Lecture 8. HIV Replication 2 Flashcards
What is Tat?
Gene is essential for HIV infection
HIV LTR linked to a reporter gene showed up to 1000x increase in expression when Tat supplied
What is Tar and where is it located?
Tat-response element, immediately downstream of txn start site
What is TAR function dependent on?
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
What part of TAR RNA does tat bind to?
Tight stemloop bulge
What other factor must also bind to TAR for transcription to be efficient (besides Tat)?
“Loop-specific factor” which is a cellular protein
What happens in the absence of Tat?
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
What happens in the presence of Tat?
The RNA polymerase complex is converted to a fully processive mode
A high proportion of initiation events lead to full-length transcription
What makes up the “loop factor”?
Two protiens
Cyclin T1 : cdk9 complex (a.k.a. Tak – Tat-associated kinase)
How is the loop factor activated?
By association with Tat/TAR and phosphorylates C terminal of RNA polII (RNA pol2 not 3)
Summary of the action of Tat
- Intitiation, TFIIH phosphorylates C-terminal domain to initiate transcription
- RNA Pol II starts to transcribe the RNA and clears the promoter region
- 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)
- 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
What are the properties of the unspliced RNA specie in HIV and what does it encode?
~9 kb
Incorporated into new virus particles as a genomic RNA
Also acts as mRNA that encodes Gag and Pol
What are the properties of the singly spliced RNA specie in HIV and what does it encode?
~4 kb
Spliced at splice donor site near the end of the U5 LTR
4kb RNAs encodes Vif, Vpr, Vpu and Env
What are the properties of the mutliply spliced RNA specie in HIV and what does it encode?
~2 kb
Encode Tat, Rev and Nef
What is Rev?
Regulator of virion protein expression
What happens in the absence of Rev?
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)
Why can the 2 kb class of viral RNAs use the host cell nuclear export pathway?
Because they are fully spliced (9 kb and 4 kb get stuck)
Where does Rev bind?
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
What is the function of RRE?
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
In vitro, what does Rev coat?
In vitro, Rev coats RNA to form long fibrils
Does Rev work by occluding splice sites?
No, because of existence of transdominant negative mutants in the ‘Activation’ domain
Therefore, RNA binding and multimerisation is not enough
What is the Rev ‘Activation’ domain?
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
What does 5S RNA export compete with?
Splicing, but splicing inhibition is not the mechanism of Rev action
How can a viral RNA that contains the envelope coding region (not very spliced) cross into the cytoplasm?
Rev binds to viral RNA and recruits Crm-1 and RAN-GTP, allowing for nuclear export
What is Rev essential for?
Nuclear export of un- or singly spliced viral transcripts
How are Tat and Rev translated?
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
How are Gag and Pol translated?
Gag and Pol are translated from the full length HIV RNA
The two proteins are in different, overlapping reading frames
Because of the overlapping reading frames between Gag and Pol, how is Pol produced?
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
How many more Gag molecules are needed to make virus particles as Pol?
At least 10 times as many Gag molecules
How many nucleotides overlap between Gag and Pol?
There are 200 nucleotides of overlap between Gag and Pol, coding for different proteins in the two reading frames
How does frame-shifting occur between Gag and Pol?
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
How is the Env protein produced?
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
What does HIV particle assembly require?
2 copies of full length HIV RNA
Approx. 2000 molecules of Gag protein
Lipid envelope containing Env glycoproteins
How does HIV ensure only the full length RNAs are packed into new particles?
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
Whats is the role of the dimerisation signal?
Palindromic region that allows interaction between two viral RNAs
End up with a dimer of RNA molecules coated in Gag
Occurs in the cytoplasm
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?
Cellular proteins called ESCRT (endosomal sorting complex required for transport) complexes, which usually function in vesicle trafficking
What happens when the viral protein covered in Gag ends up at the membrane?
The ESCRT complexes catalyse the budding of an immature particle at the cell membrane
What is the maturation step for HIV?
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
What is the role of Vif?
Inhibits host cell APOBEC3G, an antiviral enzyme that causes hypermutation of the HIV genome resulting in weakening defences
What is the role of Vpu?
Inhibits host cell tetherin, a cell surface molecule preventing the release of viral particles resulting in weakening defences
What is the role of Nef?
Causes the downregulation of MHC Class 1 and CD4 from the cell surface
Prevents expression of antigen on cell surfaces
