What are the 9 key phases of the HIV life cycle? 01:23
1) Attachment
2) Fusion (envelope virus)
3) Uncoating (inside cell)
4) Reverse transcription (only retroviruses have reverse transcriptase)
5) Integration (ligation of viral dsDNA into human chromosome)
6) Replication
7) Assembly
8) Budding
9) Maturation
What cells do HIV preferentially infect? What happens to the cell w/successful infection?
- CD4+ T cells (produce important cytokines that coordinate immune response)
- Lysed if a productive infection; leads to progressive reduction of CD4+ T cells during infection
- Symptoms appear when CD4+ T cell count drops below a threshold; prone to secondary infection if immunocomprimised
What other cells do HIV infect? What is the difference with infecting CD4+ T cells?
- NK cells (natural killer)
- CD8+ killer T cells
- Macrophages
- CNS cells; astrocytes, neurons, glial cells, brain macrophages
- Dendritic cells
> > > These cells are not lysed (unlike CD4+ T cells; though budding still occurs)
Describe the main structural features of HIV.
Enveloped virus:
- SU (surface, gp120; glycoprotein 120kD)
- TM (transmembrane, gp41; under the SU, important for fusion/invasion)
- MA (matrix, p17)
Capsid (p24, inside):
- 2 x ssRNA (HIV genome)
- PR (protease, p10)
- INT (integrase, p32)
- RT (reverse transcriptase, p66)
- Vpu, Vif, Vpr, Nef
What shape is the HIV capsid?
- Internal capsid is butternut squash-shaped
|»_space;> NOT isohedral
How does the TM (gp41) interact with the HIV-1 matrix protein, p17?
- Cytoplasmic tail of gp41 transmembrane protein interacts through envelope w/HIV-1 matrix protein p17
- Nucleocapsid protein interacts w/RNA strands too
What problems does HIV face as a virus?
- Only space for limited number of nucleotides in the capsid; limited number of genes encoded (like other viruses)
- Genome is RNA; needs to be reverse transcribed to DNA by reverse transcriptase (RT); RT has low fidelity, where if RNA genome is too long, there is high likelihood for lethal mutations (for the virus)
»> RT is prone to mistakes; advantageous to gain mutations for survival
How does HIV overcome the issue of limited space for nucleotides to code the genome, in the capsid?
Polyprotein precursors:
- Polyprotein = large precursor protein from which smaller proteins are generated by proteolytic cleavage (snipped up into key smaller proteins)
- Allowing for a more compact genome by eliminating additional genetic features e.g. promoter/enhancer elements required to express each protein individually
How many genes does HIV have for polyproteins? What are they?
Three genes:
- Gag (group specific antigen); capsid and matrix
- Pol (polymerase); reverse transcriptase, integrase, protease (maturation)
- Env (envelope); gp120 and gp41; attachment and fusion
How can the unusual butternut squash-like shape of the HIV capsid be explained?
- One gene codes for the whole capsid; the use of a single protein
- Formed by 1000 repeats of the p24 capsid protein (CA)
- Organised in: hexamers, dimers, and some pentamers
How does HIV enter cells? (HIV Tropism)
- Via a cellular receptor (CD4 on T-lymphocytes and in lower density on macrophages)
- And a co-receptor; CXCR4 for T-lymphocytes, CCR5 for macrophages
> > > CCR5 = chemokine receptor 5
CXCR4 = chemokine receptor 4
How does what cells HIV preferentially infects change as the infection progresses? What is AIDs progression attributed to?
- Initially; macrophages (M-tropic or R5 subtypes)
- Later infection; preference switches to CD4+ T-cells (T cell tropic or X4 subtypes); with rapid progression to AIDs associated with this switch in co-receptor preference (lose CD4+ cells)
What’s the play-by-play of HIV attachment (1) w/CD4+ T cells?
- gp120 (SU, surface) binds to CD4 of host T cell
- Conformational change in gp120; allowing interaction with CXCR4 co-receptor w/gp120
What’s the play-by-play of HIV fusion (2) w/CD4+ T cells?
- Further conformational change
- Allows gp41 fusogenic tip insertion into cell membrane “fork”
- FUsion of membranes occur, with envelope proteins gp41 and 120 left at cell surface membrane, but entry of capsid
What is Enfuvirtide? Why is it not used much?
- Fusion inhibitor; linear, 36 AA peptide that blocks gp41 interaction
- Preventing virus from entering cell
- Expensive, inconvenient (SC BD; is a biomimetic peptide); reserved for multiple resistant HIV strains (salvage therapy)
Give examples of 3 different entry inhibitors and their respective mode of actions.
- Ibalizumab; blocks gp120-CD4 binding (mAb)
- Maraviroc; blocks gp120-coreceptor (e.g. CXCR4) binding (second receptor)
- Enfuvirtide; blocks gp41-mediated membrane fusion
How can natural resistance to HIV infection arise?
- Mutated co-receptors e.g. CCR5 of macrophages; HIV does not enter as a result = immunity
- Mutated co-receptor is ‘defective’ and not displayed
Why does the HIV capsid not uncoat (3) immediately after cytosol release? What steps occur instead?
- Capsid does not disintegrate immediately as if it does the infectious cycle could be aborted:
• Fusion
• Reverse transcriptase complex (RTC)
• Pre-integration complex (PIC)
What are the 3 rules for RT (4)?
1) RT can only start from ds-nucleic acid and add nucleotides in the 5’ to 3’ direction
2) RT has polymerase (adding nucleotides from DNA/RNA template) and RNA cell activity
3) RNA II will delete RNA in RNA/DNA duplex
What is RT an ideal therapeutic target? What categories of antiretrovirals target it?
- Absence of RT in host cell makes it an ideal target
- First antiretroviral drug (AZT) targeted RT
- Two of the four categories of antiretrovirals:
• Nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTI)
• Nonnucleoside analog reverse transcriptase inhibitors (NNRTI)
What is the mechanism of integration (5) of the newly formed viral dsDNA?
- Cut & paste into human chromosome
- Site of integration in human genome not random; preferential for regions that are actively transcribed, high G/C content, high gene density, other specific features
- Cytosolic host cell proteins + viral proteins involved; only one needed from the virus;
»> INTEGRASE (encoded in Pol gene)
Give an example of an integrase inhibitor (5). How does it work?
- Raltegravir
- PO as part of combination therapy
- Targets integrase viral DNA complex in the PIC (pre-integration complex)
- Blocks active site of integrase; inhibiting viral replication
- Chelates Mg2+ close to catalytic triad, inhibiting phosphodiester bond cleavage
What are steps 6-9 of the HIV infection cycle?
6) Replication (HIV has 3 reading frames)
7) Assembly
8) Budding (immature virus)
9) Maturation
Explain the role of Gag polyprotein in the HIV life cycle.
- Gag (Group-specific Antigen) gene encodes structural proteins of the capsid
- Expressed as single chain polyprotein w/5 cleavage sites for HIV protease
- Polyprotein inserted into cell membrane and cleaved in subsequent steps during maturation
- Cleavage by HIV protease results in:
• Release of matrix protein (MA)
• Capsid protein (CA)
• Protein NC (part of capsid)
• p6 (role in initiating budding)
