General Virology
VIRUS STRUCTURE
Virion vs virus • Virion is the infectious particle – composed of nucleic acid, protein capsid, +/ envelope – may be extracellular or intracellular • Virus is any stage of infection
How do we know that NA is genetic material?
HersheyChase
FraenkelConrat Experiment
TRANSFECTION EXPTS
TRANSFECTION FAILS FOR SOME VIRUSES WHY?
Capsid Functions – Protection of NA – Attachment for naked viruses – Enzyme • Helical 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. •
How do helical viruses differ? • Helical one axis of symmetry down center • Multiple structural units
Icosahedral symmetry • 20 identical equilateral triangles • Structural units on faces to give morphological capsomers – Pentons (5 fold axis of symmetry) – Hexons • 3 fold through face • 2 fold through edge
How do spherical viruses differ?
Envelope
• Attachment • Entry • Assembly matrix proteins • Release • Proteins are viral • Lipids are host • Rare in plants or bacteria why?
• If the membrane envelope is destroyed, the virus becomes noninfectious. Why?
Herpesvirus complexity • • •
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Tegument proteins 12/84 viral proteins in HSV Potential role? Virion mRNA – DNAase virion nucleic acids – RTPCR – probe genome array Potential role?
Genome DNA or RNA Genome How do we experimentally show that DNA or RNA is the virus genetic material?
• strandedness - (single) (double) • linear or circular, partial double stranded circle • number (single, segmented, multicomponent)
RNA Genomes • sense (positive-sense, negative-sense, ambisense) • presence or absence of 5'-terminal cap or 5'-covalently-linked protein • presence or absence of 3'-terminal poly (A) tract • Retroviruses - replication strategy
Some viruses have high degree of secondary structure • Poliovirus 5’ internal ribosome entry site (IRES)
Guest et al. 2004. J. Virol. 78: 11097.
SARS/coronaviruses have conserved 3’region • • •
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Robertson et al. 2005. PLOsBiology:3.
SARS s2m in red a green = 530 loop of 16S RNA Similar binding properties: – b blue = S12 – magenta = IF1 Possible role for s2m – Hijacks protein synthesis from cell(binding cell factors) – Needed to bind to similar viral protein for transcription Potential drug target in red tunnel
DNA Viruses may be large genomes
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PolyDNAvirus (PDV) contain many DNA segments
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Mimivirus larger than small bacteria
Hostinduced modification • Viral property that varies depending on the host • Phage DNA hydroxymethyl cytosine (HMC) replaces C – Viral enzymes: C to HMC – Viral DNA polymerase: adds HMC not C – What is advantage of HMC? • Glucose is attached to HMC – Host enzyme needed to prepare glucose – Protects 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 • nonstructural virion proteins – transcriptase, – protease – integrase
How to identify virion proteins • • •
Purify KSHV virions Run on SDS PAGE Excise bands, digest get sequence and compare to database
Chemical synthesis of poliovirus: What are the implications? • Small genome positive strand RNA sequence known • Synthesized small DNA segments (~ 69 nucleotides) with overlapping complementary segments • Added a T7 phage promotor to DNA • Used DNA to make genome RNA in HeLa cell lysate with T7 polymerase • Results: How do you show success?
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International Congress on Taxonomy of Viruses http://www.ncbi.nlm.nih.gov/ICTV/ • Morphology – virion size – enveloped or naked nucleocapsid – capsid symmetry and structure
• Genome characteristics • Replication strategy • Antigenic Properties
Baltimore classification
WHY TRANSFECTION FAILS
ONE STEP GROWTH CURVE • •
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1939 Ellis and Delbruck: Infection with a high multiplicity of infection (MOI): ratio of virus to host cell – Simultaneous infection – Single replication cycle Sample at time intervals by plaque count for plaqueforming units (PFU), Identification of latent phase Determination of burst size/viral yield
Measuring Intracellular Events • Sample at time intervals after lysing cells (1952 Doermann) – Chloroform – Lysis from without • Identification of eclipse and maturation phases
Maturation phase
• Strategy of replication – Lytic – Temperate
Biologic Properties • natural host range • mode of transmission in nature • vector relationships • geographic distribution • pathogenicity, association with disease • tissue tropisms, pathology, histopathology
How can you identify these viruses?