Human Biology 534

1

• Human Biology 534
• Introduction to Virology and Human Retroviruses
• 26/27 March 2001
• James I. Mullins, Ph.D., Professor and Chairman,
  Department of Microbiology, jmullins_at_u.washington.
  edu
• Spring Quarter, 2001
• Copies of lectures can be downloaded (pdf) from
  http//ubik.microbiol.washington.edu/Index.html

KEYWORDS are in bolded red
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Death Rate (per 105) from Infectious Diseases in
the US 1900-1996
3

• Recognition of viruses
• F How long viruses have been within our midst?
• 1500 BC Leg deformities indicative of
  poliomyelitis, pock marks indicative of smallpox.
  

"Virus" is from the Greek meaning for "poison"
and was initially described by Edward Jenner in
1798.
During the 1800's, all infectious agents were
considered to be viruses until Koch developed
pure culture techniques which allowed the
separation and growth of bacteria. In the late
1800's Bacteria were purified and established as
disease causing agents. It then became possible
to distinguish them from the "filterable agents",
those able to pass through special filters
designed to prevent the passage of bacteria. The
first viruses discovered included Foot and mouth
disease (picornavirus), 1898 Yellow fever
(flavivirus), 1900 Rous sarcoma virus (oncogenic
retrovirus), 1906
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Viral diseases have played a major role in human
history Over the past 1000 years Smallpox and
measles were brought to North and South America
by early European explorers/conquerers. These
diseases, for which the native American
populations had no acquired (partial) immunity,
killed large fractions of the populations, and
were a major factor in the decimation of these
societies.
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Over the past 10 years As the global HIV
epidemic continues, sporadic outbreaks of viral
diseases spread to humans by non-human hosts,
such as Ebola (from chimps?) and Hanta (from
rats) raise the concern about future epidemics.
Four Corners Virus (Hanta)
Ebola
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THE DISCIPLINE OF VIROLOGY The study of virology
inherently involves a merging together of what
has traditionally been thought of as two separate
"kinds" of science basic and applied. We want to
figure out how viruses are transmitted, how they
replicate, and how the host organism responds. We
also want to figure out how to prevent
transmission, how to interfere with virus
replication, and how we can confer immunity on
the host. The "applied" follows from, and is
dependent upon, the "basic" in a quite direct
way. Virology as it is studied today, is
therefore an outgrowth of research
in Infectious diseases - which provides
recognition of viral pathogens and their
sequelae, and Molecular Biology- because of the
usefulness of viruses as probes of cellular
metabolic processes and as vectors with
potential for gene therapy.
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WHERE WE STAND IN 2001 PREVENTING CONTROLING
CURING VIRAL DISEASES
Smallpox effective vaccine this is the only
viral disease that has been wiped out worldwide
Measles effective vaccine since 1963 this
disease could be eliminated with a world-wide
effort Influenza effective strain-specific
vaccine, but new variant strains emerge
periodically Polio effective vaccine will soon
be the second viral disease wiped out HIV no
vaccine effective drugs, but they are costly and
toxic, plus resistant strains appear. World-wide
spread continues via intimate contact. 50 million
infected thus far Ebola no vaccine important
host species unknown (found recently in chimps
and rodents) outbreaks controllable because
people die quickly and human-human transmission
is via blood Hanta no vaccine rodent host
easy transmission to humans, but outbreaks
controllable We also share the world, and our
bodies, with viruses that cause hepatitis,
respiratory disease, mononucleosis, diarrhea,
genital warts, genital herpes, and some forms of
cancer
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WHAT IS A VIRUS? Viruses may be defined as
acellular organisms whose genomes consist of
nucleic acid, and which obligately replicate
inside host cells using host metabolic machinery
to different extents, to form a pool of
components which assemble into particles called
Virions.
F A virus differs from a cell in three
fundamental ways i A virus usually has only a
single type of nucleic acid serving as its
genetic material. This can be single or double
stranded DNA or RNA ii Viruses contain no
enzymes of energy metabolism, thus cannot make
ATP iii Viruses do not encode sufficient
enzymatic machinery to synthesize their component
macromolecules, specifically, no protein
synthesis machinery.
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Viruses are distinguished from other obligate
parasites, some of which are even simpler than
viruses MYCOPLASMA Small bacterium that grows
only in complex medium or attached to eucaryotic
cells. CHLAMYDIA Obligate intracellular
bacterial parasite which depends on eucaryotic
cell for energy. PROTOZOA Obligate intracellular
parasite of eucaryotes that replicate within
eucaryotic cells. VIROID Infectious agents that
exist as naked nucleic acid. Found in
plants. HEPATITIS DELTA VIRUS (HDV) Viroid-like
agent whose replication is dependent upon HBV.
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PRION (proteinacious infectious agent)
Hypothesized identity of the unconventional slow
viruses (such as the Kuru and Scrapie agents). No
nucleic acid is known to be required for prion
function. They are thought by many to consist
solely of protein and perhaps lipids.
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Virus naming and classification Usually based on
data available at the time of discovery i Diseas
e they are associated with, e.g. Poxvirus,
Hepatitis virus, HIV, measles virus ii Cytopathol
ogy they cause, e.g. Respiratory Syncytial
virus, Cytomegalovirus iii Site of isolation,
e.g. Adenovirus, Enterovirus,
Rhinovirus iv Places discovered or people that
discovered them, e.g. Epstein-Barr virus, Rift
Valley Fever v Biochemical features,
e.g. Retrovirus, Picornavirus, Hepadnavirus
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These naming conventions can lead to confusion
later, e.g., viral hepatitis is caused by at
least 6 different viruses
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Viral Hepatitis - An Overview

Type of Hepatitis
A
B
C
D
E
Source of
feces
blood/
blood/
blood/
feces
virus
blood-derived
blood-derived
blood-derived
body fluids
body fluids
body fluids
Route of
fecal-oral
percutaneous
percutaneous
percutaneous
fecal-oral
transmission
permucosal
permucosal
permucosal
Chronic
no
yes
yes
yes
no
infection
Prevention
pre/post-
pre/post-
blood donor
pre/post-
ensure safe
exposure
exposure
screening
exposure
drinking
immunization
immunization
risk behavior
immunization
water
modification
risk behavior
modification
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Typical Serological Courses of Infection
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Outcome of HBV Infection by Age at Infection
100
100
Symptomatic Infection ()
80
80
60
60
Chronic Infection ()
40
40
20
20
0
0
1-6 months
7-12 months
Older Children and Adults
Birth
1-4 years
Age at Infection
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Virus Classification is now based principally on
analysis of the particle Common
morphology observed by electron
microscopy Common serology antigenic
cross-reactivity Related genetic
material form of nucleic acid ssDNA ( or -
strand) dsDNA ssRNA ( or -
strand) dsRNA segmented RNA sequence
homology DNA sequence Hybridization
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Cellular and Viral Genomes
Type of Genome Number of Gene Equivalents Human
cell (higher eukaryotes) 1,000,000 E. coli
(bacteria) 5,000 Poxvirus, Herpesvirus (large
DNA virus) 70-200 Influenza, human
immunodeficiency virus 8-12 Papilloma, Hepatitis
B virus 3-5 Potato spindle tubor
"viroid" 1 The number of average-sized genes
assuming that the entire genome encodes sequence
information for the synthesis of proteins.
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Virus Life Cycle Early Phase i Attachment to
and entry of the virion into the host
cell ii Disassembly of the infectious
particle iii Replication of the viral
genome Late Phase iv Replication of virus
structural components v Reassembly of the
replicated pieces into progeny virus
particles vi Release from the host cell.
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STEPS IN VIRAL REPLICATION 1. Attachment
(adsorption) 2. Penetration 3. Uncoating 4.
Genome replication 5. Assembly 6. Release
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ATTACHMENT AND PENETRATION INTO CELLS The first
steps in the replication process for all viruses
involve the attachment of the virus particle (the
virion) onto the cell surface and then the entry
of at least a portion of the particle (including
the genome) into the cytoplasm. Both of these
steps are specific, and are the main reason for
the species-specificity of viruses. For all
viruses, some aspect of membrane disruption must
occur to achieve entry. For enveloped viruses,
this almost always involves membrane fusion
between the viral envelope and a cell membrane
(either the plasma membrane or an internal
vesicle membrane). For non-enveloped virions, the
viral surface protein(s) usually play a role in
causing some very localized membrane
disruption. The details of attachment and entry
for many viruses are still somewhat unclear, but
there are some viruses for which the process has
been studied in depth. We will take a look
today at three of these Picornaviruses
(poliovirus and "common cold" rhinoviruses),
Influenza virus and HIV (retrovirus)
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Icosahedral virion entry Picornavirus Poliovirus
(and rhinoviruses, which cause about 50 of
"colds") have an icosahedral structure which has
been highly characterized. The icosahedral
surface of the virion consists of 60 copies each
of three proteins designated VP1, VP2, and VP3. A
groove, or "canyon", in the VP1 structure
provides an annulus around each pentameric vertex
that provides the specific attachment site to
cells. A subsequent interaction of the protein
with the cell's plasma membrane leads to the
entry of the viral RNA genome into the cytoplasm.
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Enveloped virion entry Influenza virus
Enveloped viruses gain entry to cells by use of
specific viral proteins that have membrane fusion
- inducing properties. Influenza virions bind
to the cell surface and are endocytosed intact
into the cell. The endocytic vesicle then fuses
with an acidic vesicle. At pH 5, the hemaglutinin
glycoprotein molecules in the influenza envelope
undergo a structural transition that causes the
amino terminal end of HA2 to flip outward and be
exposed to the molecular environment. This highly
hydrophobic segment interacts with the vesicle
membrane and causes fusion. This fusion event
dumps the viral genome into the cell's cytoplasm.
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Attachment and entry of HIV Studies on HIV over
the past 3 years have elucidated its entry
process to the degree that it is the best
understood of any human virus. The two viral
envelope glycoproteins, gp120 and gp41, are
responsible for attachment and membrane fusion,
respectively. The cell surface glycoprotein CD4
is used as the primary receptor, and one of a few
other proteins are used as co-receptors
(typically CCR5 and CXCR4).
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Information Flow in cells
DNA to DNA (Replication) DNA to RNA
(Transcription) RNA to Protein (Translation) The
Central Dogma
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Viral Genome Replication
Viruses utilize the flow of information of
eucaryotic cells, as well as novel pathways, some
which violate Central Dogma.
The replication strategy of the virus depends on
the nature of its genome. Viruses can be
classified into seven (arbitrary) groups
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I Double-stranded DNA (Adenoviruses
Herpesviruses Poxviruses, etc) Some replicate in
the nucleus e.g., adenoviruses, using cellular
proteins. Poxviruses replicate in the cytoplasm
and make their own enzymes for nucleic acid
replication. II Single-stranded ()sense DNA
(Parvoviruses) Replication occurs in the nucleus,
involving the formation of a (-) sense strand,
which serves as a template for () strand RNA and
DNA synthesis.
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III Double-stranded RNA (Reoviruses
Birnaviruses) These viruses have segmented
genomes. Each genome segment is transcribed
separately to produce monocistronic mRNAs. IV
Single-stranded () sense RNA (Picornaviruses
Togaviruses, etc) a) Polycistronic mRNA e.g.
Picornaviruses Hepatitis A. Genome RNA mRNA.
Means naked RNA is infectious, no virion particle
associated polymerase. Translation results in the
formation of a polyprotein product, which is
subsequently cleaved to form the mature proteins.
b) Complex Transcription e.g. Togaviruses. Two or
more rounds of translation are necessary to
produce the genomic RNA.
V Single-stranded (-)sense RNA
(Orthomyxoviruses, Rhabdoviruses, etc) Have a
virion associated RNA directed RNA polymerase.
a) Segmented e.g. Orthomyxoviruses. The first
step in replication is transcription of the (-)
sense RNA genome by the virion RNA polymerase to
produce monocistronic mRNAs that serve as the
template for genome replication. b) Non-segmented
e.g. Rhabdoviruses. Replication occurs as above
and monocistronic mRNAs are produced.
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VI Single-stranded () sense RNA with DNA
intermediate in life-cycle (Retroviruses) Genome
is () sense but unique among viruses in that it
is diploid, and does not serve as mRNA, but as a
template for reverse transcription within the
newly infecting virion.
VII Double-stranded DNA with RNA intermediate
(Hepadnaviruses) Also rely on reverse
transcription, but this occurs inside the virus
particle on maturation. On infection of a new
cell, the first event to occur is repair of the
gapped genome, followed by transcription.
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MATURATION AND RELEASE Maturation proceeds
differently for naked, enveloped, and complex
viruses
Naked icosahedral viruses - Preassembled
capsomers are joined to form empty capsids
(procapsid) which are the precursors of virions.
They are released from infected cells in
different ways. Poliovirus is rapidly released,
with death and lysis of infected cells. DNA
viruses tend to mature in the nucleus tend to
accumulate within infected cells over a long
period and are released when the cell undergoes
autolysis, and in some cases, may be extruded
without lysis.
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Enveloped Viruses - Viral proteins are first
associated with the nucleic acid to form the
nucleocapsid, which is then surrounded by an
envelope. In nucleocapsid formation, the proteins
are all synthesized on cytoplasmic polysomes and
are rapidly assembled into capsid components. In
envelope assembly, virus-specified envelope
proteins go directly to the appropriate cell
membrane (the plasma membrane, the ER, the Golgi
apparatus), displacing
host proteins. In contrast, the carbohydrates and
the lipids are produced by the host cell. The
viral envelope has the lipid constitution of the
membrane where its assembly takes place (eg. the
plasma membrane for orthomyxoviruses and
paramyxoviruses, the nuclear membrane for
herpesviruses on right). A given virus will
differ in its lipids and carbohydrates when grown
in different cells, with consequent differences
in physical, biological, and antigenic
properties.
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VIRAL PATHOGENESIS Results from Transmission
to a new host Replication and spread within
the host (a function of viral tropism) Cell
damage and dysfunction (can be mediated by the
virus or by immune defense mechanisms) Diseas
e symptoms and abnormal laboratory test values
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KOCH'S POSTULATES Proof of etiology for
infectious agents 1) The organism must always
be found in the diseased animal but not in
healthy ones
2) The organism must be isolated from diseased
animals and grown in pure culture away from the
animal 3) The organism located in pure culture
must initiate and reproduce the disease when
reinoculated into susceptible animals 4) The
organism should be reisolated from the
experimentally infected animals Although not all
of these criteria can be met when evaluating the
etiology of human disease, and some etiologic
agents still cannot be cultured, these general
principles guide the establishment of etiology of
all infectious agents
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West Nile virus Transmission By mosquitoes that
have fed on infected birds. In 10-14 days the
virus reaches the salivary gland and can be
transmitted to humans and animals. Host At least
17 native bird species, including the house
sparrow. Once infected they can pass the virus to
mosquitos for up to 5 days. Carrier Several
species can carry the virus, Culex pipiens is
most common carrier in the NE US. They take 3-4
blood feedings in their 2-3 weeks life
span. Incidental hosts Humans and horses can be
infected but most people do not become sick.
Ordinary human contact will not spread the virus.
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Viral Pathogenesis Viral pathogenesis is an
abnormal situation of no value to the virus - the
vast majority of virus infections are
sub-clinical, i.e. asymptomatic.. For pathogenic
viruses, there are a number of critical stages in
replication which determine the nature of the
disease they produce 1) Entry into the
Host The first stage in any virus infection,
irrespective of whether the virus is pathogenic
or not. In the case of pathogenic infections, the
site of entry can influence the disease symptoms
produced. Infection can occur via
Skin - dead cells, therefore cannot support
virus replication. Most viruses which infect via
the skin require a breach in the physical
integrity of this effective barrier, e.g. cuts or
abrasions. Many viruses employ vectors, e.g.
ticks, mosquitos or vampire bats to breach the
barrier. Respiratory tract - In contrast to
skin, the respiratory tract and all other
mucosal surfaces possess sophisticated immune
defence mechanisms, as well as non-specific
inhibitory mechanisms (cilliated epithelium,
mucus secretion, lower temperature) which
viruses must overcome. Gastrointestinal tract
- a hostile environment gastric acid, bile
salts, etc Genitourinary tract - relatively
less hostile than the above, but less frequently
exposed to extraneous viruses (?) Conjunctiva
- an exposed site and relatively unprotected
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2) Primary Replication Having gained entry to a
potential host, the virus must initiate an
infection by entering a susceptible cell. This
frequently determines whether the infection will
remain localized at the site of entry or spread
to become a systemic infection, e.g Localized
Infections Virus Primary Replication Rhinovirus
es U.R.T. Rotaviruses Intestinal
epithelium Papillomaviruses Epidermis
Systemic Infections Virus Primary
Replication Secondary Replication Enteroviruses
Intestinal epithelium Lymphoid tissues,
C.N.S. Herpesviruses Oropharynx or
G.U.tract Lymphoid cells, C.N.S.
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3) Spread Throughout the Host Apart from direct
cell-cell contact, there are 2 main mechanisms
for spread throughout the host via the
bloodstream via the nervous system Virus
may get into the bloodstream by direct
inoculation - e.g. Arthropod vectors, blood
transfusion or I.V. drug abuse. The virus may
travel free in the plasma (Togaviruses,
Enteroviruses), or in association with red cells
(Orbiviruses), platelets (HSV), lymphocytes (EBV,
CMV) or monocytes (Lentiviruses). Primary viremia
usually proceeds and is necessary for spread to
the blood stream, followed by more generalized,
higher titer secondary viremia as the virus
reaches other target tissues or replicates
directly in blood cells. As above, spread to
nervous system is preceded by primary viremia. In
some cases, spread occurs directly by contact
with neurons at the primary site of infection, in
other cases via the bloodstream. Once in
peripheral nerves, the virus can spread to the
CNS by axonal transport along neurons (classic -
HSV). Viruses can cross synaptic junctions since
these frequently contain virus receptors,
allowing the virus to jump from one cell to
another.
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4) Cell/Tissue Damage Viruses may replicate
widely throughout the body without any disease
symptoms if they do not cause significant cell
damage or death. Retroviruses do not generally
cause cell death, being released from the cell by
budding rather than by cell lysis, and cause
persistent infections, even being passed
vertically to offspring if they infect the germ
line. Conversely, Picornaviruses cause lysis
and death of the cells in which they replicate,
leading to fever and increased mucus secretion in
the case of Rhinoviruses, paralysis or death
(usually due to respiratory failure) for
Poliovirus. 5) Cell/Tissue Tropism Tropism -
the ability of a virus to replicate in particular
cells or tissues - is controlled partly by the
route of infection but largely by the interaction
of a virus attachment protein (V.A.P.) with a
specific receptor molecule on the surface of a
cell, and has considerable effect on
pathogenesis. Many V.A.P.'s and virus receptors
are now known.
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6) Persistence vs. Clearance The eventual outcome
of any virus infection depends on a balance
between two processes i) Persistence Long
term persistence of virus results from two main
mechanisms a) Regulation of lytic potential The
strategy followed is the continued survival of a
critical number of virus infected cells -
sufficient to continue the infection without
killing the host. For viruses which do not
usually kill the cells in which they replicate,
this is not usually a problem, hence these
viruses tend naturally to cause persistent
infections, e.g. Retroviruses. For viruses
which undergo lytic infection, e.g.
Herpesviruses, it is necessary to develop
mechanisms which restrict virus gene expression,
and consequently, cell damage. b) Evasion of
immune surveillance - Includes antigenic
variation immune tolerance, causing a reduced
response to an antigen, may be due to genetic
factors, pre-natal infection, molecular mimicry
restricted gene expression down-regulation
of MHC class I expression, resulting in lack of
recognition of infected cells e.g. Adenoviruses
down-regulation of accessory molecules
involved in immune recognition e.g. LFA-3 and
ICAM-1 by EBV. infection of immunocompromised
sites within the body e.g. HSV in sensory ganglia
in the CNS direct infection of the cells of
the immune system itself e.g. Herpes viruses,
Retroviruses (HIV) - often resulting in
immunosuppression.
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ii) Clearance 2 mechanisms allow influenza virus
to alter its antigenic constitution Antigenic
Drift The gradual accumulation of mutations
(e.g. nucleotide substitutions) in the virus
genome which result in subtly altered coding
potential and therefore altered antigenicity, and
decreased recognition by the immune system. This
process occurs in all viruses all the time, but
at greatly different rates, e.g. RNA viruses gtgtgt
DNA viruses. The immune system constantly adapts
by recognition of and response to novel antigenic
structures - but is always one step behind. In
most cases however, the immune system is
eventually able to overwhelm the virus, resulting
in clearance. Antigenic Shift Is a sudden and
major change in the antigenicity of a virus due
to recombination or reassortment of the virus
genome with another genome of a different
antigenic type. This process results initially in
the failure of the immune system to recognize a
new antigenic type.
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Determinants of cell damage and
dysfunction 1. Direct destruction of cells by
virus Poliovirus - poliomyelitis Herpes simplex
- cold sores Rotavirus - diarrhea HIV -
AIDS 2. Immune mediated destruction of
virus-infected cells (CTL and Ab) Hepatitis B -
hepatitis Dengue - Dengue shock Measles -
post-measles encephalitis SSPE 3. Indirect
effector mechanisms Influenza - interleukins and
interferons released Rhinovirus - kinin release,
vascular changes Respiratory syncytial - IgE
Ab-mediated effects Hepatitis B and C - AbAg
complex deposition 4. Virus-encoded immune
alteration Adenovirus - E3 alters class I MHC
expression Vaccinia (and other poxviruses) - TNF
analogue Epstein-Barr - Interleukin 10 5. Allow
access to other pathogens
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Subtle effects of viruses 1. Chronic/persistent
infection Long term viral growth without disease,
more important in immunocompromised
hosts Retroviruses, herpesviruses, hepatitis B,
adenoviruses 2. Latent infection Lifelong
presence of viral genome with potential for
reactivation Retroviruses, papillomaviruses,
herpesviruses 3. Abortive infection Virus
persists with partial replication Measles
subacute sclerosing panencephalitis 4. Oncogenic
transformation Retroviruses, hepatitis B,
papillomaviruses (5. No viruses produce toxins)
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Host Cell-Virus Relationships
Explanations for the lack of replication in
non-permissive cells No attachment to
cell (no receptor) Virus can enter but not
uncoat
Virus can uncoat but not express genes (mRNA or
protein) Virus can express genes but produce no
particles Progeny particles form but fail to
mature Mature particles accumulate in the cell
but are not released Released viruses are not
infectious for other cells
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LATENCY When virus infection fails to result in
an immediate production of progeny. Rather, the
virus enters a latent state in which the viral
genome may become incorporated into the host's
genome or maintained as an extrachromosomal
element. Latent viral genomes are passively
replicated along with host chromosomes. Not all
viral genes remain silent during latency and
there may be a perceptible change in the
phenotype of the cell. The viral genome may
become reactivated, potentially up to decades
after the initial infection. Herpes,
papilloma, Hepadnaviruses Latent forms exist as
plasmids (nuclear, extrachromosomal
element) Virus Site of latent infection Herpes
simplex - sensory neurons Varicella-zoster -
dorsal root ganglion Epstein-Barr - B
lymphocytes Papilloma - basal epithelium e.g.
Development of "cold sores" by herpes viruses
after the host is exposed to large doses of
unfiltered sunlight. Retroviruses Latent forms
exist as "proviruses" integrated into the host
chromosome HTLV 1 - T cells HIV - CD4 T cells,
macrophages Latency can be associated with
tumorigenic transformation of the cell.
Infrequently, latent viral nucleic acid may be
passed not only to the progeny of the cells
within the host, but also through successive
generations in the germ line.
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A brief review of host immune responses to viral
infection
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Macrophage vs. Virus-infected cell
CTL vs.Virus- infected cell
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RETROVIRUSES F The first infectious agents
implicated in tumors (chicken sarcomas,
identified by Peyton Rous 1906) F The enormous
current interest in retroviruses can be
attributed to Their etiologic role in AIDS and
certain forms of cancer Their potential utility
as vectors in gene therapy, and, Due to several
aspects of a unique replication mechanism and a
close relationship with the genome of its host,
including that they are the only animal viruses
that integrate into the host cell's genome during
the normal growth cycle. F Retroviruses (and
transposable elements) appear to be part of every
cell's genome (bacteria, yeast, flies, mice and
humans (0.25 of the mouse genome). Some
endogenous viruses can be activated to replicate
and induce tumors however, the great majority of
sequences in eukaryotic genomes that are related
to retroviruses seem innocuous. F In most
instances retrovirus replication is
non-cytopathic (AIDS is one exception) and
persistent
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Three retrovirus subfamilies Oncovirus
subfamily is also subdivided according to
morphological criteria Subfamily Morph.
group Examples Oncoviruses Type A Intracisternal
A Particles (non-infectious) Type B Mouse
Mammary Tumor (MMTV) Type C Avian Sarcoma and
Leukemia Murine Sarcoma and Leukemia
Human T-cell leukemia virus (HTLV)
Type D Mason Pfizer monkey (MPMV) Lentiviruse
s Visna, HIV, SIV Spumaviruses Human Foamy
virus (cytopathic in vitro, no disease
association)
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All retroviruses have three essential genes which
encode polyproteins precursors essential for
virus replication gag (group specific antigen)
gene encodes the viral matrix (MA), capsid (CA)
and nucleoproteins (NC). The protease (PR) orf
encodes a product that cleaves the Gag
polyprotein precursor. It can be encoded as part
of Gag or a Gag-Pro-Pol polyprotein, sometimes
following a frame shift or a stop codon, and
read-through about 5 of the time by a ribosomal
frame shifting mechanism or by stop codon
suppression The major read-through product is
derived from the pol gene which encodes the RT
(including RNaseH) and an integrase (IN) that is
involved in provirus integration The envelope
gene encodes the surface glycoprotein (SU) -
transmembrane (TM) polyprotein.
Open Reading Frame map of HIV-1
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HIV Transcription Initiated by cellular factors
and greatly accelerated by Tat
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AUXILIARY GENES The human oncoviruses and the
lentiviruses are more complex than typical animal
oncoviruses. HTLV-1 and 2 encode regulatory
sequences located between env and the 3' LTR.
The primate lentiviruses are more complex and
encode an as yet poorly understood array of
expression regulators encoded by auxiliary
genes found in the central (between pol and env)
and 3 (within env and between env and U3)
regions of the viral genome. These genes are
expressed from a series of differentially spliced
mRNAs. Tat and Rev are the only essential
auxiliary genes
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All auxiliary gene products modulate infectivity.
Except for Nef, the additional auxiliary genes of
HIV described below are expressed late in
infection and are not essential for virus
growth. Nef (for negative effector) causes
downregulation of the HIV receptor, CD4, from the
cell surface. It may also be capable of
activating resting cells and does increase virus
levels in vivo, thus it may actually be a
positive regulator of virus. Nef also
downregulates expression of Class I HLA
molecules. This may help explain why CTL
(cytotoxic T lymphocytes), that recognize peptide
antigens presented by HLA molecules, are
ineffective at controlling HIV infections.
Permanent disruption of the SIV nef gene by
deletion results in 106 lowered levels of
expression in experimentally infected monkeys. In
contrast, point mutations are easily overcome by
this virus. Prior infection with the nef mutant
can prevent subsequent fulminant infection with a
virulent form of the virus. In Australia, a
cohort of hemophiliacs infected by transfusion
with a strain of HIV with multiple deletions in
nef have remained asymptomatic for almost two
decades. Thus, attenuated mutant forms of SIV
have been explored for potential use as vaccines.
However, recent results have shown both that SIV
can in at least some cases fix these deletions,
and that multiply deleted (nef plus other
auxiliary genes) viruses can be fully virulent in
newborns and can occasionally become virulent in
adults. Finally, the CD4 count of the
Australians have begun to drop and some adult
monkeys infected with the attenuated viruses have
gotten disease. Hence, attenuated HIV and SIV
strains can still cause disease.
65
Attacking HIV with antiretroviral drugs
Protease inhibitors
Attachment inhibitors
Assembly inhibitors
RT inhibitors
Integration inhibitors
66
Viral Oncogenic Transformation of Cells Cancer
can be viewed as a genetic disease. It is due to
discrete changes in the cellular genome which in
some cases are heritable. Transformation can be
defined as "the introduction of inheritable
changes in a cell causing changes in the growth
phenotype and immortalisation".
Virus are probably responsible for about 15 of
human cancers and as a risk factor are second
only to tobacco. We will discuss the process of
transformation, RNA tumour viruses, DNA tumour
viruses and viruses which cause tumours in humans
e.g. EBV, papilloma, Hepatitis B and HTLV-1.
67
Cell Transformation The G0/G1 boundary of the
cell cycle (G0 is the resting or stationary
phase, G1 is the phase in which the cell gears up
for division) is a particularly important control
point because this acts as a commitment to cell
division. Tumor formation results from a failure
of regulatory mechanisms which control this
boundary Tumor cells continue to divide under
circumstances in which their normal cellular
counterparts do not. Transformation of cells in
culture is the in vitro counterpart of the
process by which tumor induction in animals (by
viruses) occurs. Transformed cells are atypical
in many ways. For example Growth
high/indefinite saturation density
different, usually reduced serum requirement
tumor formation when injected into animals
no contact inhibition of growth/movement
Surface changes in glycoproteins and
glycolipids loss of tight junctions fetal
antigen expression increased rate of nutrient
transfer increased secretion of proteases
Intracellular disruption of cytoskeleton
altered amounts of signalling molecules
(cyclic nucleotides, phosphoinositides)
68
Mechanism of Transformation In vivo and
epidemiological studies indicate that
transformation is a multi-step process involving
initiation, promotion and progression Transformat
ion involves gene mutations, amplification of
cells containing these mutations and further
changes leading to transformation How do viruses
transform cells ? By subverting the normal cell
control mechanisms of a critical cellular
gene(s) There is a class of cell genes which,
generally speaking, promote cell replication.
These are sometimes called cellular oncogenes
(c-oncogenes) or proto-oncogenes. Viruses,
especially retroviruses can affect the activity
of these genes. Indeed retroviruses sometimes
carry their own versions of these genes, called
v-oncogenes. Another class of genes suppress
cell replication, these are called tumor
suppressors and many viruses especially DNA
viruses prevent their proper functioning. Cell
transformation by viruses is accompanied by the
persistence of all or a part of the viral genome.
It can also accompanied by the continual
expression of a limited number of viral genes.
69
The signal transduction pathway is responsible
for altering cellular gene expression in response
to a wide range of external and internal signals.
It is a complex net of regulatory proteins which
means that a given gene product can be
(de)activated by many different stimuli and that
a single stimuli can (de)activate many different
genes. Viral oncogenes disrupt the normal
functioning of this net. Components of the
pathway can be divided into 4 broad groups
1. Cell surface where receptors interact with
growth factors and components of the
extracellular matrix. 2. Transmembrane
signalling apparatus including the cytoplasmic
domain of receptors and submembranous components
that are functionally linked to surface
receptors, conveying signals from the outside of
the cell to the interior.
3. Cytosolic elements i.e. soluble proteins and
second messengers. 4. Nuclear proteins including
DNA binding proteins and tumour suppressors,
factors which control directly and indirectly
gene regulation and replication.
70
RETROVIRAL ONCOGENESIS Most retroviruses were
discovered by virtue of their association with
naturally occurring tumors or leukemias in
animals, including humans. It soon became
apparent that they could be divided into two
groups according to their ability to induce
tumors. Virus Type and characteristics Examples
Acutely transforming Avian, murine and feline
sarcoma cause tumors in 1 to a few weeks high
efficiency of disease induction carry
oncogenes transform cells in vitro nearly all
are defective Slowly transforming Avian,
murine and feline leukemia cause tumors in 6-12
months Mouse Mammary Tumor low efficiency HTLV
I do not carry oncogenes no visible effects on
cells in culture most are replication
competent some are endogenous viruses
71
FAST TRANSFORMING RETROVIRUSES High efficiency
(or "acute") transforming retroviruses are
usually defective, needing a helper virus to
provide necessary replication functions. Acute
transforming retroviruses incorporate or
transduce cellular genes, "oncogenes", which in
the context of being expressed from the viral
genome cause malignant growth.
Oncogene-carrying retroviruses will also
transform cells in culture so that these cells
will form tumors when injected into animals.
Oncogenes need not be incorporated into a virus
for that virus to cause a tumor. Yet, as we
study the genetic mechanisms leading to tumor
induction associated with oncogene-bearing
viruses, non-oncogene bearing viruses, and even
tumors with no known viral etiology, a recurring
theme is noted. In virtually all cases,
individual cellular genes can be identified,
referred to as proto-oncogenes, which are
subverted from their normal functioning,
resulting in some manner in tumor formation.
This subversion occurs as a result of
mutational change of coding sequences and/or
alteration of their normal regulation of
expression.
72
SLOW TRANSFORMING RETROVIRUSES In certain
retrovirus-induced tumors of chickens, proviruses
were found to integrate upstream of the c-myc
gene. Transcription, initiated within the viral
LTR, caused enhanced expression of the c-myc
gene. Thus, once again proto-oncogenes were
implicated in the cause of retrovirus-induced
tumors. A partially overlapping, yet larger
group of proto-oncogenes have been found to be
activated by provirus insertion (without
transduction). Activation of cellular
proto-oncogenes by slowly transforming viruses
occurs primarily as a result of provirus
insertion and is most often LTR mediated.
Different types of tumors are induced by the
same virus in different host genetic backgrounds.
Activation of c-myc is associated primarily with
B-cell tumors in chickens and with T-cell tumors
in mice and cats. The tumorigenic potential of
retroviruses is not constant. Viruses which
replicate more quickly or to higher levels in the
animal are generally more oncogenic, a property
due in large part to LTR enhancer properties.
However, other portions of the genome provide
essential contributions to tumorigenicity.
73
TUMOR INDUCTION BY NON-PRIMATE RETROVIRUSES Tumor
s induced by acutely transforming viruses are
polyclonal in nature, that is, many cells lose
growth control as a result of infection with an
oncogene-bearing virus. Tumors caused by slow
transforming viruses are mono or possibly
oligoclonal in nature, they occur as a result of
an outgrowth of a single rare cell with virus
integrated into a specific site near or within a
proto-oncogene. Tumor clonality therefore
reflects the mechanisms of induction.
74
Tumor induction by HTLV-1 presents a third face
The tumors are oligo or monoclonal cell
outgrowths, yet virus integrations occurred at no
preferred sites within the genome.
Furthermore, these viruses are wholly exogenous
to the species they infect, hence they harbor no
classic oncogenes. Unlike animal oncoviruses,
HTLV-1 encodes a transactivator, Tax, in addition
to Gag, Pol and Env. Tax transactivates
expression of the viral LTR and stimulates
expression of genes involved in cellular gene
regulation including the interleukin 2 (IL-2) and
IL-2 receptor (IL-2R) genes which are known to
effect T-cell growth regulation. These
observations therefore support (albeit not
convincingly to date) an autocrine model for
tumor induction in which, for example, activation
of IL-2 and IL-2R leads to continuous cell
proliferation.