Natural antivirals

Innate immune response

Innate immune response to HIV infection:
-Retroviral restriction factors 
- Intracellular inhibitors of HIV replication 
TRIM5 alpha, APOBEC3, SAMHD1, MxB, Tetherin, SERINC3/5

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Retroviral restriction in mice

Friend strain of Murine Leukaemia Virus
2 alleles: N tropic (infectious for NIH-Swiss mice) and B tropic (infectious for Balb / C mice)

Friend virus susceptibility factor-1: Fv1
2 alleles :Fv1-N and Fv1-B
Mediated post entry block to viral replication 

N-MLV can get into cell but cannot replicate

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Fv1 restriction

Protein that has 60% homology to MLV capsid protein
Present in limited amounts in murine cells 
- Can be overwhelmed by high input virus
Single AA change in capsid antigen can alter tropism 
- Residue 100: Arganine--> Glutamate
- Mechanism is unknown 

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Endogenous retroviruses

Present in all vertbrate species, it makes up 8% of the human genome 
Result from infection of the germ line cells by replication competent retroviruses
Time of insertion can be estimates, in humans 10-25 million years (most recent <200K years)
Generally defective- mutation and/or deletions- unable to produce infectious virus 
Important evolutionary, physiological and pathological implications

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Cellular mechanisms to block HIV replication

HIV-1 doesnt replicate efficiently in non human cells (except chimps and gibbon apes)
Virus enters but blocked before reverse transcription 
Absence of positive factor of presence of negative factor??

Cell fusion experiment
Fused human cells with Rhesus monkey cell. 
This cell fusion doesnt allow HIV-1 to replicate.

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A protein present in non human primate cells can restrict HIV-1 replication.

Method for identifying the HIV-1 restriction factor 
- From rhesus monkey cell, a cDNA library was made
- Used this to transfect human cells
- Infect with HIV-GFP, those that didnt glow were not infected
-Select GFP negative cells for FACS
-Repeated 7 times 
- Expand GFP negative population
- PCR with primer specific for cDNA vector plasmid
-Clone and sequence 

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HIV-1 restriction factor

Identified as TRIM5 alpha
TRI partite Motif family of proteins 
- RING domains, B-boxes and coiled coils
- Function is unknown
- Localise to cytoplasmic bodies
-RING domain- ubiquitin ligase
Large family with currently 68 known genes. 

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TRIM5 alpha

RING and B box: Auto ubiquitinylation 
--> Rapid proteosomal degradation
Coiled coil: Trimerisation
SPRY domain: Binding to capsid antigen 

Mutating Arg110 of capsid to Glu prevents TRIM5 alpha block.
TRIM5 alpha forms a net around the capsid and then targets it to the proteosome. 

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2 stage mechanism of TRIM5 restriction

TRIM5 alpha binds to capsid.
Then follows ubiquitination, blocking reverse transcription and causing capsid disruption 

Species specific retroviral restriction
HIV-1 is blocked by Rhesus and African green monkey 
HIV-1 is not blocked by human TRIM5
SIVcpz is not blocked by human TRIM5

Human TRIM5a has mutation in SPRY domain preventing the interactio with HIV-1 CA. 

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Evolutionary conserved barried to retrovirus infection
This limits the cross species transmission.
Recognise retroviral capsid structure. 
Considered a component of innate immune response, this is a 2 stage mechanism of action

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192 AA protein-cytoplasmic 
- Affects virus particle maturation- enhances infectivity 
-Binds to virion RNA in cytoplasm, incorporated in virions.
They performed reverse genetic analysis:
- Mutated to introduce stop codon: TAC (Tyr) --> TAA (stop)
-Resulted in block of Vif protein 

The phenotype of a HIV-1 vif mutant is cell type dependent.
- It infects permissive cells (no change), so they believed vif was not important in HIV replication
- In non permissive cells, it produces non infectious virus. This showed that vif required in these cells. 

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Vif counteracts a cellular antiviral factor

Non permissice phenotype is dominant 
Unique cellular gene- APOBEC3G
Only expressed in non permissive cells 
- Primary human T cells, some T cell lines (EM)
Expression of APOBEC3G in permssive cell lines converted them to non permissive 
In the absence of Vif, APOBEC3G incorporated into the virus particle 

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A new cellular antiviral factor 
Cytosine deanimase-related to APOBEC1
- Enzyme that deaminates a single cytosine in APOB mRNA
- Premature stop codon- truncated protein in gastrointestinal cells 

Cytosine--> Uracil 

Mode of action 
When virus replicates its genome ( reverse transcription) it changes all its C's to U's
This causes hypermutation 
- Hypermutation leads to misreplication (the virus does not work)
- Also as the virus does not like U as DNA base, this leads to the degradation of it. 

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How does the vif block the action of APOBEC3G?

Vif binds to APOBEC3G, causing polyubiquitination 
This polyubiquitin causes APOBEC3G to be degraded by the proteosome

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81 AA integral membrane protein 

Bind to and induce the degradation of CD4 by ubiquitin- proteasome which allows Env to escape trapping in ER
Enhanced virion release 

The phenotype of a HIV-1 Vpu mutant in cell type dependent 
- If vpu removed, in non permissive cells, the virus is retained on the cell surface
- Its stuck on the surface so is re-endocytosed. 

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Cell surface expressed
Expression stimulated by inferon treatment 
Blocks envelope virus release
- HIV, MLV and Ebola 
Vpu interacts with tetherin 
- Direct ubiquitin-dependent degradation 
Artificial tetherin functional. 

HIV1-M Vpu down regulates tetherin

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Sterile alpha motif and HD domain 1

Highly expressed in myeloid cells- Macrophages and DC
Induced by IFN
Acts at an early stage to block HIV-1 replication in myeloid cells 
Only functions in non cycling ( quiescent cells) 
HIV 2 has a vpx protein instead of a vpr, which targets SAMHD1 to induce proteosomal degradation.
Mutated in Aixardi- Goutiere Syndrome- Autoimmune condition associated with dysregulation of IFN system 

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Mode of action of SAMHD1

dGTP activates SAMHD1
SAMHD1 then removes 3 phosphates from dNTP which reduces the levels of dNTP for reverse transcription. (It is dephosphatased)

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Myxovirus resistance proteins (MxA and MxB)
Identified nearly 25 years ago, MxA is a potent inhibitor of influenza
IFN-induced dynamin like large GTPases
- Could block by stopping nuclear uptake of dsDNA
- Could block chromosomal intergration of DNA 
- Could both be correctin different cell types 

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Serine incorporators
Transmembrane proteins that play a role in the incorporation of serine into lipids
Nef interacts with protein so that SERINC3 doesnt get incorporated into new proteins so they become infectious 
This enables efficient delivery of virus to target cells 
-SERINC stop virus attaching to new cells. 

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