What is a virus?
A non-cellular micro-organism that can only replicate within cells
Genome can be RNA or DNA, single stranded or duplex, circular or linear
Simplified virus life cycle?
- Cell entry
- RNA/protein synthesis
- Genome replication
- Protein synthesis
- Assembly of infectious virus particles
- Release from the cell
What does a virus need from the host cell?
Raw materials for biomolecular synthesis
- Nucleotides (dNTP/NTP)
- Amino acids
Machinery for biomolecular synthesis
-Protein synthesis
Enveloped viruses need membranes
Intracellular transport
The cell cycle of non-diving cells?
G0 = Inactivity G1 = RNA + protein synthesis S = DNA synthesis G2 M = Mitosis
What is the dUTP problem?
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
How does dUTP –> TTP?
dUTP –> dUMP + PPi via dUTPase
dUMP –> TMP via Thymidylate synthase
TMP –> TTP via Thymidine Kinase 2ATP
Why is dUTPase esstential for viral replication
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
Role of viral uracil-DNA glycosylase (UNG) and how does it work?
- 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
Role of viral ribonucleotide reductase and how does it work?
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)
How does APOBEC3G create an anti-retroviral response?
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
How does HIV Vif blocks the action of APOBEC3G?
• 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
What is ZAP + how does it protect cells?
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
Structure of ZAP?
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
What is Viperin + how does it protect cells?
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
What is cGAS + how does it protect cells?
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:
- cGAS binds dsDNA
- Activated to produce cGAMP
- cGAMP binds to and activates STING
- Activates transcription factors IRF3 and NF-kB
- Shuttle to nucleus and activate transcription of IFN-b
