8 - Viral proteins, their role and methods of their investigation. Viral lipids and carbohydrates

Question 1

Role of viral proteins

Answer 1

• Defense and targeting of the genome
• Shape of the virion
• Enzymes for multiplication
• Receptors

Question 2

Grouping of viral proteins

Answer 2

• Structural proteins:
  
  • ​Surface proteins:
    
    • Capsid, envelope
    • Shape, antigenicity, adsorption, HA activity, enzymes for penetration or release of virions
  • Core proteins:
    
    • ​NA protection
    • Stabilization
    • Enzymes for replication
• Non-structural proteins:
  
  • ​Coded in the viral genome
  • Not integrated in the viron
  • Present only in the the vegetiative virus
  • Early, immediate-early proteins
    
    • Regulation of the cell, inhibiton of cellular defense
    • Enzymes for replication
  • Late proteins:
    
    • Enzymes for structural protein maturation, virus assembly
    • The majority of late proteins are structural

Question 3

List the methods of protein investigation

Answer 3

1. SDS-Polyacrylamide-gel electrophoresis (PAGE)
2. Immunoblotting: western blot
3. Immunoperoxidase staining
4. Monoclonal antibody production

Question 4

Methods of protein investigation

1. SDS-Polyacrylamide-gel electrophoresis (PAGE)

Answer 4

• Needs concentrated virus suspension that contains only virons
  
  • Tread with SDS (Sodium Di-Sulphate) and mercapto ethanol
  • Creates a polypeptide map of virus
• ​Staining (silver, Coomassie Brillant Blue dye)
  
  • → Polypeptide map
  • → Characteristic to the virus (~ Restriction endonuclease analysis for nucleic acids)
• Shows:
  
  • Number of proteins
  • Size of proteins in virus

Question 5

Methods of protein investigation

2. Immunoblotting

Answer 5

• Western blot
• Transfer of viral proteins from PAGE into nitrocellulose filter

Question 6

Methods of protein investigation

3. Immunoperoxidase staining

Answer 6

• Types:
  
  • SDS-PAGE
  • Western blot
  • Histology sections
• Detections of antigens, steps:
  
  1. Detect antigens: cover with antibodies (specific to the antigen)
  2. Cover with conjugate: labeled with peroxide antibody
  3. Covered with substrate + H₂O₂ → O₂
  • A color reaction
• Diagnostics and identification
• Mechanisms and timing of protein production
• Epitope investigation

Question 7

Methods of protein investigation

3. Monoclonal antibody production

Answer 7

• Principle: immunoglobulins produced by clones of B-lymphocytes
• MAb: produced by a single clone of B cells
• Specific for a particular epitope
• Mouse immunization with viral antigen, lymphocytes isolated in spleen
• B cells: Ab production, but no mitotic activity
• Creates: Can mix with tumor cells → mitosis but no antibody production
• Clone testing: by ELISA, IF, IPO → epitope specific MBa’s in mice (large scale)

Question 8

Viral lipids

Answer 8

• Enveloped viruses (30-35%), form a lipid bilayer
• Acquired lipid membrane from host (budding)
  
  • Contains:
    
    • Phospholipid
    • Cholesterol
    • Virus specific proteins
    • Glycoproteins
• Envelope viruses are quite fragile, but gives camouflage
  
  • Especially: pox virus and african swine fever

Question 9

Viral carbohydrates

Answer 9

• Found in the nucleic acid:
  
  • Ribose
  • Deoxyribose
• Found on the surface:
  
  • Glycoproteins
• NB: Pox has glycoproteins in its core too