HIV- the innate immune response

Question 1

What are retroviral restriction factors?

Answer 1

Specific proteins produced by the cell to combat retrovirus infection (they are natural antivirals- could help in treatment)

Question 2

Retroviral restriction factors have been studied in mice for the Murine Leukaemia Virus. There are two strains/alleles in this virus. What are they called?

Answer 2

N-tropic- This is only infectious for NIH-Swiss mice

B-tropic- this is only infectious in Balb/ c mice

Question 3

Charlotte Friend researched Murine Leukaemia Virus in mice and concluded that there was a ‘Friend virus susceptibility factor-1’ aka ‘Fv1’. There were two alleles to this too, Fv1-N and Fv1-B. What did these alleles do/prevent? When abouts in the life cycle does this act?

Answer 3

Mediated post-entry block to viral replication

When there is an Fv1-B susceptibility factor, B-MLV can replicate but it seems to block N-MLV (this would also happen the other way round)

Acts after reverse transcription and before entrance to the nucleus

Question 4

What was found about the genome of the Fv1 factors and how it was homologous to another protein?

What was required to change in order for the tropism of the virus to?

Answer 4

Protein has 60% homology with MLV capsid protein (so probably derived from a retrovirus)

Only one base change is needed to change in order for the virus to be able to infect other strains of mice
e.g arginine (N) to glutamate (B)

Question 5

HIV-1 was found not to grow well in non-human cells (apart from chimpanzees and gorillas). This was also due to restriction factors, but what differed from this blocking in non-human cells or positive factor in human cells to the Fv1 block?

Answer 5

It acted before reverse transcription, not after as with Fv1

It was not known whether this was due to the fact that non-human cells lacked a positive factor or whether they had a negative factor that blocked it at this point

Question 6

How was it discovered that there must be a negative factor in the non-human cells that prevents the reverse transcription of HIV-1?

Answer 6

Cell fusion experiments where a rhesus monkey cell and human cell were fused using polyethylene glycol.

The virus didn’t replicate implying there was a negative factor present in non-human cells

Question 7

Describe the method that was used for identifying HIV-1 restriction factors.

Answer 7

A cDNA library was formed from a rhesus monkey cell. A human cell was the transfected with this. Infect with HIV-GFP (flourescent protein to mark) so all infected cells glowed green.

Most of the replicated cells expressed GFP so they had the virus but some did not, suggesting replication was blocked in these cells. These were selected and amplified several times and then carried out PCR, cloned and then sequenced the plasmid

Question 8

A restriction factor that was identified was TRIM5α which is a family of proteins. What is the structure?

Answer 8

RING domain, B-box, coiled coils and a SPRY (B30.2) domain

Question 9

What is the function of TRIM5α? How does that relate to their structure?

Answer 9

SPRY domain interacts with the capsid antigen
RING and B-box targets the virus to proteosomal degradation (ubiquitination)
Coiled coil induces trimerisation

So the mechanism is as follows: the trimer of the TRIM5α protein is interacting with the three-fold symmetry of the capsid antigen (could be that each of the three SPRY domains are interacting with two of the six capsids which form the structure) then targetting to proteosomal degradation

Question 10

The capsid protein (p24) of the HIV is made up of hexamers and pentamers. What needs to be mutated to prevent the TRIM5α block?

Answer 10

Arg110 of capsid to Glu (like the MLV capsid!)

Question 11

What are the two different mechanisms that TRIM5α is thought to carry out in order to block the virus?

Answer 11

Virus enters cytoplasm and capsid is released. Capsid is recognised. TRIM5α then may:
Break open the capsid to release the content, but the capsid needs to be intact in order for transcription to occur
Could get proteosomal degradation due to auto-ubiquitation

Question 12

TRIM5α is actually also present in human cells but does not block HIV-1 virus. Why is this the case? What other virus is not blocked by human TRIM5α?

Answer 12

There is a mutation in the SPRY domain of human TRIM5α preventing interaction with HIV-1 capside antigen hence why it can replicate in human cells much more efficiently than in non-human cells

HIVcpz

Question 13

Is TRIM5α recognising the individual capsid antigen or the whole structure of the capsid?

Answer 13

The whole structure- when the capsid antigen monomer is present only, it is not recognised

Question 14

What does the Vif (virion activity infectivity factor) do? How was this discovered?

Answer 14

A protein involved in making the virus infectious by affecting the virus particle maturation and binds to virion RNA in cytoplasm, incorporated into virions

Reverse genetic analysis

Question 15

What was found about the phenotype of Vif mutant/ p HIV delta vif (basically those which had been mutated so that vif doesn’t work/is not expressed anymore) viruses?

What did this prove/show/what was concluded?

Answer 15

That the phenotype of an HIV-1 Vif mutant was cell dependent- in permissive cells, nothing happened but in non-permissive cells, a non-infectious virus was found

Vif counteracts a cellular antiviral factor which is only expresesed in non-permissive cells (primary human T-cells and some T-cell lines like CEM)- APOBEC3G

Question 16

What is APOBEC3G and what is it related to?

Answer 16

An enzyme called cytosine deaminase related to APOBEC1.

Question 17

How does APOBEC3G work?

What two outcomes are there of its mechanisms for the virus?

Answer 17

It deaminases a cytosine to uracil before reverse transcription (around 10% of Cs). This mean during reverse transcription, many As are matched in the complementary strand. This hypermutation can lead to either misreplication so the viral proteins aren’t formed but also, a ‘U’ does not belong in the genome, only in the mRNA so it is degraded

Question 18

How does Vif counteract the actions of APOBEC3G?

Answer 18

Targets it for proteosomal degradation by tagging with ubiquitin

Question 19

Vpu is another viral protein produced in the multiply spliced RNA (like Vif). What are its functions?

Answer 19

Helps with virus release
Degrades CD4 by ubiquitin-proteasome pathway
Associated with the membrane (has membrane spanning domain)

Question 20

How did Vpu also show cell-type dependency?

Answer 20

In permissive cells, no abnormal phenotype in comparison to wildtype (virus replicated as normal)

In non-permissive cells, the virus was retained on the cell surface and endocytosed

Question 21

Why was the virus retained and endocytosed in the non- permissive cells in the absence of Vpu?

Answer 21

Tetherin molecules of the cell surface interacted with the one present on the virus and pulled it back in. Tetherin blocks other viruses such as MLV and Ebola (so Tetherin= antiviral factor)

Question 22

Apart from virus retaining and degrading, what other function does Tetherin play a role in?

Which viral protein interacts with Tetherin in these situations? Why is this different to humans?

Answer 22

Cross-species transmission

In SIVcpz and SIVgor, Nef interacts with tetherin, not Vpu
This is due to a mutation in the tetherin molecule so this is why Vpu has evolved to interact with it in HIV-1 Group M- hence why it’s so widespread
O, P and N have not been able to do this hence their limited distribution

Question 23

Another restriction factor is SAMHD1. Where is this expressed (in which cells)? What targets it in the HIV-2 (note the 2!) genome?

Answer 23

It is found in non-cycling (quiescent) myeloid cells.

Target for HIV-2 Vpx protein which binds to it and induces proteosomal degradation

Question 24

What is SAMHD1’s mode of action?

Answer 24

It is a dimer that binds to dGTP to become active, then it it takes dNTPs (building blocks of DNA) and it cleaves the phosphates from it to reduce the dNTP concentration in the cytoplasm so reverse transcription can’t occur

Question 25

MxB is another antiviral restriction factor. There are two, MxA and MxB. Not much is known about how it blocks the virus but what are two hypotheses?

Answer 25

Either blocks nuclear uptake

Blocks chromosomal integration