General Virology

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General Virology
VIRUS STRUCTURE
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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

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How do we know that NA is genetic material?
Hershey-Chase
Fraenkel-Conrat Experiment
TRANSFECTION EXPTS
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TRANSFECTION FAILS FOR SOME VIRUSES

• WHY?

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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.

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How do helical viruses differ?

• Helical- one axis of symmetry down center
• Multiple structural units

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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?
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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?

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Herpesvirus complexity

• Tegument proteins - 12/84 viral proteins in HSV
• Potential role?
• Virion mRNA
• DNAase virion nucleic acids
• RT-PCR
• probe genome array
• Potential role?

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Genome - DNA or RNA

• strandedness - (single) (double)
• linear or circular, partial double stranded
  circle
• number (single, segmented, multicomponent)

How do we experimentally show that DNA or RNA is
the virus genetic material?
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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

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Some viruses have high degree of secondary
structure

• Poliovirus - 5 internal ribosome entry site
  (IRES)

Guest et al. 2004. J. Virol. 78 11097.
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SARS/coronaviruses have conserved 3region

• 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

Robertson et al. 2005. PLOsBiology3.
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DNA Viruses may be large genomes

• PolyDNAvirus (PDV) - contain many DNA segments
• Mimivirus - larger than small bacteria

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Host-induced 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

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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 proteins
• transcriptase,
• protease
• integrase

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How to identify virion proteins

• Purify KSHV virions
• Run on SDS PAGE
• Excise 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 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

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Baltimore classification
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WHY TRANSFECTION FAILS
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ONE STEP GROWTH CURVE

• 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
  plaque-forming units (PFU),
• Identification of latent phase
• Determination of burst size/viral yield

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Measuring Intracellular Events

• Sample at time intervals after lysing cells (1952
  - Doermann)
• Chloroform
• Lysis from without
• Identification of eclipse and maturation phases

Maturation phase
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• Strategy of replication
• Lytic
• Temperate

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Biologic Properties

• natural host range
• mode of transmission in nature
• vector relationships
• geographic distribution
• pathogenicity, association with disease
• tissue tropisms, pathology, histopathology

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How can you identify these viruses?