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Flashcards in HIV- molecular biology Deck (26):
1

Describe the structure of the HIV particle.

ssRNA in the middle with p7 (nucleocapsid) attached on
Nef and reverse transcriptase along with the ssRNA found in a capsid (p24 antigens)
p6 and viral protein R (Vpr) found outside
This is all encapsulated in Matrix antigens (p17)
A lipid bilayer surrounds this with gp41 (transmembrane glycoproteins) attached onto gp 120 (surface glycoproteins) on it

2

In the genome of HIV-1, There is a 'Gag' reading frame,
a 'Pol' one and an 'Env'. What do these encode?
Are 'vif', 'vpr', 'vpu' found in all viruses?

Gag- p17 matrix antigen, p24 capsid antigen, p6/7 nucleocapsid
Pol- Reverse transcriptase, protease and integrase enzymes
Env- gp120 surface gylcoprotein, gp41 transmembrane glycoprotein

vif, vpr and vpu are unique to the HIV virus

3

What difference is there between the HIV-1 genome and the HIV-2 one?

HIV-2 has an additional 'vpx' open reading frame instead of a vpu one

4

Why is the HIV genome so complicated?

It encodes a lot of proteins because it needs to combat a normal immune system and transmitted across species-
Enables replication and persistence in adult host (immunocompetent)
Enabled cross-species transmission

5

HIV life-cycle: How does the virus enter the cell? Which parts interact with which receptors on the cell?

gp120 (transmembrane domain) interacts with CD4 and second receptors (CCR5/CxCR4)
gp41 mediates fusion of cellular and viral membranes and entry of capsid into cytoplasm occurs

6

HIV life-cycle: Reverse transcription occurs in the plasmid that is in the cytoplasm. Which enzyme catalyses this process?

Reverse transcriptase catalyses this- viral DNA is reverse transcribed into proviral DNA to form a positive and negative strand

7

HIV life-cycle: After reverse transcription, the proviral DNA is then imported into the nucleus for integration into the cellular chromosomal DNA. What enzyme catalyses this?

Integrase catalyses this process by cutting and ligasing the proviral DNA into the cell's DNA. There is also involvement of the host cell factors

8

How much of our genome consists of retrovirus DNA?

8%

9

HIV life-cycle: The integrates provirus functions as a normal gene locus. What acts as an enhancer/promoter?

The 5' LTR

10

HIV life-cycle: Regulation of HIV-1 transcription depends on the host cell but also two proteins from the virus are essential. One is Tat. What is its function?

Is extracellular Tat efficiently taken up into cells?

It is an 86-101 amino acid nucleolar protein that acts as a transcriptional activator- RNA binding protein.
It has many other functions to such as activation of quiescent T-cells and induction of apoptosis

Extracellular Tat is efficiently taken up into cells

11

HIV life-cycle: How does Tat regulate the transcription of the HIV-1 genome? Start off with what would happen in the absence of Tat.

Naturally, TAR RNA loop occurs (around 50 nucleotides long) and so the RNA polymerase cannot transcribe the rest of the genome which is bad news for the virus. This means in the absence of Tat, RNA polymerase 2 only makes short transcripts

Tat binds to the loop, recruits cellular proteins cyclin T1 and cdk9 which is a a Tat-associated kinase, phosphorylating the C-terminal tail of RNA pol II. This allows productive elongation (generation of full-length transcripts) as it can carry on

12

HIV life-cycle: Retrovirus mRNA splicing is carried out by the host cell splicing machinery. What is the problem with the way the ribosomes translate the genome? (Clue:it is to do with the overlapping of ORF of the HIV virus genome).

Because they overlap, the ribosome will start translating GAG and then drop off because Pol does not follow straight after, it overlaps

13

HIV life-cycle: How does the whole virus genome therefore get translated in most viruses?

Splicing makes shorter RNA without most of the genome so that when the ribosome comes across the next ORF, it translates that.

For example, gag and pol are spliced out so that env can be translated

14

HIV life-cycle: Knowing that splicing patterns can help with making sure the entire genome is translated, how many splice variants are there in HIV-1?

50 splice variants-
Some singly spliced- e.g Vif, Vpr and Gag, Pol
and some multiply spliced- Tat, Rev and Nef

Not understood why it is spliced only once sometimes and multiple times at others

15

HIV life-cycle: Once the mRNA has been spliced, it needs to get out of the nucleus into the cytoplasm to get translated by the ribosomes. What protein regulates this?

Rev- controls the expression of these RNAs
The Rev response element (RRE) is an RNA sequence found in the envelope protein of the genome

16

HIV life-cycle: Is the RRE sequence found in every spliced variant?

No, it is found in the full one and the singly spliced but not multiply spliced RNA

17

HIV life-cycle: The RRE blocks export of the RNAs from the nucleus. So only the multiply spliced RNAs can get out. Which proteins can be translated from the multiply spliced RNAs?

Tat, Rev and Nef

18

HIV life-cycle: After the multiply spliced RNAs become translated, which protein goes back into the nucleus and why does it do this?

The Rev protein goes back into the nucleus and binds to the RRE found in the whole and singly spliced RNA. This allows these to be exported through the nuclear pore into the cytoplasm for translation. This means all the viral proteins can be translated- basically HIV regulates its expression in a temporal fashion

19

HIV life-cycle: Where does Rev bind to the RRE?

On the primary binding site and then you get multimerisation- lots of Revs cover the RRE (maybe how it helps the RNA get out the nucleus)

20

HIV life-cycle: How does the ribosome translate both Gag and pol if they are found on the same RNA but overlap?

There is a ribosomal frameshift- before reaching the end of gag, it moves a base and this enables it to translate a whole length, creating a fusion protein. This happens every 20 lengths

21

HIV life-cycle: Which proteins are considered accessory genes? (not essential for survival of the virus)

Vif and Vpu (and Vpx in HIV-2)- counteract innate anti-viral responses
Vpr- indices G2 cell cycle arrest
Nef- many functions

22

What is reverse genetics?

Which protein did they do this on?

A method to create mutations to study the functions of certain genes
Can use a plasmid to produce complete copy of a viral genome which can undergo replication if transfected into something
They did this with Nef by inducing a stop codon before it

23

What did they find was the function of the Nef protein in SIV in monkeys?

Reduction by 1000-fold reduction in viral load
low pathogenicity
revertion back to wild type- mutated back to have a selective advantage

So basically Nef is involved with replication and pathogenicity

24

Were the results in human studies the same as the SIV monkey ones?

Yes, had the virus but at very low levels for 29 years in 3 recipients

Also used transgenic mice to support this

25

How does Nef actually achieve its functions?

Down-modulation of cell surface glycoproteins such as CD4, MHC class I. This is done by pulling them into clatherin-coated pits and induces endocytosis
Enhancement of virus infectivity
Perturbation of cell signalling

26

Why would Nef want to destroy CD4?

To help its spread- doesn't hold the virus to the cell
Prevent CD4 incorporation into its own envelope
Blocks CD4 signalling- this then doesn't signal to IL16 which seems to block LTR activity (virus transcription)
Block to lethal superinfection? (two interfere with eachother's replication so not good for virus)