General VirologyVIRUS STRUCTURE

Virion vs virusVirion is the infectious particlecomposed of nucleic acid, protein capsid, +/- envelopemay be extracellular or intracellularVirus is any stage of infection

How do we know that NA is genetic material?Hershey-ChaseFraenkel-Conrat Experiment TRANSFECTION EXPTS

TRANSFECTION FAILS FOR SOME VIRUSESWHY?

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.

Helical- one axis of symmetry down centerMultiple structural unitsHow do helical viruses differ?

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?

EnvelopeAttachmentEntryAssembly- matrix proteinsReleaseProteins are viralLipids are hostRare in plants or bacteria - why?If the membrane envelope is destroyed, the virus becomes noninfectious. Why?

Herpesvirus complexityTegument proteins - 12/84 viral proteins in HSVPotential role?

Virion mRNA DNAase virion nucleic acidsRT-PCR probe genome arrayPotential role?

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?

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

Some viruses have high degree of secondary structurePoliovirus - 5 internal ribosome entry site (IRES)Guest et al. 2004. J. Virol. 78: 11097.

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.

DNA Viruses may be large genomesPolyDNAvirus (PDV) - contain many DNA segments

Mimivirus - larger than small bacteria

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

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

Proteins

structural proteins non-structural virion proteinstranscriptase, proteaseintegrase

How to identify virion proteinsPurify KSHV virionsRun on SDS PAGEExcise bands, digest - get sequence and compare to database

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?

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

Baltimore classification

WHY TRANSFECTION FAILS

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

Measuring Intracellular EventsSample at time intervals after lysing cells (1952 - Doermann)ChloroformLysis from withoutIdentification of eclipse and maturation phases

Maturation phase

Strategy of replicationLyticTemperate

Biologic Propertiesnatural host rangemode of transmission in naturevector relationshipsgeographic distributionpathogenicity, association with disease tissue tropisms, pathology, histopathology

How can you identify these viruses?