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11/4/13
Chapter 13-Viruses. Viroids, and
Prions
Viruses: Obligate Intracellular Parasites
§  Viruses simply genetic information: DNA or RNA
contained within protective coat
•  Inert particles: no metabolism, replication, motility
•  Genome hijacks host cell’s replication machinery
•  Inert outside cells; inside, direct activities of cell
•  Infectious agents, but not alive
•  Can classify generally based
on type of cell they infect:
eukaryotic or prokaryotic
•  Bacteriophages (phages)
infect prokaryotes
•  May provide alternative
to antibiotics
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History began with the Tobacco
Mosaic Virus (TMV)
•  1886 Aldolf Mayer
showed that a virus
was transmissable
between plants
•  1892 Iwanowski tried
to isolate it by filtering
with porcelain filter
General Characteristics of all viruses
•  Contain a single type of nucleic acid
•  Contain a protein coat
•  Obligate intracellular parasites
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General Characteristics of Viruses
§  Most viruses notable
for small size
•  Smallest:
~10 nm
~10 genes
•  Largest:
~500 nm
Virion (viral particle) is nucleic acid with a protein coat
Contain either RNA or DNA
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2,288 species
348 genera
6 orders
Common Shapes
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Icosahedral
§  Three shapes:
Icosahedral
Helical
Complex
Protein coat
(capsid)
Nucleic acid
Adenovirus
75 nm
(a)
Helical
Nucleic acid
Protein coat
(capsid)
Tobacco mosaic virus (TMV)
100 nm
(b)
Complex
Protein coat
(capsid)
Nucleocapsid
Head with
nucleic acid
(DNA)
Tail
Base plate
Tail spike
Tail fibers
T4 Bacteriophage
100 nm
(c)
Two different types of Viruses
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•  Naked viruses lack
envelope; more resistant to
disinfectants
•  Enveloped viruses
have
lipid bilayer envelope
Protein
capsid: protects
nucleic
acids
Made of identical subunits -capsomers
Capsomere
subunits
Nucleic acid
Nucleocapsid
Capsid (entire
protein coat)
Spikes
(a) Naked virus
Spikes
Matrix protein
Nucleic acid
Nucleocapsid
Capsid (entire
protein coat)
Envelope
(b) Enveloped virus
•  Capsid + nucleic acids =
nucleocapsid
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DNA or RNA
Genome may be linear or circular
Double- or single-stranded
§  Viruses have components for attachment
•  Phages have tail fibers
•  Many animal viruses have spikes
•  attach to specific receptor sites
A complex virus showing attachment fibers
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Relationship of virus with host cell
Bacterial viruses
• 
• 
Known as bacteriophages or phages
Two different life cycles
1.  Lytic cycle (lytic or virulent phage)-results in
lysis of the cell
2.  Lysogenic cycle (temperate or lysogenic
phage)-may result in lysis of the cell or
becomes a permanent part of the chromosome
by integrating
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T4 phage replication
1. Attachment
Phage uses bacterial receptors
2. Genome entry
T4 lysozyme degrades cell wall, Tail
contracts, injects genome
3. Synthesis of proteins and genome
4. Assembly--Some components
spontaneously assemble, others require
protein scaffolds
5.  Release
Lysozyme produced late in infection;
digests cell wall--Cell lyses, releases
phage
Burst size of T4 is ~200
Lambda Phage replication
•  Lytic infection or incorporation of DNA
into host cell genome
•  Lysogenic infection
•  Infected cell is lysogen
•  Lambda (λ) phage as model
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Lambda integrates into the chromosome
•  Site specific
recombination
•  Integrated phage DNA
termed prophage
phage-encoded
enzymes
•  A repressor prevents,
maintains lysogenic state
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§  Temperate Phage Infections (continued...)
•  Lambda (λ) phage: DNA excised from chromosome only about
once per 10,000 divisions of host bacterium
•  If DNA damaged (e.g., UV light exposure), SOS repair system
turns on, activates a protease
•  Protease destroys repressor, allows prophage to be excised,
enter lytic cycle
•  Called phage induction; allows phage to escape damaged host
Properties conferred by prophage
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Some phage are filamentous
13.2. Bacteriophages
§  Filamentous Phages
•  Single-stranded DNA phages
•  Used to produce only single-
stranded recombinant DNA
•  Look like long fibers
•  Cause productive infections
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Filamentous
phage
F pilus
Phage
DNA
•  Host cells not killed, but grow more slowly
•  M13 phage as model
•  Attaches to protein on F pilus of E. coli
•  Single stranded DNA genome
enters cytoplasm
Phage attaches to the
F pilus of a bacterial
cell and injects its
single-stranded DNA.
Phage DNA replicates;
phage capsomeres are
synthesized and
embedded in the
host cell membrane.
Outside
environment
Carrier cell
Carrier cell
Phage nucleic acid gains its capsid
as it extrudes through the membrane.
The bacteria do not lyse.
Phage
DNA
Capsomeres
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Replication of filamentous phage
•  DNA polymerase synthesizes complementary strand
Replicative form (RF)—one strand for mRNA synthesis, the other for genome
•  M13 phage coat protein
molecules inserted into
cytoplasmic membrane
•  Other proteins form pores
•  As phage DNA
excreted through pores,
coat proteins coat the
DNA, form
nucleocapsids
M13 is ssDNA…how does it replicate the ssDNA?
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ssDNA (+) strand
Host enzyme synthesizes
complementary strand.
dsDNA (±) strand (RF)
Replication
(–) strand DNA
transcribed
Into mRNA
(+) strand DNA
functions as
phage genome
mRNA translated
into phage
coat protein
•  DNA polymerase synthesizes
complementary strand
•  Replicative form (RF); one strand
used as template for synthesis of
mRNA, copies of genome
•  M13 phage coat protein molecules
inserted into cytoplasmic
membrane
•  Other proteins form pores
•  As phage DNA excreted through
pores, coat proteins coat the DNA,
form nucleocapsids
Virion
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How do bacteria protect
themselves against phage?
•  Prevent phage attachment
•  Attacking foreign DNA with
restriction enzymes, protecting
native DNA with methylation
•  CRISPR system degrades
incoming viral nucleic acid
CRISPR defense system against
phage
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Methods to study bacteriophage
•  Plaque Assay used
to quantitate phage
How do animal viruses differ
from bacterial viruses?
•  Attachment or entry into the cell
•  Replication of viral nucleic acid (remember
eukaryotic cells have a nucleus)
•  Uncoating step is required by animal viruses
•  Exit the host cell by budding or shedding
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Animal Virus Replication
§  Five-step infection cycle
•  Attachment
• 
• 
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Viruses bind to receptors
Usually glycoproteins on plasma membrane
Often more than one required (e.g., HIV binds to two)
Normal function unrelated to viral infection
Specific receptors required; limits range of virus
E.g., dogs do not contract measles from humans
Effects of animal virus on cells
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Entry of animal virus
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1
Membrane
fusion
.
2
Adsorption
Protein
spikes
3
Nucleocapsid
release
4
Uncoating
Fusion of virion and
host cell membrane
Envelope
Receptors
Nucleocapsid
Capsid
Host cell
plasma membrane
Nucleic acid
(a) Entry by membrane fusion
2
Adsorption
1
Endocytosis
3
Release from
vesicle
Uncoating
Nucleic acid separates
4
from capsid.
Membrane surrounds
Attachment to
the virion, forming an
receptors
endocytic vesicle.
triggers endocytosis.
(b) Entry
by endocytosis
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Replication strategies
•  Depends on type of
nucleic acid
•  What enzymes are
needed for the process?
•  There are DNA and
RNA viruses. •  These can be either
double stranded (ds) or
single stranded (ss)
Animal Virus Replication
§  Five-step infection cycle (continued...)
•  Synthesis
•  Expression of viral genes to produce viral structural and catalytic
genes (e.g., capsid proteins, enzymes required for replication)
•  Synthesis of multiple copies of genome
•  Most DNA viruses multiply in nucleus
•  Enter through nuclear pores following penetration
•  Three general replication strategies depending on type of
genome of virus
–  DNA viruses
–  RNA viruses
–  Reverse transcribing viruses
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Animal Virus Replication
•  Replication of DNA
viruses
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•  Usually in nucleus
DNA viruses
•  Poxviruses --exception:
ss (+)
ss (–)
replicate in cytoplasm,
DNA
DNA
encode all enzymes for
ds (±)
DNA, RNA synthesis
DNA
ds (±) DNA
ds (±) DNA
•  dsDNA replication
straightforward
ss (+) RNA
ss (+) RNA
ss (+) RNA
(mRNA)
(mRNA)
(mRNA)
•  ssDNA similar except
complement first
protein
protein
protein
synthesized
(a)
Pox viruses are large: carry enzymes with them for
initial transcription
•  Replication of RNA viruses
•  Majority single-stranded;
replicate in cytoplasm
•  Use virally encoded RNA
polymerase (replicase)
lacks proofreading
allows antigenic drift
•  ss (+) RNA used as mRNA
•  ss (–) RNA, dsRNA viruses
carry replicase to
synthesize (+) strand
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RNA viruses
ss (+)
RNA
(mRNA)
ss(–)RNA
protein
ss (+) RNA
(mRNA)
ss (–)
RNA
protein
ds (±)
RNA
ss (+) RNA
(mRNA)
protein
(b)
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•  Replication of reverse-transcribing viruses
•  Encode reverse transcriptase: makes DNA from RNA
•  Retroviruses have ss (+) RNA genome (e.g., HIV)
•  Reverse transcriptase synthesizes single DNA strand
•  Complementary strand synthesized; dsDNA integrated into
host cell chromosome
•  Can direct productive
infection or remain latent
•  Cannot be eliminated
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Reverse transcribing viruses
ss (–) DNA
ds (±) DNA
ss (+)
RNA
(mRNA)
protein
(c)
Release of enveloped viruses
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1 Viral proteins
(spikes) insert
into membrane.
Viral proteins
Host plasma
membrane
Capsid
Nucleocapsid extrudes from cell,
3
becoming coated with matrix proteins
2 Viral matrix
protein coats
and envelope with protein spikes.
inside of
4 New virus is
membrane.
released.
Enveloped
virus
Matrix
protein
Intact host
membrane
Nucleic acid
(a)
(b)
b: © Dr. Dennis Kunkel/Visuals Unlimited
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13.7. Categories of Animal Virus Infections
§  Acute and Persistent Infections
•  Acute: Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
•  Some viruses
exhibit both
(e.g., HIV)
State of Virus
Virus disappears
after disease ends.
Time (days)
(a)
Chronic infection (hepatitis B)
Appearance of
symptoms and
infectious virions
•  Continue for
years or lifetime
•  May or may not
have symptoms
Infectious virions
Disease
Influenza
Hepatitis B
Days
State of Virus
After initial infection with
or without disease
symptoms, infectious virus
is released from host with
no symptoms.
Release of virus
Time
Years
(b)
Latent infection (cold sores)
Appearance of
symptoms and
infectious virions
•  Persistent:
Appearance of
symptoms and
infectious virions
Acute infection (influenza)
•  Rapid onset
•  Short duration
Cold
sores
Virus
activation
Cold
sores
Non-infectious
Days
Time
State of Virus
After initial infection, virus
is maintained in neurons
in non-infectious state.
Virus
activated to produce new
disease symptoms.
Years
(c)
Acute viral infections
•  Usually short in duration
•  Host develops long lasting immunity
•  Infection with the virus results in a
productive infection…host cells die as a
result of infection
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General Steps of Acute Viral
infection
• 
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Attachment
Entry into host cell
Targeting where it will reproduce
Uncoating of the capsid
Synthesis of proteins, replication of nucleic acid
Maturation
Cell lysis—Cells lyse due to induction of cell
death. This is different from the ‘bursting’ that
happens in bacteria infected with phage.
Persistent infections
•  Virus is continually present in the body, released
by budding (inserted into host genome as a
provirus)
•  Three types
Latent infections
Chronic infections
Slow infections
Slow release of virus from cells
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Persistent: Latent Infections
•  Persistent infection with symptomless
period followed by reactivation of virus and
symptoms
•  Example of latent viruses are found in the
family Herpesviridae –  Herpes simplex virus -1
–  Herpes simplex virus -2
–  Varicella—chicken pox
Latent Viral infections
•  All of these viruses are in the Herpesviridae
family
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Herpesviridae Family
•  Double stranded DNA (dsDNA),
enveloped viruses
-herpes simplex virus type 1(cold sores)
-herpes simplex virus type 2 (genital
herpes)
-Varicella-zoster virus (chicken pox,
shingles)
-Epstein-Barr (infectious mono and
Burkitt’s lymphoma)
Herpes Simplex virus-1
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HSV-1 reactivation
•  HSV-1 causes fever
blisters, HSV-2 genital
herpes
Varicella (chickenpox) and
Herpes Zoster (Shingles)
•  HSV-3 causes chicken
pox and latent
activation known as
shingles
•  Acquired by
respiratory route, 2
weeks later see
vesicles on skin
•  Vaccine established in
1995 for chickenpox
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Epstein Barr
•  Causes infectious
mononucleosis
•  Acquire by saliva,
incubation period is 4-7
weeks
•  Identify by -lobed lymphocytes
-heterophile antibodies
-fluorescent antibody tests
Chronic infections
•  Infectious virus present at all times
•  Disease may be present or absent
•  Examples are Hepatitis Type B and Type C
viruses
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Type Hepadnaviridae family: Hepatitis B
•  dsDNA virus, enveloped
•  Hepatitis B
-passes through
intermediate stage (RNA)
for replication
-three particles found in
blood sample
1. Dane
2. filamentous
3. sphericle
-exposure through blood/
body fluids
Hepatitis Type B
•  Incubation period is ~12 weeks
•  10% of cases become chronic, mortality rate
is less than 1% •  About 40% of the chronic cases die of liver
cirrhosis 25
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Flaviviridae Family: Hepatitis Type C
Not the same as Hep B. Different type of virus
•  Hepatitis C virus
–  (+) ssRNA virus, enveloped
–  Obtain from blood/body fluids
–  Incubation period averages 6 weeks
–  Hard to screen blood for the virus
–  85% of all cases become chronic (high rate)
What other types of Hepatitis viruses are
known to infect humans?
•  Hepatitis Type A
–  Found in the Picornaviridae family (+) ssRNA
-obtain through fecal-oral route, enters GI tract
and multiplies
-incubation period is ~4 weeks
-symptoms include: anorexia, malaise, nausea,
diarrhea, abdominal discomfort, fever, and chills
lasting 2-21 days
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Slow Infections
•  Infectious agent increases in amount over a
long time during which there are no
symptoms
•  Examples are HIV found in the Retroviridae
family
•  Retroviruses use reverse transcriptase to
replicate ssRNA
Retroviridae-multiple strands of (-)RNA
•  HIV
-infects Helper T cells
-requires the enzyme
reverse transcriptase
-integrates as a provirus
-is released by budding,
or lyses the cell
Provirus: DNA in chromosome
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HIV replication
Viruses associated with cancers
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Viruses and tumors
•  dsDNA viruses are most common to cause
viral-induced tumors
•  Cancer is result of integration of viral genes
into the host chromosome
•  Transforming genes are called oncogenes
•  Examples: papillomavirus, herpesvirus
Orthomyxoviridae-multiple
strands of (-)RNA
•  Influenza virus
–  Consists of 8 segments of RNA
–  Envelope has H spikes (hemagglutinin) and N
spikes (neuraminidase)
–  Incubation is 1-3 days
–  Symptoms include: chills, fever, headache,
muscle aches, may lead to cold-like symptoms
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Influenza virus
If multiple forms infect one
cell…reassortment can
occur
That means some of the
segments mentioned in
previous slide can end up
in the capsid of the other
virus. They mix genes.
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Antigenic shift vs antigenic drift
Ways to study viruses
•  Since viruses grow in living cells….need a
live cell to culture them
–  Cell culture/tissue culture
–  Embryonated chicken eggs
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Cell Culture
Proteinaceous infectious
particles: PRIONS
•  1982 Stanley Prusiner proposed that there
were infectious proteins
•  Caused the disease “scrapie” in sheep
•  Caused the “mad-cow”disease in 1987
•  Human forms suggest a genetic component
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Prions
•  Contain no nucleic acid
•  Abnormal protein promotes conformational
change to normal protein
•  Results in damage to neurons…
transmissible spongiform encephalopahthies
Brain with spongiform
encephalopathy
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Infections caused by prions
Mechanism
of prion
replication
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