Transcript General Virology - California State University, Fullerton

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?
Hershey-Chase
Fraenkel-Conrat
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
• Tegument proteins - 12/84 viral
proteins in HSV
• Potential role?
• Virion mRNA
– DNAase virion nucleic
acids
– RT-PCR
– probe genome array
• Potential role?
Genome - DNA or RNA
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
• PolyDNAvirus (PDV) - contain
many DNA segments
• Mimivirus - larger than small
bacteria
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
Host enzyme makes
• 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?
Proteins
• structural proteins
• non-structural 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?
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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 plaque-forming 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