1 UNIVERSITY OF ABERDEEN SCHOOL OF MEDICAL SCIENCES THE BIOCHEMISTRY OF AIDS

2 II VIRUS BINDING AND ENTRY Initial interaction is between 2 glycoproteins: virion surface gp120 and cell surface CD4 Evidence: HIV replication is blocked by exogenous CD4 HIV replication is blocked by exogenous CD4 anti-CD4 antibodies; CD4-gp120 complexes are detected in infected cell lysates; CD4-gp120 complexes are detected in infected cell lysates; HIV only replicates in CD4 + cells, but most human cells HIV only replicates in CD4 + cells, but most human cells support replication if transfected with provirus; CD4 transfer to CD4 - cells makes them HIV susceptible. CD4 transfer to CD4 - cells makes them HIV susceptible. (This does not work for CD4 - mouse cells.)

3 How CXCR4 and CCR5 were discovered 2 sets of mouse cells were set up, to act as: mock human CD4 + cells mock HIV virions cells contain vector cells contain vector with CD4 gene with gp120/gp41 genes cells contain vector cells contain vector with CD4 gene with gp120/gp41 genes They do not fuse. Selections from human cDNA library introduced into cells on the left. Some now fuse with cells on the right. The cDNA in these cases contains gene for CXCR4 or CCR5. How was the fusion detected? Cells on left contain T7 RNA polymerase gene. Cells on right contain galactosidase gene behind T7 promoter. Only when cells fuse is galactosidase detected.

4 gp41 at the cell surface (just one gp41 molecule shown) cell membrane N virion envelope C pre-hairpin intermediate triple hairpin pre-hairpin intermediate triple hairpin

5 HIV entry into the body: dendritic cells as Trojan horses Dendritic cells occur at vulnerable mucosal surfaces. They detect pathogens, internalise and degrade them, and display fragments on their surface. They migrate to lymph nodes and present the fragments to CD4 + T cells, which initiate an immune response to the pathogen. gp120 non-specifically binds DC-SIGN (dendritic cell-specific ICAM grabbing non-integrin). HIV is internalised, carried to lymph nodes and, displayed intact on the dendritic cell surface, presented to CD4 + T cells.

6 Virion +RNA 5 3 cap ______________________________________________ poly A tail R U5 PBS U3 R

7 The product of reverse transcription The product of reverse transcription Virion +RNA 5 3 cap______________________________________________poly A tail R U5 PBS U3 R R U5 PBS U3 R Double-stranded DNA - _________________________________________________________ - _________________________________________________________ +_________________________________________________________ +_________________________________________________________ U3 R U5 PBS U3 R U5 U3 R U5 PBS U3 R U5 5-LTR 3-LTR 5-LTR 3-LTR

8 The product of integration The product of integration The product of reverse transcription - _________________________________________________________ - _________________________________________________________ +_________________________________________________________ +_________________________________________________________ U3 R U5 PBS U3 R U5 U3 R U5 PBS U3 R U LTR 3-LTR 9298 The product of integration _________________________________________________________ _________________________________________________________ cell DNA cell DNA U3 R U5 PBS U3 R U5 U3 R U5 PBS U3 R U

9 Cell activation For T cells, this involves presentation of foreign antigens, interaction with cytokines, infection with particular viruses, oxidant stress or DNA damage. stimulates cell protein kinase C, which catalyses phosphorylation and inactivation of I B. I B is normally complexed to NF B, a constitutive cytoplasmic protein. When I B is inactivated, NF B is able to move to nucleus (it has a nuclear signal sequence, like those in p17, Vpr, Tat and Rev Sections III, VII). In the nucleus, NF B binds to enhancers at the start of different genes in different tissues. For T cells, these include genes for cytokines and cell-surface adhesion molecules. Their expression allows T cells to influence other cells of the immune system.

10 Positions of TATA box, enhancer and NRE at the 5LTR Only +DNA shown cell DNA U3 R U NRE enhancer TATA NRE enhancer TATA transcription of -DNA

11 HIV has additional genes, shown below, in 3 frames, on the +DNA vif rev2 tat gag tat1 vpu gag tat1 vpu pol env nef pol env nef U3 R U5 U3 R U5 U3 R U5 U3 R U5 vpr rev1 vpr rev1

12 Current model of Tat activity Current model of Tat activity Tat recruits histone acetyl transferases that remodel chromatin around the provirus and initiate transcription; this leads to TAR formation. Tat-TAR interaction recruits transcription factors that covalently modify RNA pol II, making it more processive. Independently of TAR, cytoplasmic Tat aids NF B migration to the nucleus. These 3 activities account for Tat transcriptional activation. Further, secreted Tat remains attached to the cell surface, and, by interacting with receptors on the same or other cells, affects signalling pathways.

13 The function of Nef 4 activities have been defined (in cultured cells). Nef: decreases cell surface [CD4], thus stopping superinfection of cells; decreases cell surface [CD4], thus stopping superinfection of cells; inhibits HLA I expression, making the cell resistant to cytotoxic T cell attack; inhibits HLA I expression, making the cell resistant to cytotoxic T cell attack; assembles a cell-surface signalling complex that primes the cell for fast HIV replication when the cell is activated; assembles a cell-surface signalling complex that primes the cell for fast HIV replication when the cell is activated; activates expression of cytokines that cause apoptosis in nearby cytotoxic T cells. activates expression of cytokines that cause apoptosis in nearby cytotoxic T cells.

14 In the absence of Rev, transcripts are eventually spliced. This removes CRSs, and RNA exits the nucleus, becoming early viral mRNAs that encode Tat, Nef and Rev. In the presence of Rev, Rev-RRE interaction prevents activity of CRSs. Complexed with Rev, unspliced or singly spliced transcripts move to the cytoplasm, becoming late viral mRNAs (Section X). Rev, prompted by its nuclear signal sequence, moves back to repeat the process.

15 gag gag UUUUUUA UUUUUUA Gag polyprotein N aa Phe Leu aa C Occasionally the loaded leucyl tRNA slips back, from UUA to UUU: UUUUUUA Gag-Pol polyprotein N aa Phe Leu aa new C Leu aa new C

16 4 Inhibitors of gp41-mediated membrane fusion (Section II) N enfuvirtide cell membrane 36-mer peptide; licensed 2003; homologous to gp41 C-terminal region; competes for N terminal binding virion envelope virion envelope C 5-helix 5-helix is 5 of the 6 helices is 5 of the 6 helices of the triple hairpin. of the triple hairpin. Acquires 6 th helix from gp41 C region, Acquires 6 th helix from gp41 C region, thus preventing the C region from interacting with the N region. thus preventing the C region from interacting with the N region.

17 HOH 2 C thymine N= N= N N= N= N AZT is triphosphorylated by cell kinases and then inhibits reverse transcription by: competing with dXTPs for rtase; competing with dXTPs for rtase; chain terminating. chain terminating.

18 Inhibitors of the viral proteinase (p10; Section XI) Inhibitors of cell aspartyl proteinases, like renin, are well explored as drugs for lowering blood pressure. p10 cleaves bonds (e.g. X-Pro) resistant to cell proteinases. X-ray pictures of p10 have enabled transition state analogues to be developed. Saquinavir was licensed in There are now 10 such drugs in use, often in HAART combination with rtase inhibitors.

19 XX HOW DO SOME PEOPLE ESCAPE AIDS? Some people, repeatedly exposed to HIV, fail to progress to AIDS (long-term non-progressors) or even to register HIV + (elite controllers). It is not clear what allows such protection, but two probable factors have emerged. 1 Often, such individuals mount a very potent CD8 + cytotoxic T cell response to HIV. This has focussed attention on vaccines that promote this response (Section XVII). As regards the origin of the natural protection, mutations in HLA I of cells that present (HIV) antigens to CD8 + cells seems to be a factor. The mutations variously: lead to enhanced presentation; more complicatedly, discourage HIV escape, by gag mutation, from the CD8 + cell response, because, in such HLA I mutants, two successive gag mutations are needed for escape.

20 2 Some individuals are homozygous for a natural 32bp deletion in the CCR5 (Section II) gene. They are largely resistant to infection by CCR5-binding HIV strains (Section II), and have no detrimental phenotype. The condition is present in 1-2% Caucasians. Heterozygotes (~20% Caucasians) show some delayed progression to AIDS. Other mutations affecting expression of CCR5 and/or of its natural ligands are associated with accelerated or delayed progression.