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@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
Death Rate (per 105) from Infectious Diseases in the US 1900-1996
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
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.
Over the past 100 years: A newly emerged strain of
influenza killed 20 million people in 1918-1919 in the
immediate aftermath of World War I (It was the most
lethal combatant of the war). A decade later, polio
became one of the most feared infections of children
and young adults (Franklin D. Roosevelt, the U.S.
President throughout the Depression and World War II,
had polio).
As the last century entered its final 20 years, a new
>90% lethal virus, HIV, spread rapidly around the
world via body fluid transmission.
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.
Ebola
Four Corners Virus (Hanta)
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.
Mouse primary spleen cells transduced with a
GFP (green fluorescent protein) -retrovirus vector
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
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 Viruses cannot be
grown on sterile media,
but require the presence
of specific host cells.
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.
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.
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.
Kuru
Scrapie
BSE
vCJD
BSE
in
Britian
80
70
CJD,
nvCJD
in
Britian
60
50
40
30
20
10
0
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000
Virus naming and classification
Usually based on data available at the time of discovery:
i
Disease they are associated with, e.g.:
Poxvirus, Hepatitis virus, HIV, measles virus
ii
Cytopathology 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
Rotavirus
These naming conventions can lead to confusion later, e.g.,
viral hepatitis is caused by at least 6 different viruses
“Infectious”
Viral
hepatitis
“Serum”
A
E
F, G,
? Other *
NANB
B
Enterically
transmitted
C
Parenterally
transmitted
D
* 10-20% of cases of presumed viral hepatitis are still not accounted for
Viral Hepatitis - An Overview
Type of Hepatitis
A
Source of
virus
Route of
transmission
Chronic
infection
Prevention
B
C
D
E
feces
blood/
blood/
blood/
blood-derived blood-derived blood-derived
body fluids
body fluids
body fluids
feces
fecal-oral
percutaneous percutaneous percutaneous
permucosal
permucosal
permucosal
fecal-oral
no
yes
pre/postexposure
immunization
pre/postexposure
immunization
yes
yes
blood donor
pre/postscreening;
exposure
risk behavior immunization;
modification risk behavior
modification
no
ensure safe
drinking
water
Acute Hepatitis B Virus Infection with Recovery
Typical Serological
Courses of Infection
Symptoms
HBeAg
anti-HBe
Total anti-HBc
Titer
IgM anti-HBc
HAV Infection
HBsAg
anti-HBs
Symptoms
Titer
Total anti-HAV
0
ALT
4
8
12 16 20 24 28 32 36
Weeks after Exposure
IgM anti-HAV
Acute (6 months)
Titer
1
100
Progression to Chronic HBV Infection
Fecal
HAV
0
52
2
4
5
6
3
Months after exposure
Chronic (Years)
anti-HBe
HBeAg
HBsAg
Total anti-HBc
12
IgM anti-HBc
0
4
12
24
36
Weeks after Exposure
52
Years
Outcome of HBV Infection by Age at Infection
100
100
Symptomatic
Infection (%)
80
60
Chronic
Infection (%)
Chronic Infection
80
60
40
40
20
20
Symptomatic Infection
0
Birth
1-6 months
7-12 months
Age at Infection
1-4 years
0
Older Children
and Adults
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
Cellular and Viral Genomes
Type of Genome
Number of Gene Equivalents*
Human cell (higher eukaryotes)
1,000,000
E. coli (bacteria)
Poxvirus, Herpesvirus (large DNA virus)
Influenza, human immunodeficiency virus
5,000
70-200
8-12
Papilloma, Hepatitis B virus
Potato spindle tubor "viroid"
*The number of average-sized genes assuming
that the entire genome encodes sequence
information for the synthesis of proteins.
3-5
1
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.
STEPS IN VIRAL REPLICATION
1. Attachment (adsorption)
2. Penetration
3. Uncoating
4. Genome replication
5. Assembly
6. Release
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)
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.
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.
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).
Information Flow in cells:
DNA to DNA (Replication)
DNA to RNA (Transcription)
RNA to Protein (Translation)
The Central Dogma
QuickTime™ and a
GIF decompressor
are needed to see this picture.
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:
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.
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.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
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.
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.
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.
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)
Disease symptoms and abnormal laboratory test values
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
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.
Viral Pathogenesis
Viral pathogenesis is an abnormal situation of no value to the virus - the vast majority of virus infections are subclinical, 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
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:
Rhinoviruses
U.R.T.
Rotaviruses
Intestinal epithelium
Papillomaviruses
Epidermis
Systemic Infections:
Virus:
Primary Replication:
Enteroviruses
Intestinal epithelium
Herpesviruses
Oropharynx or G.U.tract
Secondary Replication:
Lymphoid tissues, C.N.S.
Lymphoid cells, C.N.S.
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.
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.
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.
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
>>> 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.
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 - Ab:Ag 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
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)
Host Cell-Virus
Relationships
A cell exposed
to a virus
Explanations for the lack of
replication in nonpermissive cells:
Permissive Cell
Non-Permissive Cell
• No attachment to cell
(no receptor)
• Virus can enter but not
Replication
No Replication
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
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.
A brief review of host immune responses to viral infection
QuickTime™ and a
Video decompressor
are needed to see this picture.
Macrophage vs.
Virus-infected cell
QuickTime™ and a
Video decompressor
are needed to see this picture.
CTL vs.Virusinfected cell
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
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 DMason Pfizer monkey (MPMV)
Lentiviruses
Visna, HIV, SIV
Spumaviruses
Human Foamy virus
(cytopathic in vitro, no disease association)
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:
v pr r e v
t at
env
• •
v if • •
pol
gag
1
1000
2
2000
3
3000
4
4000
5
5000
•
ne f
•
6
6000
t a t vpu
7
7000
8
8000
rev
9kb
9000
HIV Transcription
Initiated by cellular factors and
greatly accelerated by Tat
Unspliced and partially
spliced mRNA produced
through action of Rev
“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
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.
Attacking HIV with antiretroviral drugs
Protease
inhibitors
Assembly
inhibitors
Attachment
inhibitors
RT
inhibitors
Integration
inhibitors
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.
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)
Mechanism of Transformation
In vivo and epidemiological studies indicate that transformation is a multi-step process
involving: initiation, promotion and progression
Transformation 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.
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.
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
cause tumors in 1 to a few weeks
high efficiency of disease induction
carry oncogenes
transform cells in vitro
nearly all are defective
Avian, murine and feline sarcoma
Slowly transforming
cause tumors in 6-12 months
low efficiency
do not carry oncogenes
no visible effects on cells in culture
most are replication competent
some are endogenous viruses
Avian, murine and feline leukemia
Mouse Mammary Tumor
HTLV I
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.
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.
TUMOR INDUCTION BY NON-PRIMATE RETROVIRUSES
Tumors 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 protooncogene.
Tumor clonality therefore reflects the mechanisms of induction.
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.