only Hepatitis delta virus). o Single stranded OR double stranded. o Single stranded can be either (+) or (-) polarity or both (ambi-sense) o Monopartite (single segment) or multipartite (multiple genome segments) Viral Genomes – Conventions (Important!)  mRNA is defined as positive strand (+) because it contains immediately translatable information.  A strand of DNA that contains the equivalent polarity is also designated as (+) strand.  The RNA & DNA complement of the positive strand is the minus strand.  (+) strands are synthesized using (-) strands as a template and vice versa. Virus Genomic Variety – Examples  Genome may be DNA or RNA but not both  Genome may be single stranded or double stranded  Genome may be monopartite (single segment) or multipartite (multiple segments make up genome.  Genome may be linear or circular (ALL RNA IS linear except Hepatitis D)  Single stranded can be either positive or negative sense  May be double stranded with cross-linked ends  Linear RNA in virions is diploid  Virus Example DNA ss or ds? Monopartite/ Linear or Sense or multipartite? circular RNA? Parvoviruses DNA ss ________ Linear (Parvoviridae) Herpesviruses DNA ds Linear (Herpesiridae) Adenoviruses DNA ds Linear (Adenoviridae) Vaccinia DNA ds Smallpox DNA ds cross- (Poxviridae) linked ends Polyomaviruses DNA ds Closed circular Papillomaviruses DNA ds Closed circular Baculoviruses DNA Ds cross- Closed (Baculoviridae) linked ends circular Picornaviruses RNA ss Linear (+) (Picornaviridae) Rhabdoviruses RNA ss Linear (-) (rabies) Paramyxoviruses RNA ss Monopartite Linear (-) (Paramyxoviridae) (single seg.) Retroviruses (HIV) RNA ss Segmented Linear (+) (Retroviridae) (diploid) Orthomyxoviruses RNA ss 6-8 segmented Linear (-) (Orthomyxoviridae) Reoviruses RNA ds 10-12 Linear (Reoviridae) segmented The polarity 5’  3’ on upper strand in diagram of strand. Genome Diversity and Intracellular Replication  Most DNA viruses replicate in the cell nucleus, whereas most RNA viruses replicated in the cytoplasm of the infected cell.  There are, however, important exceptions! o Poxvirus genomes are DNA, yet they replicate in the cytoplasm. o Influence virus genomes are RNA, yet they replicate in the nucleus. Virus Classification and Taxonomy  New viruses constantly being identified.  There are vertebrate viruses, invertebrate viruses, plant viruses, bacterial viruses, archaea viruses, fungal viruses and viruses of unknown host. Even viruses may have viruses (“virophage”)!  Virus taxonomy constantly evolving, under the direction of the International Committee on Taxonomy of Viruses (ICTV) o Virus taxonomic units: Order, Family, Genus, Species  Viruses we work with in the lab are isolates of species.  To date, over 6,000 viruses classified in 2818 species have been identified o 7 orders, 96 families, 420 Genera  To develop a firm basis for answering all questions related to modern virology, we need a common language for discussion (taxonomy) and a way to group viruses into “classes” (classification) How are viruses classified?  Two classification schemes o Classical system (Fig. 25.2)  The Baltimore classification system o Based on central dogma – DNA makes RNA makes proteins Table 25.1 The Classical System  Viruses grouped according to their shared physical properties rather than their hosts.  Four characteristics used in classification: o Nature of the genetic material in the virion (DNA or RNA) o Symmetry of the capsid (helical, icosahedral or complex) o Naked or enveloped o Dimensions of the virion and capsid The Baltimore Classification System  Need to know examples of the viruses (the underlined ones) and how they replicate and divide up by the classification system.  A molecular biologists view of virus classification: o Based on the Central Dogma DNA  RNA  Protein o All viruses must produce mRNA that can be translated by cellular ribosomes.  The unique pathways from various viral genomes to mRNA define specific virus families on the basis of the nature and polarity of their genomes.  Knowledge of these designations provides virologists with immediate insight into the steps that must take place during virus replication and gene expression. Taxonomic Criteria  Genome organization and replication o Intracellular location of replication, presence or absence of DNA intermediated.  Nucleic acid characteristics o DNA or RNA, size, strandedness, linear or circular, (+) or (-) sense, number and size of segments.  Genomics (gene/genome sequence) o Development of sequence databases for prototype viruses of nearly all taxa, assisting identification, epidemiology and diagnosis. -RNA virus genomes are often smaller than DNA -Less stable than DNA so they’re more susceptible to hydrolysis. Review: Important Concepts in Molecular Virology (and life!)  Replication: to make new genome copies (DNA or RNA)  Transcription: DNA or RNA template is transcribed into mRNA  Translation: mRNA is translated into protein NOTE: there are exceptions in the world of viruses since may have RNA genomes. Protein comes from mRNA; mRNA from RNA or DNA (going the other way) The virus must present a recognizeable virus mRNA to the host replication system. All viruses will eventually present their mRNAs and present them to the host cell that they’ve infected. Key concepts:  Virus must present a recognizable mRNA to host translation machinery.  Virus mRNA synthesis (transcription) can be directed by host (nucleus only) or virus transcription factors (nucleus or cytoplasm)  Virus mRNA must be translocated into the correct cell compartment (cytoplasm only) to be translated into virus protein. Animal Viruses with DNA genomes Animal Viruses with RNA genomes Role of the Host Cell Essential cell machinery that the viruses need  Required for processes not directed by the virus o Nuclear transcription by host RNA polymerase II and RNA splicing. o Intracellular transport of viral components to the appropriate cell sites. o Protein synthesis system. Outline of Baltimore Classification classes: based on viral genome and process to synthesize RNA ??????????  I: herpesviruses and many others  II: parvoviruses  III: reoviruses (+/- RNA)  IV: picornavirus, many others,  V: orthomyxoviruses, many others, (-RNA)  VI: retroviruses (RNA viruses that go through DNA intermediate)  VII: hepadnaviruses, (DNA viruses that go through RNA intermediate) Both have to go through a synthesis of the opposite polarity strand in order to make genome. Positive sense genome: same as a messenger RNA. Can be translated directly into protein. Negative sense genome: Virus Replication Cycle  Why should you care about virus replication strategies? o Studying virus life cycles exposes vulnerabilities that may be exploited in the treatment of the disease.  However, because viruses are obligate intracellular parasites, they rely on the host cell biosynthetic machinery for their propagation. o Problem – drugs that are effective at blocking virus replication are often toxic to cells, and thus also to he host. This is reflected in the relative paucity of the antiviral chemotherapies available today. Solution?  Identify important virus specific activities o Characterize virus replication patterns o Devise ways to interfere with pathways through the development of antiviral drugs. o Develop vaccines that protect against viral infection  Prevention is the best method! Can’t cure once you’ve got it. Viral Replication Patterns: Single-Step Growth Curve  Protocol: performed in cultured cells (homogenous system for studying virus replcation)  Every cell is infected – normally use a multiplicity of infection (MOI) of 10. o Theoretically; 10 infectious virions per cell o Practically; virtually ever cell infected with at least 1 PFU  Virus is first adsorbed (added) to cells at 4C, hen shifted to 37C in order to synchronize the infection. This is just to get the virus to contact the host cell, at a low temperature. Penetration of the virus into the cell cannot occur at 4C because penetration is an energy-dependent process. Adsorption is usually not energy dependent. o Use a plaque assay (important) to monitor how much infectious virus is being produced at different times during infection. Initial Stages of Virus Replication Cycle  Eclipse period: o Time between absorption of virus to the cell and the appearance of the infections virus within the cell.  Latent period o Time between absorption of virus and release of new infectious virus from the cell.  For some viruses the eclipse period = the latent period o Example: those viruses where maturation and release form the cell coincide. Virus Replication Cycle  Lots happening inside the cell during latent period o Attachment/absorption of the virus to the cell o Penetration of virus into the cell o Upcoating of the viral genome o Viral gene expression o Virus genome replication o Assembly of new viruses and egress or release of virus from the cell 1. Attachment and Adsorption Specific binding of virion protein (anti-receptor) to a cell component (receptor) Virus attachment usually energy-independent process Receptors may be proteins (usually glycoproteins) or carbohydrates on glycoproteins. Anti-receptor examples:  Polovirus and rhinovirus – canyon at the bottom of a surface depression around vertices accessible by receptor projection on cell surface by not by antibodies  Adenovirus – fibre extending from the penton base  Influenza virus – hemagglutinin and neuraminidase spikes on the envelop.  Retrovirus – interaction of HIV glycoproteins gp120 and gp41 with cell surface receptors CD4 AND CCR-5 Both proteins and carbohydrates can serve as receptors for viruses Carbohydrate receptors less specific than protein receptors because same configuration of carbohydrate side-chains may occur on many different glycosylated membrane-bound proteins. Many viruses use multiple receptors Multiple receptors used by viruses to gain access into different tissue types. Sometimes multiple receptors are utilized for the entry virus into a single cell.  E.g. co-receptors  Curiously, a number of very different viruses use the same cellular receptor.  Remember that viruses are constantly evolving along with their hosts. Mechanisms of viral attachment/adsorption into host cells (a) Influenza viruses (b) Herpes Simplex Virus (c) HIV/AIDS virus Summary  Discovery of new viruses requires a system/process for classification.  Two different ways to classify viruses o Their physical properties (“classical”) o Their replication strategies (“Baltimore”)  Understanding virus replication cycles has led to insights into both virus and host processes.  Viruses must present a recognizeable mRNA to the host cell so that virus- specific proteins are made.  Virus replication cycle is a continuum but can be broken down into discrete steps. o The first step is attachment but remember this is all very dynamic. Virus Replication Cycle A Virus infecting a virus? Sputnik virophage (not that important)  “Satellite virus”: infection requires host cell to be co-infected with Mimivirus.  Non-enveloped, icosahedral capsid. Virion can be incorporated into helper Mimivirus capsid particle. Ciruclar, dsDNA genome. Virus Replication Cycle  Attachment/adsorption of virus to the cell.  Penetration of virus into the cell.  Uncoating of the viral genome.  Viral gene expression.  Virus genome replication.  Assembly of new viruses and egress or relase of virus from the cell. 2. Penetration (cont. from 2 pg earlier)  Energy dependent step occurring immediately after attachment (cell must be metabolically active).  Occurs by two general pathways: o Receptor-mediated endocytosis  E.g. influenza virus (Orthomyxoviridae) o Surface fusion of virion envelope with cellular membrane (only with enveloped viruses)  Requires presence of specific fusion protein in virus envelope  E.g. mumps virus (Paramyxoviridae) o May also occur directly by translocation through cytoplasmic membrane (rare!)  E.g. Polio virus (Picornaviridae) o ULTIMATE GOAL: get viral genome into approprate cell compartment. Virus-Host Cell Membrane Fusion Mechanism  Virus membrane-fusion proteins drive the fusion reaction by undergoing a major conformation change triggered by interactions with the target cell (coupling of attachment and penetration).  Specific trigger depends on the virus: o E.g. influenza viruses, alphaviruses & flaviviruses are classic examples of viruses that fuse upon exposure to low pH in endocytic pathway. o By contrast, fusion of HIV-1 occurs at neutral pH, triggered by the sequential interaction o fthe virus fusion protein env (gp120 ad gp41) with the receptor CD4 and a co-receptor such as CCR5 or CXCR4.  Other variations include viruses with fusion reactions triggered by: o Interaction with a single receptor o The binding of the receptor to a separate attachment protein o A combo of receptor binding plus low pH or by endosomal proteolysis.  Probably that there are additional interesting twists yet to be discovered. Variety of Penetration Methods Entry of enveloped virus by fusing with plasma membrane 1) Virus’s envelope spikes bind to receptors on surface of host cell. 2) Lipid bilayer of viral envelope fuses with host cell membrane. 3) Nucleocapsid is released into the cytoplasm. Entry of eveloped virus by endocytosis 1) Viruse’s envelope spikes bind to receptors enriched in the membrane of a coated pit of the cell’s surface. 2) Binding to the receptor triggers receptor mediated endocytosis. 3) Increased acidity allows nucleocapsid to escape from the endosome and enter the cytoplasm. Entry of noneveloped virus by endocytosis 1) Virus’s capside proteins bind to receptors on cell surface and trigger receptor mediated endocytosis. 2) Nucleic acid is extruded from the endosome into the cytoplasm. 3. Uncoating  Events occurring following or together with penetration, setting stage for viral gene expression. o Remember attachment, penetration and uncoating are dynamic and related processes!  Ordered removal of capsid an/or release of nucleic acid.  Can occur at many stages of viral replication and at different sites in the cell (surface to nuclear pore).  Molecular process is poorly understood. Three general strategies for uncoating 4. Initiation of Virus Gene Expression Attachment  Adsorption  Penetration  Uncoating  Once uncoated, viral genome is accessible to host cell machinery including nucleotides, amino acids, enzyme complexes and transport pathways.  The genome serves as a template for all of the molecules that viruses require to replicate and produce new virions. Early Gene Expression  Key event in viral replication: synthesis of viral proteins by host protein- synthesizing machinery.  These viral proteins are: enzymes necessary for replication of genome.  Therefore, a recognizable mRNA coding for these proteins must be presented by the virus in the cytoplasm. Late Gene Expression  Some viruses have a cascade of ordered gene expression which is temporally regulated o Early genes code for proteins that are required for genome replication and expression of late genes. o Late genes usually code for viral structural proteins.  Once the virus produces some of its essential regulatory proteins, it can more easily direct the cell to produce more virions. Intracellular Problems Confronting Viruses  RNA viruses must code for their own RNA-dependent RNA polymerases o Because host eukaryotic cells do not have enzymes necessary to synthesize mRNA from a (viral) RNA genome.  DNA viruses that replicate in the cytoplasm (Poxviruses) must code for their own DNA-dependent RNA polymerases o Because host eukaryotic cells do not have enzymes capable of transcribing viral DNA in the cytoplasm. Gene Expression – Transcription  DNA viruses o Must produce viral mRNA using the (-) strand of the DNA enome as a template. For most DNA viruses the enzyme that performs this function is the host RNA polymerase II. This takes place in the nucleus of infected cells where RNA polymerase II is located.  Notable exceptions are the poxviruses which replicate in the cytoplasm of infected cells and encode their own viral RNA polymase.  (+) stranded ssRNA viruses: o Genomes of these viruses act as mRNA, can be translated directly bu cellular ribosomes: important exception are the retroviruses.  (-) stranded ssRNA viruses and double-stranded RNA viruses o mRNA must be transcribed from the (-) sense genome. This function is performed by viral RNA-dependent RNA polymerases. (Animal cells do not contain this enzymatic function so these viruses must bring this enzyme into the infected cell).  Must package a virus-encoded RNA-dependent RNA polymerase into the virion (KEY CONCEPT).  (+) strands must be coded by (-) strands and (-) strands must be coded by (+) stands (KEY CONCEPT).  Retroviruses (including HIV), circuitous system to make viral mRNA  Genome is (+) diploid RNA. o Replicate RNA genome through a DNA intermediate. o RNA genome is “reverse transcribed: by the viral enzyme reverse transcriptase (RT) into double stranded DNA.  Animal cells do not contain an enzyme that can perform this function. o Double stranded DNA transports to nucleus and integrates into the genome of the host cell (by viral intergrase). o Integrated DNA (provirus) then serves as a template for the synthesis of viral mRNAs using host RNA polymerase II. o This enzyme also synthesizes genome length (+) RNAs (new genomes) using the provirus as a template. Summary  Uncoating can occur at many stages of viral replication and at different stages in the cell (surface to nuclear pore)  Only viruses with DNA genomes that reach nucleus and contain appropriate cis-acting signals can use host transcriptase (RNA polymerase II) to make viral mRNA.  Viruses with RNA genomes must have their own mechanisms for presenting a recognizable mRNA to host translation system (RNA-dependent RNA polymerase).  Viruses with DNA genomes which replicate in the cytoplasm must produce DNA polymerases which function in that compartment and encode their own RNA polymerase.  Viruses have evolved strategies to either confer competitive advantage to viral mRNAs or abolish the synthesis or translation of cellular mRNAs.  Although viruses can trigger an apoptotic response in host cells, some have evolved mechanisms to counteract this effect and repress apoptosis. Virus Replication Cycle 2 Virus Modification of Host Systems: Examples  Use RNA splicing to compress more genetic information into genomes (e.g. Adenoviridae, Papillomaviridae)  Modify host-cell transcription o “Cap snatching” e.g. Orthomyxoviridae  Modify host-cell translation o Degradation of cap-binding complex (host shut-ff) and use of IRES (internal ribosome entry site) e.g. Picornaviridae  Stimulate host-cell macromolcular synthesis (cell cycle S phase) e.g. Polymavirida  Inhibit cell apoptosis (programmed cell death) e.g. Baculoviridae Variation in Virus Genetic Information Remember diversity in viral genomes: they must all replicate in eukaryotic cell. o RNA vs DNA o Single stranded vs double stranded o Monopartite vs segmented o Positive vs negative stranded DNA Genome Replication  Double-stranded DNA viruses  Two groups: o Replication exclusively nuclear. The replication of these viruses is relatively dependent on cellular factos. o Replication occurs in cytoplasm (Poxviruses). These viruses have evolved all the necessary factors for replication of their genomes and are therefore largely independent of the cellular machinery.  Single stranded DNA viruses  Replication occurs in nucleus, involving the formation of a double-stranded intermediate that serves as a template for the synthesis of single stranded genomic DNA. Double strand (ds) DNA viruses  Reminder: Virus family names end in –viridae  Examples: Polyomaviridae, Baculoviridae, Papillomaviridae, Adenoviridae and Herpesviridae o Genome transcribed and replicated in the nucleus and can use the host transcriptional machinery. However, they have at least two or three cycles of transcription (immediate early, early , late cascade)  Poxviridae o Transcribe and replicate their genomes in the cytoplasm and thus encode all factors necessary for transcription and replication of their genomes in cytoplasm. Single strand (ss) DNA Viruses  Genome one small single stranded DNA molecule o Examples: Parvoviridae, Anelloviridae, Geminivirida  Relies on host cell enzymes for replication, producing a dsDNA replicative intermediate.  Only single stranded DNA is packaged but It can be either positive or negative sense: o Adeno-associated virus, human parvovirus B10, torque teno virus 1 Single Strand (ss) RNA viruses positive (+) sense  Plus strand RNA genome serves as mRNA o Examples: Coronavirida, PIcornaviridae, Picornaviridae, Togaviridae, Caliciviridae, Flaviviridae  Genome is infectious as purified RNA  Replicate in cytoplasm  Synthesize large polyprotein which is subsequently cleaved to produce final protein products: o E.g. poliovirus, Norwalk, West Nile, SARS, Dengue, Yellow fever ssRNA Viruses Negative (-) Sense  Negative strand RNA genome – serves two functions o First, template for transcription (transcriptase) o Then for replication (replicase)  Examples: Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Arenaviridae and Rhaboviridae.  Virion brings in transcriptase to make its mRNA o No proof-reading function  high mutation rate (antigenic drift)  Genomic negative strand RNA is not infectious  Segmented genomes can lead to antigenic shift o Orthomyxoviridae  Viral mRNAs are gene unit length, coding for single polypeptides o E.g. Influenza
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