general virology virus structure. virion vs virus virion is the infectious particle –composed of...
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Virion vs virusVirion 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 How do we know that NA is genetic material?material?
Hershey-Chase Fraenkel-Conrat Experiment
TRANSFECTION EXPTS
CapsidCapsid
• 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.
• Helical- one axis of symmetry down center
• Multiple structural units
How do helical viruses differ?How do helical viruses differ?
Icosahedral symmetryIcosahedral 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?
EnvelopeEnvelope• 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 complexityHerpesvirus complexity
• Tegument proteins - 12/84 viral proteins in HSV
• Potential role?
• Virion mRNA
– DNAase virion nucleic acids
– RT-PCR
– probe genome array
• Potential role?
Genome - 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?
RNA GenomesRNA 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 Some viruses have high degree of secondary structurestructure
• Poliovirus - 5’ internal ribosome entry site (IRES)
Guest et al. 2004. J. Virol. 78: 11097.
SARS/coronaviruses have conserved 3’regionSARS/coronaviruses have conserved 3’region
• 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 tunnelRobertson et al. 2005. PLOsBiology:3.
DNA Viruses may be large genomesDNA Viruses may be large genomes
• PolyDNAvirus (PDV) - contain many DNA segments
• Mimivirus - larger than small bacteria
Host-induced modificationHost-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
• 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
ProteinsProteins
• structural proteins • non-structural virion
proteins– transcriptase, – protease– integrase
How to identify virion proteinsHow 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 Chemical synthesis of poliovirus: What are the implications?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?
International Congress on Taxonomy of VirusesInternational 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
ONE STEP GROWTH CURVEONE 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
Measuring Intracellular EventsMeasuring Intracellular Events
• Sample at time intervals after lysing cells (1952 - Doermann)
– Chloroform
– Lysis from without
• Identification of eclipse and maturation phases
Maturation phase