Replication

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Chapter 4

• Replication

2
Learning Objectives

• Understand how the nature of a virus genome
  determines its pattern of replication
• Describe a typical, generalized replication cycle
  of a virus
• Compare the patterns of replication of each of
  the seven major virus groups

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Classification of Viruses

• The way in which viruses are classified has
  altered as our perception of them has changed
• By disease early civilizations
• not good as many have the same symptoms
• By morphology 1930s-1950s
• similar shapes but different clinical symptoms,
  use serology
• Functional classification
• replication strategies

4
Investigation of Virus Replication

• Bacteriophages have long been used by virologists
  as models to understand the biology of viruses
• Two particularly significant experiments which
  illustrate the fundamental nature of all viruses
  were performed on bacteriophages
• Ellis and Delbruck in 1939
• Hershey and Chase in 1952

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The Single-Burst Experiment or One-Step
Growth Curve

• This was the first experiment to show the three
  essential phases of virus replication
• Initiation of infection
• Replication and expression of the virus genome
• Release of mature virions from the infected cell

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The Single-Burst Experiment

• Bacteriophages were added to a culture of rapidly
  growing bacteria
• After a few minutes, the culture was diluted,
  preventing further interaction between the phage
  particles and the cells.
• This step synchronizes the infection of the cells
  and allows the subsequent phases of replication
  in a population of individual cells and virus
  particles to be viewed as if it were a single
  interaction.
• Repeated samples of the culture were taken at
  short intervals and analysed for bacterial cells
  by plating onto agar plates and phage particles
  by plating onto lawns of bacteria.

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The Single-Burst Experiment
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The Single-Burst Experiment

• There is a stepwise increase in the concentration
  of phage particles with time, each increase
  representing one replicative cycle of the virus
• However, the data from this experiment can also
  by analysed by plotting the number of
  plaque-forming units (p.f.u.) per bacterial cell
  against time

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Single-Burst Experiment

• Total number of pfu per bacterial cell (dotted
  line)
• Lyse bacteria with chloroform and can determine
  the total number of pfu (solid line)

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Additional Terms

• Eclipse period 10-15 minutes no phage
  present, virus breaking down and releasing
  genome, not infectious at this tiem
• Latent period 20-25 minutes before start to see
  extracellular particules
• Release/Rise period
• The yield (number) of particles produced per
  infected cell can be calculated from the overall
  rise in phage titer

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Virus Replication

• Following the development of plaque assays for
  animal viruses in the 1950s, 'single burst'
  experiments have been performed for eukaryote
  viruses
• Major difference between these viruses and
  bacteriophages is the much longer time interval
  required for replication, which is measured in
  terms of hours or days rather than minutes
• difference reflects the slower growth rate of
  eukaryotic cells and the complexity of virus
  replication in compartmentalized cells

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Eukaryotic Virus Replication
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Biochemical Analysis of Virus Infection

• Use radioisotopes to follow the rate of growth of
  eukaryotic cells and viruses
• rate of incorporation determines amount of growth

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The Hershey-Chase Experiment

• Bacteriophage T2 was propagated in Escherichia
  coli cells which had been 'labelled' with one of
  two radioisotopes
• either 35S, which is incorporated into
  sulphur-containing amino acids in proteins
• or 32P, which is incorporated into nucleic acids
  (which do not contain any sulphur)
• Particles labelled in each of these ways were
  used to infect bacteria

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The Hershey-Chase cont

• After a short period to allow attachment to the
  cells, the mixture was homogenized in a blender
  which did not destroy the bacterial cells but
  removed the phage coats from the outside of the
  cells
• Analysis of the radioactive content in the cell
  pellets and culture supernatant (containing the
  empty phage coats) showed that most of the
  radioactivity in the 35S-labelled particles
  remained in the supernatant, while in the
  32P-labelled particles, most of the radiolabel
  entered the cells

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Hershey-Chase Experiment
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The Hershey-Chase Results

• DNA genome of the bacteriophage entered the cells
  and initiated the infection
• indicates that nucleic acid carries the genetic
  information of the phage
• At the time, it was generally believed that
  proteins were the carriers of the genes and that
  DNA was probably a structural component

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The Replication Cycle
19
The Replication Cycle

• Virus replication can be divided into eight
  arbitrary stages
• regardless of their hosts, all viruses must
  undergo each of these stages in some form to
  complete their replication cycle
• Not all the steps described here are detectable
  as distinct stages for all viruses

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Attachment

• Virus attachment consists of specific binding of
  a virus-attachment protein (or 'anti-receptor')
  to a cellular receptor molecule
• Target receptor molecules on cell surfaces may be
  proteins (usually glycoproteins), or the
  carbohydrate residues present on glycoproteins or
  glycolipids
• Some complex viruses (e.g. poxviruses,
  herpesviruses) use more than one receptor and
  have alternative routes of uptake into cells

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Virus Receptors

• Virus receptors fall into many different classes
• immunoglobulin-like superfamily molecules
• membrane-associated receptors
• transmembrane transporters and channels
• Viruses have subverted molecules required for
  normal cellular functions
• Plants enter thru breach, usually caused by
  vector cant penetrate the waxy coat, pectin or
  cell wall

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Virus Receptors

• Many examples of virus receptors are now known.
• Schematic representation of some virus receptors
• arrows indicate virus attachment site

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Virus-Receptor Interactions

• The major human rhinovirus (HRV) receptor
  molecule (4), ICAM-1 (intercellular adhesion
  molecule 1), is an adhesion molecule whose normal
  function is to bind cells to adjacent substrates
• ICAM-1 is similar to an immunoglobulin molecule,
  with constant (C) and variable (V) domains
  homologous to those of antibodies and is a member
  of the immunoglobulin superfamily of proteins
• The poliovirus receptor (1) is also a member of
  this family, with one variable and two constant
  domains

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Virus-Receptor Interactions

• Human rhinoviruses (HRVs) have a deep cleft known
  as the 'canyon' in the surface of each face of
  the capsid
• formed by the flanking monomers, VP1, VP2, and
  VP3
• Interaction between ICAM-1 and the virus particle
  occurs in this canyon
• unlike other areas of the virus surface, the
  amino acid residues forming the internal surfaces
  of the canyon are relatively invariant (same in
  all rhinoviruses)
• also protected from antigenic pressure because
  the antibody molecules are too large to fit into
  the cleft
• Other members of family use low-density
  lipoproteins, vascular cell adhesion molecule-1
  or glycophorin 1
• Picornoviruses use integrins, very-late
  antigen-2, fibronectin, decay accelerating factor

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Poliovirus Receptor Binding
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Influenza Virus Receptor Binding

• The influenza haemagglutinin protein (HA) is one
  of two types of glycoprotein spike on the surface
  of influenza virus particles, the other type
  being the neuraminidase protein (NA)
• HA spike is composed of a trimer of three
  molecules, while the NA spike consists of a
  tetramer
• HA spikes are responsible for binding the
  influenza virus receptor, which is sialic acid
  (N-acetyl neuraminic acid)
• result little cell-type specificity imposed by
  this receptor interaction and therefore influenza
  viruses bind to a wide variety of different cell
  types

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Influenza Virus Receptor Binding
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Neuraminidase

• Functions to detach virus from cell
• Attachment to cellular receptors is usually
  reversible and influenza may bind inappropriately
  to a variety of cells and cell debris because of
  the sialic acid wide-spread distribution
• Neuraminidase is an esterase which cleaves sialic
  acid from sugar side-chains allows for release
  of virus
• Elution from the cell surface after receptor
  binding has occurred often leads to changes in
  the virus (e.g. loss or structural alteration of
  virus-attachment protein) which decrease or
  eliminate the possibility of attachment to other
  cells

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Cell Tropism

• The expression of receptors on the surface of
  cells largely determines the tropism of a virus,
  i.e. the type of host cell in which it is able to
  replicate
• This initial stage of replication has a major
  influence on virus pathogenesis and in
  determining the course of a virus infection

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Multiple Receptors (1)

• In some cases, interactions with more than one
  protein are required for virus entry - neither
  protein alone is a functional receptor
• Adenovirus receptor-binding is a two stage
  process involving an initial interaction of the
  virion fiber protein with a range of cellular
  receptors, including MHC class I molecule and the
  coxsackievirus-adenovirus receptor (CAR)
• Another virion protein, the penton base, then
  binds to the integrin family of cell surface
  heterodimers allowing internalization of the
  particle via receptor-mediated endocytosis
• Most cells express primary receptors for the
  adenovirus fiber coat protein however, the
  internalization step is more selective

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Multiple Receptors (2)

• The primary receptor for HIV is the T cell
  antigen, CD4
• Transfection of human cells which do not express
  CD4 with recombinant CD4-expression constructs
  makes them permissive for HIV infection
• Transfection of rodent cells with human CD4 does
  not permit productive HIV infection
• If HIV DNA is added to rodent cells by
  transfection, virus is produced - no
  intracellular block to infection
• Several members of a family of proteins known as
  b-chemokine receptors play a role in the entry of
  HIV into cells, and their distribution may be the
  primary control for the tropism of HIV for
  different cell types (lymphocytes, macrophages,
  etc)

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Accidental Virus Uptake

• Can use pinocytosis, phagocytosis
• usually very low incidence
• Ab coated viruses can be taken up by FC receptor
• important in HIV uptake by macrophages and
  monocytes

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Penetration

• Penetration of the target cell normally occurs a
  very short time after attachment of the virus to
  its receptor in the cell membrane
• Unlike attachment, cell penetration is generally
  an energy-dependent process, i.e. the cell must
  be metabolically active for this to occur
• Three main mechanisms are involved
• translocation
• endocytosis
• fusion

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Translocation

• Translocation of the entire virus particle across
  the cytoplasmic membrane of the cell
• Process is relatively rare among viruses and is
  poorly understood
• Mediated by proteins in the virus capsid and
  specific membrane receptors

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Endocytosis

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

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Fusion

• Fusion of the virus envelope with the cell
  membrane, either directly at the cell surface or
  in a cytoplasmic vesicle
• Requires the presence of a fusion protein in the
  virus envelope which promotes joining of the cell
  and virus membranes, resulting in the
  nucleocapsid being deposited directly in the
  cytoplasm
• There are two types of virus-driven membrane
  fusion
• pH-dependent
• pH-independent

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Uncoating

• Uncoating is a general term for the events which
  occur after penetration
• relatively poorly understood
• not aggressively studied
• Product of uncoating depends on the structure of
  the virus nucleocapsid
• picornovirus uses VPg protein attached to viral
  RNA (simple)
• retrovirus is more complex with diploid RNA
• Structure and chemistry of the nucleocapsid
  determines the subsequent steps in replication

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Poliovirus Penetration and Uncoating

• Linked together
• Acid environment on capsid causes exposure of
  hydrophobic regions with the endosome membrane
  forms a pore for genome to exit

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Genome Replication and Gene Expression

• All viruses can be divided into seven groups
• Originally, this classification included only six
  groups, but it has since been extended to include
  the hepadnaviruses and caulimoviruses
• For viruses with RNA genomes in particular,
  genome replication and the expression of genetic
  information are inextricably linked, so both are
  taken into account

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Class I Double-stranded DNA

• 2 subgroups to this class
• Exclusively nuclear replication
• relatively dependent on cellular factors
• Cytoplasm replication such as poxviruses
• evolved (or acquired) all the necessary factors
  for transcription and replication of their
  genomes and are therefore largely independent of
  the cellular machinery

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Class I Double-stranded DNA
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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 ss ()sense RNA

• These can be subdivided into two groups
• Viruses with polycistronic mRNA
• the RNA genome forms the mRNA
• translated to form a polyprotein product, which
  is subsequently cleaved to form the mature
  proteins
• Viruses with complex transcription
• two rounds of translation (e.g. Togavirus) or
  subgenomic RNAs (e.g. Tobamovirus) are necessary
  to produce the genomic RNA

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Class IV ss ()sense RNA
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Class V ss ()sense RNA

• The genomes of these viruses can be divided into
  two types
• Non-segmented genomes (order Mononegvirales)
• 1st step is reverse transcription to mRNA and
  then becomes template for new genomes
• some are ambisense
• Segmented genomes (orthomyxoviruses)
• in nucleus
• monocistronic mRNA by viral transcriptase from
  full length

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Class V ss ()sense RNA
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Class VI ss ()sense RNA with DNA Intermediate

• Retrovirus genomes are ()sense RNA but unique in
  that they are diploid, and 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

• Relies on reverse transcription but 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
• hepadnavirus and cauliomavirus

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Class VII Double-stranded DNA with RNA
Intermediate
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Assembly

• Involves the collection of all the components
  necessary for the formation of the mature virion
  at a particular site in the cell
• during assembly, the basic structure of the virus
  particle is formed
• Site of assembly depends on the site of
  replication within the cell and on the mechanism
  by which the virus is eventually released
• in picornaviruses, poxviruses and reoviruses
  assembly occurs in the cytoplasm
• in adenoviruses, polyomaviruses and parvoviruses
  it occurs in the nucleus

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Maturation

• Stage of the replication-cycle at which the virus
  becomes infectious
• Usually involves structural changes in the virus
  particle which may result from specific cleavages
  of capsid proteins, conformational changes in
  proteins
• Virus proteases are frequently involved in
  maturation, although cellular enzymes or a
  mixture of virus and cellular enzymes are used in
  some cases

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Release

• Apart from plant viruses which have evolved
  particular strategies to overcome the structure
  of plant cell walls, all other viruses escape the
  cell by one of two mechanisms
• for lytic viruses (most non-enveloped viruses),
  release is a simple process - the infected cell
  breaks open and releases the virus.
• for 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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Summary

• Virus replication involves three broad stages
  which are carried out by all types of virus
• initiation of infection
• replication and expression of the genome
• release of mature virions from the infected cell
• At a detailed level, there are many differences
  between the replication processes of different
  viruses
• Nevertheless, it is possible to derive an
  overview of virus replication with common stages
  which are followed by all viruses