Lecture 8. HIV Replication 2

Question 1

What is Tat?

Answer 1

Gene is essential for HIV infection
HIV LTR linked to a reporter gene showed up to 1000x increase in expression when Tat supplied

Question 2

What is Tar and where is it located?

Answer 2

Tat-response element, immediately downstream of txn start site

Question 3

What is TAR function dependent on?

Answer 3

Absolutely position and orientation dependent
Can’t be moved around - suggests TAR’s RNA sequence is what Tat binds to
TAR might function as RNA, not DNA

Question 4

What part of TAR RNA does tat bind to?

Answer 4

Tight stemloop bulge

Question 5

What other factor must also bind to TAR for transcription to be efficient (besides Tat)?

Answer 5

“Loop-specific factor” which is a cellular protein

Question 6

What happens in the absence of Tat?

Answer 6

The HIV LTR assembles a poorly processive RNA polymerase complex
Once clear of the promoter, RNA pol frequently drops off the template, producing a truncated RNA
Short RNA comprising TAR can often be detected

Question 7

What happens in the presence of Tat?

Answer 7

The RNA polymerase complex is converted to a fully processive mode
A high proportion of initiation events lead to full-length transcription

Question 8

What makes up the “loop factor”?

Answer 8

Two protiens
Cyclin T1 : cdk9 complex (a.k.a. Tak – Tat-associated kinase)

Question 9

How is the loop factor activated?

Answer 9

By association with Tat/TAR and phosphorylates C terminal of RNA polII (RNA pol2 not 3)

Question 10

Summary of the action of Tat

Answer 10

1. Intitiation, TFIIH phosphorylates C-terminal domain to initiate transcription
2. RNA Pol II starts to transcribe the RNA and clears the promoter region
3. Once the viral RNA/TAR sequence gets a far enough distance along, is able to fold into stem loop structure, binding to Cyclin T1, CDK-9 and Tat (if present)
4. The binding of Tat, CDK-9 and Cyclin T1 cause the hyperpolarisation of the C-terminal domain, making the enzyme efficient and causes efficient transcription for the rest of the mRNA

Question 11

What are the properties of the unspliced RNA specie in HIV and what does it encode?

Answer 11

~9 kb
Incorporated into new virus particles as a genomic RNA
Also acts as mRNA that encodes Gag and Pol

Question 12

What are the properties of the singly spliced RNA specie in HIV and what does it encode?

Answer 12

~4 kb
Spliced at splice donor site near the end of the U5 LTR
4kb RNAs encodes Vif, Vpr, Vpu and Env

Question 13

What are the properties of the mutliply spliced RNA specie in HIV and what does it encode?

Answer 13

~2 kb
Encode Tat, Rev and Nef

Question 14

What is Rev?

Answer 14

Regulator of virion protein expression

Question 15

What happens in the absence of Rev?

Answer 15

In the absence of Rev, cytoplasmic mRNA for Gag, Pol, Env are reduced – but mRNA for Tat, Nef are unaffected
Suggests Rev has a post-transcriptional effect (has an effect on the nuclear export of viral RNAs)

Question 16

Why can the 2 kb class of viral RNAs use the host cell nuclear export pathway?

Answer 16

Because they are fully spliced (9 kb and 4 kb get stuck)

Question 17

Where does Rev bind?

Answer 17

Binds to viral RNAs in the envelope coding region - Rev-responsive element (RRE)
If RRE is not present, then viral RNAs cannot get out into the cytoplasm to be translated

Question 18

What is the function of RRE?

Answer 18

RRE confers Rev-dependent export on mRNAs that contain it and that would otherwise be retained in the nucleus
RRE has a complex secondary structure
RRE binds Rev at a high-affinity site in RRE and Rev then multimerises on the RNA

Question 19

In vitro, what does Rev coat?

Answer 19

In vitro, Rev coats RNA to form long fibrils

Question 20

Does Rev work by occluding splice sites?

Answer 20

No, because of existence of transdominant negative mutants in the ‘Activation’ domain
Therefore, RNA binding and multimerisation is not enough

Question 21

What is the Rev ‘Activation’ domain?

Answer 21

A nuclear export signal that binds the exportin, Crm1, and exits dependent on RAN (allowing for export of non-fully spliced RNAs)
Pathway of ribosomal 5S RNA export

Question 22

What does 5S RNA export compete with?

Answer 22

Splicing, but splicing inhibition is not the mechanism of Rev action

Question 23

How can a viral RNA that contains the envelope coding region (not very spliced) cross into the cytoplasm?

Answer 23

Rev binds to viral RNA and recruits Crm-1 and RAN-GTP, allowing for nuclear export

Question 24

What is Rev essential for?

Answer 24

Nuclear export of un- or singly spliced viral transcripts

Question 25

How are Tat and Rev translated?

Answer 25

Early proteins include Tat (required for efficient transcription) and Rev (required for export of mRNAs for Gag, Pol, Env etc.)
Tat and Rev translated in the cytoplasm and then transported into the nucleus

Question 26

How are Gag and Pol translated?

Answer 26

Gag and Pol are translated from the full length HIV RNA
The two proteins are in different, overlapping reading frames

Question 27

Because of the overlapping reading frames between Gag and Pol, how is Pol produced?

Answer 27

9/10 ribosomes translate Gag and terminate at the Gag Stop codon.
1 in 10 ribosomes translate most of Gag and then undergo a -1 frameshift: the ribosome slips back by 1 nucleotide and continues translating in a different reading frame to produce a Gag-Pol fusion protein

Question 28

How many more Gag molecules are needed to make virus particles as Pol?

Answer 28

At least 10 times as many Gag molecules

Question 29

How many nucleotides overlap between Gag and Pol?

Answer 29

There are 200 nucleotides of overlap between Gag and Pol, coding for different proteins in the two reading frames

Question 30

How does frame-shifting occur between Gag and Pol?

Answer 30

A stem-loop structure forms in the HIV RNA which causes the ribosome to pause during translation
The ribosome pauses with its A and P sites containing a UUUUUUA “slippery sequence”
10% of the time, the ribosome slips back 1 nucleotide, and then continues to translate = Gag-Pol fusion protein

Question 31

How is the Env protein produced?

Answer 31

Co-translationally inserted into ER membrane
Gp41 region of Env traverses the membrane, gp120 region of Env in the lumen of the ER where it is heavily glycoslyated
Env follows the route of membrane and secretory proteins in vesicles via the Golgi to the cell membrane
Env clusters in regions of the membrane that are high in cholesterol, called lipid rafts
Lipid rafts are the regions where HIV particles start to be assembled

Question 32

What does HIV particle assembly require?

Answer 32

2 copies of full length HIV RNA
Approx. 2000 molecules of Gag protein
Lipid envelope containing Env glycoproteins

Question 33

How does HIV ensure only the full length RNAs are packed into new particles?

Answer 33

By having the packing signal (Ψ) which lies just downstream of the major splice donor site
Only full length viral RNAs have packaging signal
Gag protein binds to Ψ which encourages multimerisation along the RNA

Question 34

Whats is the role of the dimerisation signal?

Answer 34

Palindromic region that allows interaction between two viral RNAs
End up with a dimer of RNA molecules coated in Gag
Occurs in the cytoplasm

Question 35

How is the Gag-RNA complex transported from the cytoplasm into the region of the cell membrane containing Env proteins to form an immature virus particle?

Answer 35

Cellular proteins called ESCRT (endosomal sorting complex required for transport) complexes, which usually function in vesicle trafficking

Question 36

What happens when the viral protein covered in Gag ends up at the membrane?

Answer 36

The ESCRT complexes catalyse the budding of an immature particle at the cell membrane

Question 37

What is the maturation step for HIV?

Answer 37

Catalysed by the protease enzyme of HIV
During or after budding of the immature virus particle from the cell, viral Protease self-cleaves from Pol, and cleaves the Gag and Gag-Pol polyproteins
Cleaving of Gag and Gag-Pol releases virus particles which then rearrange within the virus particle, becoming infectious

Question 38

What is the role of Vif?

Answer 38

Inhibits host cell APOBEC3G, an antiviral enzyme that causes hypermutation of the HIV genome resulting in weakening defences

Question 39

What is the role of Vpu?

Answer 39

Inhibits host cell tetherin, a cell surface molecule preventing the release of viral particles resulting in weakening defences

Question 40

What is the role of Nef?

Answer 40

Causes the downregulation of MHC Class 1 and CD4 from the cell surface
Prevents expression of antigen on cell surfaces