2 Structure
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Transcript of 2 Structure
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General VirologyVIRUS STRUCTURE
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Virion vs virusVirion is the infectious particlecomposed of nucleic acid, protein capsid, +/- envelopemay be extracellular or intracellularVirus is any stage of infection
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How do we know that NA is genetic material?Hershey-ChaseFraenkel-Conrat Experiment TRANSFECTION EXPTS
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TRANSFECTION FAILS FOR SOME VIRUSESWHY?
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CapsidFunctionsProtection of NAAttachment for naked virusesEnzymeHelical vs Icosahedral Symmetry - Why do most viruses look alike?Tobacco mosaic virus is a ssRNA virus composed of 6000 nucleotides. The capsid is made of 2100 copies of a single protein subunit that contain 158 amino acids. Calculate the percentage of the genome that is used for structure.
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Helical- one axis of symmetry down centerMultiple structural unitsHow do helical viruses differ?
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Icosahedral symmetry20 identical equilateral trianglesStructural units on faces to give morphological capsomersPentons (5 fold axis of symmetry)Hexons3 fold through face2 fold through edge
How do spherical viruses differ?
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EnvelopeAttachmentEntryAssembly- matrix proteinsReleaseProteins are viralLipids are hostRare in plants or bacteria - why?If the membrane envelope is destroyed, the virus becomes noninfectious. Why?
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Herpesvirus complexityTegument proteins - 12/84 viral proteins in HSVPotential role?
Virion mRNA DNAase virion nucleic acidsRT-PCR probe genome arrayPotential role?
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Genome - DNA or RNAstrandedness - (single) (double)linear or circular, partial double stranded circlenumber (single, segmented, multicomponent)
How do we experimentally show that DNA or RNA is the virus genetic material?
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RNA Genomessense (positive-sense, negative-sense, ambisense) presence or absence of 5'-terminal cap or 5'-covalently-linked proteinpresence or absence of 3'-terminal poly (A) tractRetroviruses - replication strategy
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Some viruses have high degree of secondary structurePoliovirus - 5 internal ribosome entry site (IRES)Guest et al. 2004. J. Virol. 78: 11097.
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SARS/coronaviruses have conserved 3regionSARS s2m in reda - green = 530 loop of 16S RNASimilar binding properties:b - blue = S12magenta = IF1Possible role for s2mHijacks protein synthesis from cell(binding cell factors)Needed to bind to similar viral protein for transcriptionPotential drug target in red tunnelRobertson et al. 2005. PLOsBiology:3.
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DNA Viruses may be large genomesPolyDNAvirus (PDV) - contain many DNA segments
Mimivirus - larger than small bacteria
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Host-induced modificationViral property that varies depending on the host Phage DNA hydroxymethyl cytosine (HMC) replaces C Viral enzymes: C to HMCViral DNA polymerase: adds HMC not CWhat is advantage of HMC?Glucose is attached to HMCHost enzyme needed to prepare glucoseProtects against host nuclease
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What would happen if virus without glucose enters host with RE?What would happen if virus with glucose enters host w/o enzyme to create UDP- glucose?Host enzyme makes
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Proteins
structural proteins non-structural virion proteinstranscriptase, proteaseintegrase
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How to identify virion proteinsPurify KSHV virionsRun on SDS PAGEExcise bands, digest - get sequence and compare to database
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Chemical synthesis of poliovirus: What are the implications?Small genome positive strand RNA - sequence knownSynthesized small DNA segments (~ 69 nucleotides) with overlapping complementary segmentsAdded a T7 phage promotor to DNA Used DNA to make genome RNA in HeLa cell lysate with T7 polymeraseResults: How do you show success?
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International Congress on Taxonomy of Viruses http://www.ncbi.nlm.nih.gov/ICTV/Morphologyvirion sizeenveloped or naked nucleocapsidcapsid symmetry and structure
Genome characteristicsReplication strategyAntigenic Properties
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Baltimore classification
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WHY TRANSFECTION FAILS
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ONE STEP GROWTH CURVE1939- Ellis and Delbruck: Infection with a high multiplicity of infection (MOI): ratio of virus to host cellSimultaneous infection Single replication cycleSample at time intervals by plaque count for plaque-forming units (PFU),Identification of latent phaseDetermination of burst size/viral yield
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Measuring Intracellular EventsSample at time intervals after lysing cells (1952 - Doermann)ChloroformLysis from withoutIdentification of eclipse and maturation phases
Maturation phase
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Strategy of replicationLyticTemperate
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Biologic Propertiesnatural host rangemode of transmission in naturevector relationshipsgeographic distributionpathogenicity, association with disease tissue tropisms, pathology, histopathology
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How can you identify these viruses?