MicrorobiologyPathobiology 445

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• Microrobiology/Pathobiology 445
• Lecture 4, 5Apr01
• Viral Life Cycle
• (and an overview of host-virus interactions)
• James I. Mullins, Ph.D.
• Professor and Chairman
• Department of Microbiology
• jmullins_at_u.washington.edu
• Spring, 2001
• Slides can be downloaded (ppt and pdf) from the
  course website and at http//ubik.microbiol.washin
  gton.edu/Index.html
• (Graphics and animation obtained from Murray text
  and several links from
• All the Virology on the WWW at
  http//www.virology.net/garryfavweb.html)

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The Course of Virus Infections

• Patterns of virus infection can be divided into a
  number of different types
• Abortive infection occurs when a virus infects a
  cell (or host), but cannot complete the full
  replication cycle. Therefore, this is a
  non-productive infection
• Acute infection many common virus infections
  (e.g. 'colds') - relatively brief infections,
  where the virus is usually eliminated completely
  by the immune system
• Chronic infection These are the converse of
  acute infections, i.e. prolonged stubborn. The
  best studied example is lymphocytic
  choriomeningitis virus (LCMV, an arenavirus)
  infection in mice
• Latent infections These typically persist for
  the entire life of the host, e.g. herpes simplex
  virus (HSV)

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Virus-Host Interactions

• For all viruses, pathogenic or non-pathogenic,
  the first factor which influences the course of
  infection is the mechanism site of entry into
  the body

4
The Skin

• Mammalian skin is a highly effective barrier
  against viruses
• The outer layer (epidermis) consists of dead
  cells therefore does not support virus
  replication
• Very few viruses infect directly by this route
  unless there is prior injury such as minor trauma
  or puncture of the barrier, such as insect or
  animal bites or subcutaneous injections
• Some viruses which do use this route are herpes
  simplex virus papillomaviruses, although these
  viruses probably still require some form of
  disruption of the skin such as small abrasions or
  eczema

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Mucosal Membranes

• The mucosal membranes of the eye genitourinary
  (GU) tract are much more favourable routes of
  access for viruses to the tissues of the body.
• This is reflected by the number of viruses which
  can be sexually transmitted virus infections of
  the eye are also quite common.

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The Alimentary Canal

• Viruses may infect the alimentary canal via the
  mouth, oropharynx, gut, or rectum, although
  viruses which infect the gut via the oral route
  must survive passage through the stomach, an
  extremely hostile environment with a very low pH
  high concentrations of digestive enzymes
• The intestinal epithelium is constantly
  replicating there is a good deal of lymphoid
  tissue associated with the gut which provides
  many opportunities for virus replication
• The constant intake of food fluids provides
  ample opportunity for viruses to infect these
  tissues
• However, the gut has many specific (e.g.
  secretory antibodies) non-specific (e.g.
  stomach acids bile salts) defense mechanisms

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The Respiratory Tract

• The respiratory tract is probably the most
  frequent site of virus infection
• As with the gut, it is constantly in contact with
  external virus particles which are taken in
  during respiration
• The respiratory tract also has defences aimed at
  virus infection - filtering particulate matter in
  the sinuses, and cells antibodies of the immune
  system
• Viruses which infect the respiratory tract
  usually come directly from the respiratory tract
  of others, since aerosol spread is very
  efficient 'coughs sneezes spread diseases'

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The Natural Environment is a Considerable Barrier
to Virus Infections.

• Most viruses are relatively sensitive to heat,
  drying, ultraviolet light (sunlight), etc.,
  although some are quite resistant to these
  factors
• This is particularly important for viruses which
  are spread via contaminated water or foodstuffs -
  not only must they be able to survive in the
  environment until they are ingested by another
  host, but as most are spread by the faecal-oral
  route, they must also be able to pass through the
  stomach to infect the gut before being shed in
  the faeces
• One way of overcoming environmental stress is to
  take advantage of a secondary vector for
  transmission between the primary hosts

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Virus Transmission

• Viruses without a secondary vector must rely on
  continued host-to-host transmission, have
  evolved various strategies for this
• Horizontal transmission The direct host-to-host
  transmission of viruses.
• This strategy relies on a high rate of infection
  to maintain the virus population
• Vertical transmission The transmission of the
  virus from one generation of hosts to the next.
• This may occur by infection of the fetus before,
  during, or shortly after birth (e.g. during
  breastfeeding).
• More rarely, it may involve direct transfer of
  the virus via the germ line itself, e.g.
  retroviruses
• In contrast to horizontal transmission, this
  strategy relies on long-term persistence of the
  virus in the host rather than rapid propagation
  dissemination of the virus.

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Primary Replication

• The virus must initiate an infection by entering
  a susceptible cell (primary replication)
• This initial interaction frequently determines
  whether the infection will remain localized at
  the site of entry or spread to become a systemic
  infection
• In some cases, virus spread is controlled by
  infection of polarized epithelial cells the
  preferential release of virus from either the
  apical (e.g. influenza virus - a localized
  infection in the upper respiratory tract) or
  basolateral (e.g. rhabdoviruses - a systemic
  infection) surface of the cells

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Infection of Polarized Epithelium
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Systemic Spread

• Following primary replication at the site of
  infection, the next stage may be spread
  throughout the host
• In addition to direct cell-cell contact, there
  are two main mechanisms for spread throughout the
  host
• Via the bloodstream Viruses may get into the
  bloodstream by direct inoculation, for example,
  by arthropod vectors, blood transfusion, or
  sharing of non-sterilized needles
• Via the nervous system Spread of virus to the
  nervous system is usually preceded by primary
  viremia

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The Virus Life Cycle
Note typo in handout
Early Phase i/ii Attachment to and penetration
of the virion into the host cell iii Uncoating
of the virion iv Expression of viral proteins
involved in genome replication v Replication
of the viral genome Late Phase vi Synthesis of
virus structural components vii Assembly of
progeny virus particles viii Maturation of
virus ix Release from the host cell
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The Replication Cycle

• Virus replication can be divided into nine
  stages. These are arbitrary divisions, used in
  explaining the replication cycle of a
  non-existent 'typical' virus. All viruses must
  undergo each of these stages in some form to
  successfully complete their replication cycles.
  Not all the steps described here are detectable
  as distinct stages for all viruses often they
  blur together appear to occur almost
  simultaneously

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Cell Tropism

• In many if not most cases, the expression (or
  absence) of receptors on cell surfaces largely
  determines the tropism of a virus, i.e. the type
  of host cell in which it is able to replicate.
• In some cases, intracellular blocks at later
  stages of replication are responsible for
  determining cell tropism
• Therefore, this initial stage of replication
  the very first interaction between the virus
  the host cell has a major influence on virus
  pathogenesis in determining the course of a
  virus infection

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Attachment

• Virus attachment consists of specific binding of
  a virus-attachment protein to a cell surface
  receptor molecule
• Receptors may be proteins (usually
  glycoproteins), or carbohydrate residues present
  on glycoproteins or glycolipids
• Receptor proteins are usually specific for that
  virus
• Carbohydrate receptors are usually less specific
  because the same configuration of side-chains may
  occur on many different membrane-bound molecules
• Some complex viruses (e.g. poxviruses,
  herpesviruses) use more than one receptor and
  therefore have alternative routes of uptake into
  cells
• In some instances (e.g., dengue virus), Antibody
  Fc domains facilitate virus binding to Fc
  receptors

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Virus Receptors

• Virus receptors fall into may different classes,
  e.g
• immunoglobulin-like superfamily molecules
• membrane-associated receptors
• transmembrane transporters channels
• One factor that
• unifies all virus
• receptors is that
• viruses have
• subverted molecules
• required for normal
• cellular functions

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Poliovirus Attachment
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Penetration Endocytosis

• Endocytosis into intracellular vacuoles is
  probably the most common mechanism of virus entry
• Fusion does not require any specific virus
  proteins (other than those already utilized for
  receptor binding) but relies on the normal
  formation internalization of coated pits at the
  cell membrane
• Receptor-mediated endocytosis is an efficient
  process for taking up concentrating
  extracellular macromolecules

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Influenza virus entry
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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Penetration Fusion

• Fusion of the virus envelope (enveloped viruses
  only) with the cell membrane, either directly at
  the cell surface or in a cytoplasmic vesicle.
  Fusion requires the presence of a specific

fusion protein in the virus envelope (e.g.
influenza haemagglutinin or retrovirus
transmembrane (TM) glycoproteins), which promotes
joining of the cellular virus membranes. This
results in the nucleocapsid being deposited
directly into the cytoplasm
22
HIV entry
lt- Here the virion attachment protein - gp120 -
attaches initially to the CD4 protein on a helper
T-cell.  The gp120 undergoes conformational
change due to binding, and binds a co-receptor -
CCR-5, a ß-chemokine receptor in this case.  gp41
- a cleavage product of a gp160 precursor, and a
part of the "spike protein" of the viral membrane
- is then able to bind into the cell membrane,
via a hydrophobic domain.  The juxtaposition of
cell and viral membranes promotes membrane fusion
and nucleoprotein entry into the cell.
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Poliovirus entry
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Genetic information flow in cells and viruses
DNA to DNA (Replication) DNA to RNA
(Transcription) RNA to Protein (Translation) The
Central Dogma
The replication strategy of any virus depends on
the nature of its genetic material
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Class I Double-stranded DNA
Seven classes of virus replication schemes known
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Class II Single-stranded DNA

• Replication occurs in the nucleus, involving the
  formation of a double-stranded intermediate which
  serves as a template for the synthesis of
  single-stranded progeny DNA

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Class III Double-stranded RNA

• These viruses have segmented genomes
• Each segment is transcribed separately to produce
  individual monocistronic mRNAs

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Class IV Single-stranded () sense RNA

• The genome RNA forms the mRNA
• This is translated to form a polyprotein product,
  which is subsequently cleaved to form the mature
  proteins.
• More complex transcription patterns are also
  known (e.g. Togavirus)

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Class V Single-stranded ()sense RNA
The first step in replication is transcription of
the () sense RNA genome by the virion
RNA-dependent RNA polymerase to produce
monocistronic mRNAs, which also serve as the
template for subsequent genome replication
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Class VI Single-stranded () sense RNA with DNA
Intermediate

• Retrovirus genomes are () sense RNA but unique
  in that they are diploid, do not serve directly
  as mRNA, but as a template for reverse
  transcription into DNA

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Class VII Double-stranded DNA with RNA
Intermediate

• This group of viruses also relies on reverse
  transcription
• Unlike the retroviruses (class VI), this occurs
  inside the virus particle during 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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Expression of Genetic Information

• The course of virus replication is determined by
  tight control of gene expression
• Viruses have had to achieve highly specific
  quantitative, temporal, spatial control of
  expression with much more limited genetic
  resources than that of host cells
• Viruses have evolved a range of solutions to the
  problem of gene expression
• Positive negative signals which promote or
  repress gene expression
• Highly compressed genomes in which overlapping
  reading frames are commonplace
• Control signals which are frequently nested
  within other genes
• Several strategies designed to create multiple
  polypeptides from a single messenger RNA

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Shutoff

• A number of viruses which cause cell lysis
  exhibit a phenomenon known as shutoff early in
  infection
• Shutoff is the sudden dramatic cessation of
  most host cell macromolecular synthesis
• In poliovirus-infected cells, this is the result
  of the virus 2A protein
• 2A is a protease which cleaves the p220 component
  of eIF-4F, a complex of proteins required for
  cap-dependent translation of mRNAs by ribosomes
• Since poliovirus RNA does not have a 5'
  methylated cap but is modified by the addition of
  the VPg protein, virus RNA continues to be
  translated
• In poliovirus-infected cells, the dissociation of
  mRNAs polyribosomes from the cytoskeleton can
  be observed this is the reason for the
  inability of the cell to translate its own
  messages
• A few hours after translation ceases, lysis of
  the cell occurs

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Biochemical Analysis of Virus Infection
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Assembly

• Assembly involves the collection of all the
  components necessary for the formation of the
  mature virion at a particular site in the cell
• The basic structure of the virus particle is
  formed
• The site of assembly depends on the site of
  replication within the cell on the mechanism by
  which the virus is eventually released from the
  cell varies for different viruses
• In picornaviruses, poxviruses reoviruses
  assembly occurs in the cytoplasm
• In adenoviruses, polyomaviruses parvoviruses it
  occurs in the nucleus
• As with the early stages of replication, it is
  not always possible to identify the assembly,
  maturation release of virus particles as
  distinct separate phases.

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Maturation

• Maturation is the stage of the replication-cycle
  at which the virus becomes infectious
• Maturation usually involves structural changes in
  the virus particle which may result from specific
  cleavages of capsid proteins to form the mature
  products or conformational changes in proteins
  during assembly
• Virus proteases are frequently involved in
  maturation, although cellular enzymes or a
  mixture of virus cellular enzymes are used in
  some cases.

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Release

• For lytic viruses (most non-enveloped viruses),
  release is a simple process - the infected cell
  breaks open releases the virus
• Enveloped viruses acquire their lipid membrane as
  the virus buds out of the cell through the cell
  membrane or into an intracellular vesicle prior
  to subsequent release. Virion envelope proteins
  are picked up during this process as the virus
  particle is extruded - this process is known as
  budding

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Budding
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