Introduction and nucleic acid synthesis and metabolism

Question 1

What is a virus?

Answer 1

A non-cellular micro-organism that can only replicate within cells
Genome can be RNA or DNA, single stranded or duplex, circular or linear

Question 2

Simplified virus life cycle?

Answer 2

1. Cell entry
2. RNA/protein synthesis
3. Genome replication
4. Protein synthesis
5. Assembly of infectious virus particles
6. Release from the cell

Question 3

What does a virus need from the host cell?

Answer 3

Raw materials for biomolecular synthesis

• Nucleotides (dNTP/NTP)
• Amino acids

Machinery for biomolecular synthesis
-Protein synthesis

Enveloped viruses need membranes

Intracellular transport

Question 4

The cell cycle of non-diving cells?

Answer 4

G0 = Inactivity
G1 = RNA + protein synthesis 
S = DNA synthesis
G2
M = Mitosis

Question 5

What is the dUTP problem?

Answer 5

dUTP levels high in non-dividing cells + used as building blocks for some pathways

• Misincorporation into DNA is potentially mutagenic
• Viruses have their own dUTP to build

Question 6

How does dUTP –> TTP?

Answer 6

dUTP –> dUMP + PPi via dUTPase

dUMP –> TMP via Thymidylate synthase

TMP –> TTP via Thymidine Kinase 2ATP

Question 7

Why is dUTPase esstential for viral replication

Answer 7

dUTPase converts dUTP –> dUMP + PPi. This is important to keeping a low cellular dUTP over dTTP ratio as dUTP cause DNA polymerase to incorporate uracil into DNA which is mutagenic

By make more dUMP which is a substitute for Thymidylate synthase, more TTP is made and is a safer material for DNA synthesis than UTP

Question 8

Role of viral uracil-DNA glycosylase (UNG) and how does it work?

Answer 8

• Herpesviruses and poxviruses encode the DNA repair enzyme uracil-DNA glycosylase (UNG) –> performs base excision repair pathway that removes uracil from DNA if it is incorporated via dUTP
• HIV-1 is known to package cellular UNG into virus particles
• Leaves abasic site that is repaired by cellular enzymes

Important for viral replication

Question 9

Role of viral ribonucleotide reductase and how does it work?

Answer 9

Herpesviruses and poxviruses also encode ribonucleotide reductase:

Two subunit enzyme – converts ribonucleotides (NTP) –> deoxyribonucleotides (dNTP)

Increases pool of dNTPs for DNA synthesis
Not required in cultured cells but required in vivo especially in non-dividing cells (neurons etc)

Question 10

How does APOBEC3G create an anti-retroviral response?

Answer 10

Example of a cytosine deamination antiviral response.

Can terminate HIV by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription

APOBEC3G changes bases CAA –> UAA = generates a premature termination codon

If UNG works alongside –> degradation
By itself can cause hypermutation cause of uracil –> misreplication

Question 11

How does HIV Vif blocks the action of APOBEC3G?

Answer 11

• HIV-1 encodes the Vif to induce proteasomal
degradation of APOBEC3G

• A functional Cul5-Vif-APOBEC3 ubiquitin ligase complex is required for Vif to induce APOBEC3 proteasomal degradation
• HIV-1 Vif is also ubiquitinated and degraded by the proteasomal pathway

Question 12

What is ZAP + how does it protect cells?

Answer 12

Zinc finger antiviral protein (ZAP) protects cells from infection by diverse RNA viruses through its ability to specifically detect and deplete viral RNAs that have a greater frequency of CG dinucleotides than host messenger RNAs

Because CpG is under-represented in mammalian genomes –> usually in viruses

Question 13

Structure of ZAP?

Answer 13

2 Isomers
ZAP-L: has a poly-ADP-ribose polymerase (PARP) like domain
ZAP-S : does not

Both have CCCH fingers

Important co-factors for ZAP activity:
• TRIM25 = role unknown
• KHNYN = endonuclease that is important for ZAP antiviral activity against retroviruses but not against Sindbis virus + RNA exosome

Question 14

What is Viperin + how does it protect cells?

Answer 14

Produces novel antiviral ribonucleotide.

Viperin catalyses the conversion of cytidine triphosphate (CTP) to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP) by via a SAM-dependent radical mechanism. This acts as chain-terminator for viral RNA-dependent RNA polymerases

Question 15

What is cGAS + how does it protect cells?

Answer 15

Detection of cytosolic dsDNA - essentially Cyclic GMP-AMP synthase

• Normally DNA is restricted to the nucleus (and mitochondria)
• DNA virus infection will generate cytosolic DNA

STEPS:

1. cGAS binds dsDNA
2. Activated to produce cGAMP
3. cGAMP binds to and activates STING
4. Activates transcription factors IRF3 and NF-kB
5. Shuttle to nucleus and activate transcription of IFN-b