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MICR 306
Applications of Viruses
(Part 2)
Classification ….
Prof. J. Lin
University of KwaZulu-Natal
Westville campus
Microbiology Discipline
2014
School of Life Sciences
CLASSIFICATION OF VIRUSES
INTERNATIONAL CLASSIFICATION OF VIRUSES
The internationally agreed system of virus classification is
based on the structure/composition (Primary
characteristics) of the virus particle (virion), in some cases,
the mode of replication (Secondary characteristics) is
also important in classification.
Sometimes a group of viruses which seems to be a single
group by the above criteria is found to contain a subgroup of
viruses which have a fundamentally different replication
strategy - in this case the group will be divided based on the
mode of replication.
VIRAL CLASSIFICATION
RNA or DNA
single-stranded or double-stranded
Nucleic acid
non-segmented or segmented
linear or circular
If genome is single stranded RNA, can it
function as mRNA?
whether genome is diploid (it is in retroviruses)
Virion
structure
symmetry (icosahedral, helical, complex)
enveloped or not
number of capsomers
How are viruses classified?
A universal system for classifying viruses, and a
unified taxonomy, has been established by the
International Committee on Taxonomy of Viruses
(ICTV) since 1966. The system makes use of a series of
ranked taxons with the:
•- Order (-virales) being the highest currently recognized.
- then Family (-viridae)
o- Subfamily (-virinae)
- Genus (-virus)
- Species ( eg: Tobacco mosaic virus)
The Baltimore Classification
Replication strategy
By convention the top strand of coding DNA
written in the 5' - 3' direction is + sense.
mRNA sequence is also + sense. The
replication strategy of the virus
depends on the nature of its genome.
Viruses can be classified into seven
(arbitrary) groups:
The Baltimore Classification
Central Dogma
The current (2013) taxonomy releases by
ICTV lists more than 7,400 viruses in 2827
species, 455 genera, 103 (22) families (sub)
and 7 orders.
 77 families not assigned to an order
• The definition of `species' is the most important but
difficult assignment to make with viruses.
• Problem for taxonomists:
1) Too small to be seen without EM
2) The high mutation rate of virus: Very small
changes in molecular structure may give rise to
agents with radically different properties.
 quasispecies
• Classification makes possible predictions about
details of replication, pathogenesis and
transmission.
Transfection
• The relative simplicity of virus genomes
(compared with even the simplest cell) offers a
major advantage - the ability to 'rescue'
infectious virus from purified or cloned nucleic
acids. Infection of cells caused by nucleic acid
alone is referred to as Transfection (although it
is about one million times less infectious than
virus particles).
 (+)sense RNA (Baltimore classification group 4)
 double-stranded DNA (Baltimore group 1)
Taxonomic Criteria -- name
The most important criteria are:
• Host Organism(s): eukaryote; prokaryote;
vertebrate, etc.  HIV, Phycodnavirus, phage T4
• Particle Morphology: filamentous; isometric;
naked; enveloped  Rotavirus, Corona virus
• Genome Type: RNA; DNA; ss- or ds-; circular; linear
 Picornavirus (small, RNA), Parva(small)viruses,
Hepadnaviruses (Liver, DNA),
Other criteria
• Disease symptoms  Influenza virus, pox
virus, Herpesviruses, HIV
• Antigenicity  N1H1, N1H5
• Protein profile,
• Host tissue range, etc.  Rhinoviruses,
HIV….
• Locations  New Castle Disease Virus
REPLICATION
Viral genomes contain information which:
• ensures replication of viral genomes
• ensures packaging of genomes into virions
• alters the structure and/or function of the
host cell to a greater or lesser degree - alter
the metabolism of the infected cell so that
viruses are produced
For a virus to multiply it must obviously infect a
cell. Viruses usually have a restricted host
range i.e. animal and cell type in which this is
possible.
Replication of a retrovirus, HIV-1
the main events are common to all viruses although the precise details obviously vary.
Viral Replication Pathway
•
Initiation phase:
–
–
–
•
Replication phase:
–
–
–
•
attachment
penetration
uncoating
DNA synthesis
RNA synthesis
protein synthesis
Release phase:
–
–
–
assembly
maturation
exit from cell
Initiation phase:
The genetic material of the virus is introduced
into the cell, often accompanied by
essential viral protein cofactors.
– attachment
– penetration
– uncoating
Entry Pathway of hepatitis C virus
Attachment
The virion protein (antireceptor) binds to a cell
surface receptor.
A classic example of this process is the
haemaglutinin anti-receptor of influenza virus.
Another intensively studied anti-receptor is the
gp120 envelope glycoprotein of HIV vs CCR5
and CD4:
Complex viruses such as pox or herpes may have
more than one anti-receptor.
The expression (or absence) of receptors on the
surface of cells largely determines the TROPISM
of most viruses, i.e. the type of cell in which they
are able to replicate - important factor in
pathogenesis.
Penetration
• Endocytosis of the entire particle resulting in
accumulation of virus particles inside a cytoplasmic
vesicle. The receptor bound virus is internalized
into the cell by endocytosis. Endocytosed vesicles
form bodies called endosomes.
• Fusion of the cellular membrane with the virion
envelope and direct release of the capsid into the
cytoplasm examples include paramyxo and herpes
viruses as well as HIV.
• Rarely, translocation of the virus particle directly
into the cytoplasm.
Uncoating:
This is the general term applied to events after
penetration which allows the virus to express its
genome.
In poxviruses, host factors induce the disruption of
the virus. The release of DNA from the core
depends upon viral factors made after entry.
In the influenza virus an envelope viral protein called
M2 may allow endosomal protons into the
virion particle resulting in its partial dissolution
and permitting replication.
In herpesviruses, adenoviruses and papovaviruses,
the capsid is eventually routed along the
cytoskeleton to nuclear envelope.
Replication phase:
•
•
•
DNA synthesis
RNA synthesis
protein synthesis
• The genomic size, composition and
organization of virus shows tremendous
diversity (look at the Baltimore
classification).
• In a few instances it is cellular enzymes that
replicate the viral genome, assisted by viral
proteins e.g. parvovirus.
• In most cases the opposite is true, viral
proteins are responsible for genome
replication although they utilize cellular
proteins to aid this.
• After entry into the cell, many phage
genomes are degraded and destroyed. Since
phage are so prevalent in the environment,
bacteria have specific mechanisms to protect
themselves against infection with phage "restriction/ modification" systems which
depend on the recognition and destruction of
foreign (unmodified - methylated or
glucosylated) DNA. Surviving phage
genomes sequester the cellular apparatus for
gene expression (transcription and
translation) to various extents.
• New copies of the viral genome must be
replicated and viral proteins must be
synthesized in order for viral multiplication
to occur. Since all viruses are obligated
pathogens.
• Despite the diversity of genome structure,
viruses must obey the central dogma of
molecular biology. All genetic information
flows from nucleic acid to protein. In
addition, all viruses use the host cell’s
translational machinery, and so no matter
what genome structure of virus, messenger
RNA must be generated that can be
translated on the host’s ribosomes.
Once viral mRNA is made, viral proteins can be
synthesized. The proteins synthesized as a
result of viral infection can be grouped into
two broad categories:
• Proteins (usually enzymes) synthesized soon
after infection, called the early proteins,
which are necessary for the replication of
viral nucleic acid.
• Proteins synthesized later, called the late
proteins, which include the proteins of viral
coat.
Retrovirus
SIVgsn
LTR
gag
env
vpr
pol
LTR
vif
rev
tat vpu
rev
tat
nef
HIV Structure
Retrovirus  the genome is integrated into host genome.
• The outer envelope comes from the host cell plasma
membrane. Coat proteins (surface antigens) are encoded
by env (envelope) gene. More than one surface
glycoprotein in the mature virus.
• icosahedral capsid containing proteins encoded by the
gag gene (Group-specific AntiGen)  nucleocapsid
(NCp15)
• Two molecules of plus sense genomic RNA per viron
with a 5' cap and a 3' poly A sequence.  diploid.
• a) Reverse transcriptase with RNase H activity
b) Integrase
c) Protease
MECHANISM OF VIRAL GENOME REPLICATION
• If host RNA polymerase II is used to copy the DNA
back to RNA, there are major problems with having a
DNA provirus form but an RNA genome in the mature
virus particle
• These problems include:
1) RNA polymerase II does not copy the upstream and
down stream control sequences of genes. It only copies
the information necessary to make a protein
2) The lack of proof reading by RNA polymerase II
The structure of the RNA genome of the mature and proviral retrovirus
Release phase:
•
•
•
assembly
maturation
exit from cell
Assembly
• Some viruses make morphogenetic factors
which are not structurally part of the virus but
whose presence is required for normal
assembly. These are sometimes called
molecular chaperones. Cellular chaperones
may also take part in this process.
• Other viruses self-assemble.  TMV
• Symmetry is important in viral assembly. It
reduces ambiguities in the assembly process
and makes it easier.
• In order for virus to be released from the host by
a lysis mechanism, enzymes are required that
damage both the cytoplasmic membrane and
the cell wall.
• In all instances it is viral proteins that are
responsible for this third and final stage although
host proteins and other factors may still
associate with the virus particle.
• Cells infected with polio virus can yield more
than 100,000 copies of virus per infected cell.
• Particle/infectivity ratio in picornaviruses is as
low as 0.1% i.e. only 1 in 1000 virion particles
are infectious. Virus can be defective for
innumerable reasons but it makes the study of
replication difficult because so many infections
are abortive Information about the reproductive
cycle of viruses has come from the study of
synchronously infected cells
• Shortly after infection and for a time period of
minutes to hours depending on the virus being
studied only low amounts of parental infectious
material can be identified, this is the eclipse
phase. Genome replication has been initiated
but progeny viruses have not yet formed.
There is then a maturation phase when viral
material accumulates exponentially in the cell or
surrounding medium. After a few hours cells
infected with lytic viruses become metabolically
disordered and then die, viral production ceases.
HIV - EM
Replication of a retrovirus, HIV-1
Nucleocapsid protein NCp15
Gp120
Reverse Transcriptase
Integratase
HIV Protease
Adenovirus
Episomal elements
The virally coded terminal protein (TP) acts as a primer
Adenovirus
One Step Growth Curve
the early period in which no infective virions are found even inside the infected host cells
is called the eclipse period.
Outcomes of viral infection
1) productive, i.e. entry into permissive cells
followed by virion formation;
2) abortive, i.e. entry into a non permissive cell
which does not result in virion formation; there
can be many reasons for non-permissiveness:
restringent or restrictive, the cell is transiently
permissive and a few virus are produced.
Thereafter virus production stops but the
genome remains present in the cell, examples
include Epstein Barr Virus and herpes simplex
virus. This kind of infection may still have
serious consequences e.g. cell
transformation and cancer.
Lysogenic or Temperate Phage
Either multiply via the lytic cycle or enter a quiescent
state in the cell.
In this quiescent state most of the phage genes are not
transcribed; the phage genome exists in a repressed
state. The phage DNA in this repressed state is
called a prophage because it is not a phage but it
has the potential to produce phage.
In most cases the phage DNA actually integrates into
the host chromosome and is replicated along with
the host chromosome and passed on to the daughter
cells. The cell harboring a prophage is not adversely
affected by the presence of the prophage and the
lysogenic state may persist indefinitely.
The cell harboring a prophage is termed a lysogen.
 Pathogenesis, Biofilm formation of bacteria
Events Leading to Lysogeny
1
2
3
Events Leading to Termination of
Lysogeny
Anytime a lysogenic bacterium is exposed to
adverse conditions, the lysogenic state can be
terminated. This process is called induction.
Conditions which favor the termination of the
lysogenic state include: desiccation, exposure to
UV or ionizing radiation, exposure to mutagenic
chemicals, etc. Adverse conditions lead to the
production of proteases (recA protein) which
destroy the repressor protein. This in turn
leads to the expression of the phage genes,
reversal of the integration process and lytic
multiplication.
Lytic vs Lysogenic Cycle
The decision for lambda to enter the lytic or
lysogenic cycle when it first enters a cell is
determined by the concentration of the
repressor and another phage protein called
cro protein in the cell. The cro protein turns off
the synthesis of the repressor and thus prevents
the establishment of lysogeny. Environmental
conditions that favor the production of cro will
lead to the lytic cycle while those that favor the
production of the repressor will favor lysogeny.
Bacteriophage replication
TRANSDUCTION
Lysogeny plays a significant role in bacterial genetics. On rare
occasions, lysogeny permits the transfer of bacterial genes
between cells by TRANSDUCTION.
There are two forms:
Generalized Transduction: bacterial rather than phage DNA is
packaged into a phage head. When another cell is infected, the
bacterial DNA is injected and in a proportion of cases, may be
incorporated into the chromosome by homologous recombination,
replacing the existing genes (frequency 105-108 per cell). More
than one gene may be co-transduced (limit: packaging size =
~50kbp = ~1% of bacterial chromosome). Abortive transduction
occurs when the new DNA does not integrate into the
chromosome - may affect the phenotype transiently but is not
replicated and is eventually lost.
Specialized Transduction: Results from inaccurate excision of an
integrated prophage; some phage DNA is lost and some bacterial
genes are picked up and carried to the next host - therefore phage
are usually defective (non-infectious) and require replicationcompetent helper phage to replicate, depending on which phage
genes are lost.
Significance of Lysogeny
Model for animal virus transformation - Lysogeny is a
model system for virus transformation of animal cells
Lysogenic conversion - When a cell becomes
lysogenized, occasionally extra genes carried by the
phage get expressed in the cell. These genes can
change the properties of the bacterial cell. This
process is called lysogenic or phage conversion. This
can be of significance clinically. e.g. Lysogenic phages
have been shown to carry genes that can modify the
Salmonella O antigen, which is one of the major antigens
to which the immune response is directed. Toxin
production by Corynebacterium diphtheriae is mediated
by a gene carried by a phage. Only those strains that
have been converted by lysogeny are pathogenic.
Genetic control in prokaryotes
during infection
Anti-termination: To promote the synthesis of longer
transcripts encoding additional genetic information.
Modification of RNA polymerase: Several
bacteriophages, e.g. phage SP01 of Bacillus
subtilis, encode proteins which function as
alternative sigma factors, sequestering RNA
polymerase and altering the rate at which phage
genes are transcribed. Phage T4 of E. coli encodes
an enzyme which carries out a covalent
modification (ADP-ribosylation) of the host cell
RNA polymerase.
Genetic control in prokaryotes
At a post-transcriptional level, regulated by
control of translation. Bacteriophages of the
family Leviviridae, such as R17, MS2 and Qbeta  the secondary structure of the singlestranded RNA phage genome regulates both
the quantities of different phage proteins
which are translated, but in addition, also
operates temporal control of a switch in the
ratios between the different proteins
produced in infected cells.
DNA Virus Genomes
• Bacteriophages  small
• Bacteriophage M13  7.2 kb Molecular cloning
• Parvovirus  5 kb  contains only two genes,
rep, which encodes proteins involved in
transcription & cap, which encodes the coat
proteins. The ends of the genome have
palindromic sequences of about 115nt, which
form 'hairpins'. These structures are essential for
the initiation of genome replication, once again
emphasizing the importance of the sequences at
the ends of the genome.
RNA Virus Genomes
• size of single-stranded RNA genomes  small  the
fragility of RNA & the tendency of long strands to
break.
• tend to have higher mutation rates than those
composed of DNA because they are copied less
accurately towards smaller genomes.
• Viruses with negative-sense RNA genomes are a little
more diverse than positive-stranded viruses. Possibly
because of the difficulties of expression, they tend to
have larger genomes encoding more genetic
information. Because of this, segmentation is a
common though not universal feature of such viruses.
Some RNA viruses are not strictly 'negative-sense' but
ambisense, since they are part (-) sense & part
(+)sense.
– This type of virus particles all contains a virus-specific
polymerase.
– are inherently non-infectious.
Segmented virus genomes
• Segmented virus genomes are those which are divided
into two or more physically separate molecules of nucleic
acid, all of which are then packaged into a single virus
particle.
• Multipartite genomes are those which are segmented &
where each genome segment is packaged into a
separate virus particle. These discrete particles are
structurally similar & may contain the same component
proteins, but often differ in size depending on the length
of the genome segment packaged.
• There are many examples of segmented virus genomes,
including many human, animal & plant pathogens such
as orthomyxoviruses, reoviruses & bunyaviruses. There
are rather fewer examples of multipartite viruses, all of
which infect plants.
Viral Genetics
GENERAL
• Viruses grow rapidly  usually a large number
of progeny virions per cell.
• More chance of mutations occurring over a
short time period.
• The nature of the viral genome (RNA or DNA;
segmented or non-segmented)
Viruses may change genetically due to mutation
or recombination
Outcomes of Viral Mutations
(Individual)
• Conditional lethal mutants:
e.g. Temperature sensitive (ts) mutants
e.g. Change of host range
• Plaque size:  alter pathogenicity
• Drug resistance: antiviral agents
• Enzyme-deficient mutants:
• "Hot" mutants:  host fever may have little effect
on the mutants but may slow down the replication
of wild type virions
• Attenuated mutants: cause much milder
symptoms (or no symptoms) compared to the
parental virus role in vaccine development and
useful tools in determining why the parental virus is
harmful
Outcomes of Viral Mutations
(Infections)
• COMPLEMENTATION
• MULTIPLICITY REACTIVATION: reactivation if
cells are infected with the inactivated virus at a
very high multiplicity of infection
• DEFECTIVE VIRUSES: Defective viruses lack
the full complement of genes necessary for a
complete infectious cycle and so they need
another virus (a helper virus) to provide the
missing functions.
• DEFECTIVE INTERFERING PARTICLES
Re-assortment
• If a virus has a segmented genome and if two variants of that virus
infect a single cell, progeny virions can result with some segments
from one parent, some from the other. This is an efficient process
- but is limited to viruses with segmented genomes - so far the
only human viruses characterized with segmented genomes are
RNA viruses e.g. orthomyxoviruses, reoviruses, arenaviruses.
• Reassortment may play an important role in nature in generating
novel reassortants and has also been useful in laboratory
experiments. It has also been exploited in assigning functions to
different segments of the genome. For example, in a reassorted
virus if one segment comes from virus A and the rest from virus B,
we can see which properties resemble virus A and which virus B.
Reassortment is a non-classical kind of recombination.
Life cycle and reassortment of a
segmented positive-sense RNA plant virus
Phenotypic Mixing
• If two different viruses infect a cell, progeny
viruses may contain coat components derived
from both parents and so they will have coat
properties of both parents. IT INVOLVES NO
ALTERATION IN GENETIC MATERIAL.
• Phenotypic mixing may occur between related
viruses  Pseudotype formation (pseudovirion)
e.g. a retrovirus nucleocapsid in a rhabdovirus
envelope. This results in pseudotypes having an
altered host range/tissue tropism on a
temporary basis.
Phenotypic Mixing
HIV
Exponential epidemic growth
45
40
35
30
Millions
of
infections
WHO:
25
40 M
infected
2000
Series1
20
Expon. (Series1)
15
< 2k
10
< 300k
5
0
1920
origin-5
1930
1940
1950
1960
Year
1970
1980
1990
2000
2010
Bushmeat market
photograph by Karl Ammann
Polio Vaccine in 1960s?
Mutations of HIV !!!
Since the roll-out of antiretroviral therapy
(ART) almost ten years ago, drugresistant HIV has increased
significantly in parts of Sub-Saharan
Africa in 2012.
In 2004, polio outbreaks in China were
not caused by an imported wild
poliovirus, but by a circulating vaccinederived poliovirus that had mutated
from the OPV used to prevent polio.
Applied genetics
• NEW VACCINE  influenza virus. contains 3
strains of attenuated influenza strains The
vaccine technology uses reassortment to
generate reassortant viruses which have six
gene segments from the attenuated, coldadapted virus and the HA and NA coding
segments from the virus which is likely to be
a problem in the up-coming influenza
season.
Viruses in the act of speciation
If a full complement of genes arrives in a
new host from the genomes of two
different parental viruses, a new virus can
be assembled
 Tobravirus strains I6 and N5 = RNA 1 from
Tobacco rattle virus + RNA 2 sequences from
Pea early browning virus.
Cooperation of viruses in mixed
infections
When viruses in a mixed infection share genetic
information or gene products, they are in
symbiosis, or cooperation.
Synergy (disease symptoms can be
exacerbated) -- Potato virus Y (PVY, genus
Potyvirus) and Potato virus X (PVX, genus
Potexvirus)
 Attenuation
Some viruses are totally dependent on other
viruses for parts of their life cycles, making them
obligate symbionts.
Interdependence
Several plant virus diseases are caused by
viruses that are in obligate symbiotic
relationships with other viruses. 
umbraviruses + luteoviruses, which enable the
umbraviruses to be transmitted by insects. In
groundnut rosette disease, an umbravirus, a
luteovirus and a satellite-like RNA are all required for
successful transmission by aphids. In some cases,
the umbravirus also assists the luteovirus in tissue
invasion, forming a completely mutualistic
symbiosis.
Helper Viruses; Satellites
Competition in plant viruses
Studies indicated that an exclusion
mechanism was functioning, whereby a
cell that was already infected with a virus
could not be superinfected by a related
virus.
potyviruses; mixed infection of Wheat streak
mosaic virus strains and strains of
Cucumber mosaic virus (CMV).
 Other viruses vs HIV?
The aphid-borne cucumber mosaic virus
causes infected plants to release higher
levels of volatile compounds than
uninfected plants, thus attracting more
aphids. The aphids then emigrate from the
infected plants more quickly than from
healthy plants, so increasing the chance of
virus spread.
Proc. Natl Acad. Sci. USA doi:10.1073/ pnas.0907191107 (2010)
Forming a long-term relationship with a virus
involves a tricky decision for the host. A
strong immune response might lead to better
control of the pathogen, but, at the same
time, such responses can potentially cause
substantial tissue damage, so sometimes a
little tolerance is required.
 co-evolution between the host and virus.
Summary
• There is more genetic diversity among
viruses than in all the rest of the Animal,
Plant & Bacterial kingdoms, all of whose
genomes consist of d/s DNA.
• The expression of virus genetic information
is dependent on the structure of the genome
of the particular virus concerned, but in
every case, the genome must be recognized
& expressed using the mechanisms of the
host cell.
 Viral roles in Ecology?