The Beginning and the End of the Virus Replication Cycle

1
Chapter 6

• The Beginning and the End of the Virus
  Replication Cycle

2
Outline of Replication

• Depends on many factors
• Virus recognition, attachment and entry into
  cells must get into cell by being engulfed or
  by direct injecting of genome into the cell
• Viral gene expression and genome replication
  must make viral proteins, make mRNA dependent
  on viral type
• Viral capsid formation and virion assembly need
  to form virus structure, release it from the
  cell, pick up envelope from cell membrane

3
Viral Entry

• Bacterial and animal viruses use a receptor to
  enter the cell, no evidence that this is done by
  plants
• Very many ways for viral entry as well as viral
  release
• viral release may result in death of the cell
  (cytocidal)
• may or may not involve cell lysis (cytolysis)
• also can see changes to the cell (cytopathology)

4
Animal Viral Entry

• Cellular receptor used for something other than
  virus entry
• Must get thru the lipid bilayer that has proteins
  in the upper and lower layers, some forming
  channels for ions and small molecules to pass
  thru
• Glycoproteins on surface can act as mediators of
  immunity, cell recognition, cell signaling and
  cell adhesion

5
Receptor Use

• See Table 6.1 for a list of Receptor Usage
• Ability of virus to replicate in specific cells
  is determined by the type and distribution of
  receptors it uses
• HIV uses receptors on CD4 T-cell to get in the
  cell CD4 usually involved in immunity, HIV can
  evade the immune system
• Poliovirus uses intercellular adherens molecule
  (ICAM)
• Rabies uses acetylcholine receptor that is in the
  synapse between nerves to spread in the CNS
  jumps from one nerve to the next
• Influenza virus uses sialic acid residue to
  specifically target respiratory cell

6
Tissue Tropism by Receptors

• Determined in part by whether receptor is
  available for recognition
• HIV binds a certain subset of T cells and certain
  neuronal cells
• Poliovirus can infect only primates
• Viruses use additional receptors as co-receptors
  both needed for efficient infection
• HIV also uses a chemokine receptor must bind to
  CD4 receptor and chemokine receptor for
  efficient infection
• dependent on co-receptor, can cause different
  cell tropism
• HIV binds CD4 and CCR5 and infects macrophages
• HIV binds CD4 and CXCR4 and infects T-cell

7
Other Mechanisms

• Not all virus entry is cell receptor-mediated
• Membrane changes allow adjacent cells to fuse and
  the formation of a syncytia
• HSV strains
• may not be complete fusion
• Virus may have a surface protein involved in
  receptor-mediated entry
• glycoproteins on enveloped HSV
• can remove the glycoprotein and virus can enter
  by other receptors

8
Entry of Non-Enveloped Virus

• Require translocation across the bilayer
• Involves receptor-mediated endocytosis
• endocytic vesicle acidity causes the breakdown of
  the capsid and release of genome
• Some viruses like SV40 needs intracellular
  trafficking proteins to transport to the nuclear
  membrane to dump genome into the nucleus
• process controlled by virus

9
Entry of Enveloped Virus

• Use membrane-associated viral glycoproteins that
  are made in a previously infected cells Golgi
• Enter by fusion of envelope at the cells surface
  or receptor-mediated endocytosis

10

• Interaction may be as simple as 1 glycoprotein
  and 1 receptor (Pseudorabies) or as a cascade of
  protein interactions like in HSV that uses 5-6
  viral glycoproteins with specific cellular
  receptor such as heparin sulfate which is a
  sulfated sugar receptor on host cell surface

11
Entry of Plant Virus

• Unique because of cell wall
• Requires a break in cell wall integrity so virus
  can have close proximity to plasma membrane,
  needs no specific receptors
• breaks come from mechanical force or other
  organisms feeding on plant
• virus may be brought in by pests eating on the
  plant
• mechanical damage is usually in experimental
  systems or process of agricultural harvesting
• Once inside the cell will have a replication
  pattern similar to animal systems
• Viral spread is by plasmodesmata the cell to
  cell communication systems
• Virus usually causes spread plant-wide because of
  this

12
Entry into Bacteria

• Bacteriophage interacts with receptor on bacteria
• bacterial surface has glycoproteins, LPS, amino
  acid and sugar transport complexes and
  cell-to-cell interaction apparatus (F or sex
  pilus used for conjugation and genetic material
  transfer)
• Virus must rearrange shape to attach to the
  bacteria
• not always the way it happens, different phages
  enter differently
• Some phages have only DNA and a few accessory
  proteins enter, some have entire phage particle
  entering cell

13
Entry

• Relatively weak interactions with phage tail
  fibers and LPS on surface triggering a stronger
  and irreversible interaction
• Tail pins on base plate of virion interact with
  structures in outer membrane, tail fibers change
  conformation
• Leads to compression of tails contractile sheath
  and injection of phage DNA, past cell wall but
  must still get thru cell membrane
• done by a viral protein called the pilot protein

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Non-Specific Entry Mechanisms

• Must get genome in cell to make more virus can
  get DNA or particle into bacteria using physical
  or chemical means
• Transfection used to introduce viral DNA into
  cells
• use calcium phosphate to aggregate genomes and
  then taken into cell
• use liposomes in solution that contain the DNA
  and hope they fuse with the cell membrane and
  release contents
• Both methods are inefficient and non-specific

15
Transfection
16
Late Events in Capsid Assembly

• 2 types of viral capsids
• helical
• icosahedron

17
Helical Capsid Assembly

• Capsids of helical viruses must form around the
  genome, appears to be similar for all helical
  viruses
• dependent on ss or dsRNA or DNA to form a helix
  when associated with the proper protein
• capsomeres self-assemble to make a disc and
  interacts with pac (packaging signal) in genome
• turns into a lock and washer and subsequent
  capsomeres thread on and complete capsid
• RNA is screw inside the capsid, continues
  association with genome

18
Icosahedral Capsid Assembly

• Mature from an immature pro-capsid
• Imparts capsid stability and often encapsidation
  of viral genome
• Preassembly of pro-capsid and specific covalent
  modification of virion proteins is accomplished
  by proteolytic processing
• maturational proteases are responsible for
  cleavage and are antiviral drug target
• Poliovirus (small RNA virus) can make empty
  capsid that is stable after self-assembly before
  the genome enters virion, may assemble around
  viral RNA
• Empty without non-functional by-product of
  assembly process

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Larger Icosahedral Viruses

• Capsid assembly is complex with a scaffolding
  protein mold
• assembly occurs before viral genome enters but
  can generate empty capsids
• See pilot protein ehich will be used to inject
  DNA later
• Scaffold proteins may be recycled and can make
  more than one capsid

20
Retrovirus Assembly

• Retrovirus activate virion-associated enzymes
  during final stage of assembly
• Form structural proteins
• protease inhibitors have been made to counteract
  viral infection

21
Envelopment and Viral Egress

• Envelope comes from the infected cell membrane
  no genes identified to date that may help with
  this
• However, envelope becomes virus-specific by
  insertion of 1 or more virus encoded membrane
  proteins

22
Assembly in the Cytoplasm

• Viral glycoproteins in the RER and then processed
  thru the Golgi
• C in the cytoplasm and N on outside
• Assemble in subcellular location similar
  arrangement use trafficking signals
• Glycoproteins in the membrane, virus assembles
  and packages DNA, virion buds thru the membrane
  at the site of the glycoproteins

23
Viral Egress

• Some will bud from the cell at the apical surface
  Influenza
• Some bud from basolateral surface vesicular
  stomatitis virus

24
HSV Egress

• Capsid forms in nucleus and full capsid associate
  with tegument proteins near the nuclear membrane
• Bud thru nuclear membrane and then looses
  envelope and gets another one at the plasma
  membrane
• Can get envelope on an empty capsid or one that
  just contains matrix proteins called dense bodies
• both are non-infectious without the viral genome

25
Double Membrane Theory