Infectious cycle

Question 1

Steps of the infectious cycle.

Answer 1

1. Attachment and entry
2. Uncoating
3. Translation
4. Genome replication
5. Assembly
6. Release

Question 2

What cells are required for viral replication to occur?

Answer 2

Host cells must be:
Susceptible- have viral receptors (no receptors=resistant cell)
Permissive- allow replication
Replication requires a combination of a permissive and susceptible cells

A virus can only successfully infect a cell in which it can replicate.

Question 3

Explain stages of the one step growth curve.

Answer 3

Eclipse period (up to 11mins): No active, assembled virus can be found either inside or outside the cell. During this period the viral nucleic acid is uncoated from its protective shell and the genome is replicated. Attachment, penetration and uncoating.

Latent period (up to 22mins): Quantity of intracellular virus increases marking the beginning of assembly of viral proteins to generate infectious particles. No extracellular virus is detected.

Rise period (22mins+): Viral production plateaus once every cell in the population is infected and graphs converge (total and extracellular) when lysis of the culture is complete.

Question 4

How is the Poliovirus receptor made?

Answer 4

Five VP1 protein subunits at the 5fold axis of symmetry form a canyon in the capsid which is the recognition site of the receptor.
1 poliovirus interacts with 60 receptors.

Question 5

Influenza A,B and C receptor binding

Answer 5

Influenza virus binds to negatively changed terminal sialic acid present in oligosaccharide moieties of cell surface glycoproteins using a haemagglutinin trimer.
Haemagglutinin allows the virus to attach to the cell and begin the penetration process.

Question 6

How do naked viruses enter the cell and what are some examples?

Answer 6

Direct translocation- zoonotic infections
Genome injection-bacteriophages
Endocytosis-adenovirus, polio virus

Question 7

How do enveloped viruses enter the cell and what are some examples?

Answer 7

Membrane fusion- sendai virus, measles virus, HIV

Endocytosis followed by membrane fusion- influenza

Question 8

What viruses uncoat at the plasma membrane?

Answer 8

Enveloped viruses e.g. Paramyxoviridae and Herpes virus

Question 9

What viruses uncoat at the endosome?

Answer 9

Enveloped and naked e.g. Togaviridae

Question 10

What viruses uncoat at the nuclear membrane?

Answer 10

Naked viruses e.g. Adenovirus

Question 11

Uncoating at the plasma membrane process

Answer 11

1. Adhesion of viral membrane protein H to cell surface receptors, this induces a conformational change of the H protein as well as of the adjacent protein F.
2. The hydrophobic fusion peptide is exposed and inserted into the PM of the target cell.
3. The fusion protein induces fusion and the viral nucleocapsid, genome and other proteins are released into the cytoplasm.

Question 12

By what pathways can receptor mediated endocytosis occur and what are some examples?

Answer 12

1. Clathrin dependent
2. Caveolin dependent (SV40, Coxsackie)
3. Clathrin and cave-in dependent (Influenza A, Herpes simplex)

Question 13

Uncoating of Influenza virus in the endosome process

Answer 13

1. Viral HA binds to sialic acid containing receptors and endocytosis occurs.
2. import of H+ ions acidifies the endosome causing the HA to to undergo a conformational change which converts it to a fusogenic protein.
3. The loop region of the HA becomes a coiled coil, moving the fusion peptides near to the endosome membrane. Fusion occurs.

Question 14

Role of M1 and M2 in uncoating the influenza genome.

Answer 14

1. M2 ion channel is a homotetramer which forms a pore at low pH.
2. Pores allow protons to enter the virion.
3. The drop in pH results in a conformational change which releases viral ribonucleoprotein from the matrix M1.
4. This reveals nuclear localisation signals and allows the vRNP to be imported into the nucleus.

Question 15

Poliovirus (naked) entry into the cell

Answer 15

Adhesion of the virus to the PVR, CD155 results in a conformational change to the vision at temps above 33oC. Altered particles are formed which have lost the internal viral protein VP4. The hydrophobic N terminus of VP1 is displaced on the surface.

Due to this change, the particle becomes more hydrophobic and has increased affinity for membranes. The exposed lipophilic N terminus of VP1 inserts into the membrane forming a pore which allows transport of viral genome into the cell.

Question 16

Uncoating of an Adenovirus during cell entry.

Answer 16

1. The virus binds to its receptor using the fibre protein to an integral or Ig-like cell surface receptor.
2. Interaction with the pentane base of the fibre protein with another integral leads to internalisation by endocytosis. Attachment is mediated by RGS sequences.
3. As the virus is transported towards the nuclear membrane it undergoes multiple uncoating steps by which capsid proteins are removed sequentially.
4. As the endosome becomes acidified, the pentane base is released.
5. Protein VI is then believed to trigger lysis of the endosomal membrane releasing the virus into the cytoplasm.
6. Small amounts of histone H1 bind to heron proteins.
7. Importin 7 and beta recognise the binding and imports the protein into the nucleus triggering capsid disassembly.

Question 17

3 methods of assembly of protein shells

Answer 17

1. Assembly from individual proteins (SV40)
2. Assembly from a polyprotein precursor (Poliovirus)
3. Chaperone assisted assembly (Adenovirus type 2)

Question 18

Flaws of viral assembly process from individual proteins.

Answer 18

1. Individual proteins must encounter each other to assemble.
2. Non-specific interactions can hinder assembly.
3. Necessitates production of structural proteins in excess.

Question 19

Assembly of bacteriophage T4 features.

Answer 19

Head, tail and tail fibres are formed separately then assemble with one another. The assembly can’t proceed unless the previous structure is formed. The tail joins the head first then the tail fibres join last.

Question 20

Assembly of poliovirus steps

Answer 20

1. RNA enters the cell and transcribes. Polyprotein P1,P2,P3 are produced.
2. P1 is cleaved to VP0, VP1 and VP3.
3. VP0 gives rise to VP2 and VP4.
4. Replicase copies viral +RNA to -RNA.
5. At the same time host cell protein synthesis is shut off (prevention of cellular RNA from binding ribosomes).
6. 5S unit forms spontaneously and polymerises to form the 14S penton.
7. Pentamer is stabilised by protein-protein interactions and by interactions mediated by mysterate chains on the N-terminus of VP0.
8. Pentamers associate with the new RNA in membrane vesicles resulting in the formation of 150S provirion.

Question 21

Assembly of influenza A virus steps

Answer 21

1. Envelope proteins are delivered to the PM by the hosts’ secretory pathway.
2. M1 protein binds to ribonucleoprotein which prevents further transcription and replication and targets it for assembly. It also allows NEP to bind which allows the ribonucleoprotein to exit the nucleus.
3. M1 binds to targets on the cytoplasmic face of the PM and tethers the ribonucleoprotein to HA and NA.

Question 22

Function of scaffolding proteins

Answer 22

• Establish transient intermediate structures.
• Accurate assembly of large icosahedral viruses is mediated by scaffolding proteins.
• Scaffolding proteins are not present in the mature virus because they are subject to proteolytic degradation prior to entry of the DNA genome into the capsid.

Question 23

Assembly of herpes simplex virus type 1

Answer 23

1. Pre-VP22a self assembles into a scaffold-like structure and this stimulates binding of the VP5 structural protein followed by a triplet protein formed by VP23 and VP19.
2. The scaffold guides the self assembly of pentanes and hexons.
3. The VP24 protease in the core is activated and cleaves a short C terminal sequence from the scaffold protein.
4. The scaffold disintegrates and is further degraded, promoting conformational change and allowing entry for genomic DNA.

Question 24

How does polio induce cell lysis?

Answer 24

1. Polio virus shuts down host cell protein synthesis.
   - Initiation factor eIF4G, which binds to 5’-7-methyl guanosine CAP, is cleaved by viral protease 2A.
   - The eIF4E subunit is then lost from the eIF4F complex, preventing cellular RNA from binding ribosomes so mRNA is not translated.
2. Polio alters membrane permeability of host cells. 2Bpro a tetramer of a 99aa peptide forms pores in the membrane of infected cells so things can escape.

Question 25

How do glycoproteins insert into the plasma membrane?

Answer 25

1. Glycosylation starts on the rough ER
2. Vesicle containing the viral glycoprotein transports it to the Golgi apparatus where glycosylation continues.
3. Viral glycoproteins transported to the PM in a vesicle. The nucleocapsid can then bind to it and the whole thing can bud off.

Question 26

Assembly and maturation of a retrovirus

Answer 26

1. Association of Gag molecules with the plasma membrane and the RNA genome.
2. Assembly of core completes and exocytosis can begin.
3. Fusion of the membrane releases an immature non-infectious particle.
4. Proteolytic cleavage of Gag and Gag-Pol polyproteins competes the maturation.

Question 27

How are viral genomes distinguished from cellular DNA/RNA?

Answer 27

Requires discrimination among similar nucleic acid
molecules. Discrimination is the result of packaging signals in the viral genome ‐ sequences necessary for incorporation of nucleic acid into virions; genetically defined.

Question 28

Packing of HSV type 1 DNA

Answer 28

1. Replication of herpesviruses produces concatomers containing many head to tail copies of the viral genome.
2. Individual genomes must therefore be liberated from such concatomers.
3. Pac1 and pac2 packaging signals recognise viral DNA and cleave it within adjacent DR repeats.
4. Proteins which recognise pac endonuclease to release DNA from concatomers.
5. A terminase complex (which includes an
   endonuclease) forms on pac and is bound
   by the portal. DNA is cleaved.
6. DNA is reeled into the capsid until a head full threshold
   Is reached and a sequence of the same orientation
   encountered. DNA is then cleaved.