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 • strandedness - (single)

experimentally show that (double)
DNA or RNA is the virus
genetic material? • 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

• 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. PLOsBiology:3.
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
 • 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
Host enzyme makes 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 • Genome characteristics

– virion size • Replication strategy
– enveloped or naked • Antigenic Properties
nucleocapsid
– capsid symmetry and
structure
Baltimore classification
WHY TRANSFECTION FAILS
 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
 Measuring Intracellular Events

• Sample at time intervals

after lysing cells (1952 -
Doermann)
– Chloroform
– Lysis from without
• Identification of eclipse Maturation phase

and maturation phases