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UNIVERSITY OF ABERDEENUNIVERSITY OF ABERDEENSCHOOL OF MEDICAL SCIENCESSCHOOL OF MEDICAL SCIENCES

THE BIOCHEMISTRY OF AIDSTHE BIOCHEMISTRY OF AIDS

http://www.abdn.ac.uk/~bch118/index.htmhttp://www.abdn.ac.uk/~bch118/index.htm

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II VIRUS BINDING AND ENTRYII VIRUS BINDING AND ENTRY

Initial interaction is between 2 glycoproteins:Initial interaction is between 2 glycoproteins:

virion surface virion surface gp120 andgp120 andcell surface cell surface CD4CD4

Evidence:Evidence:

• HIV replication is blocked by HIV replication is blocked by exogenous CD4exogenous CD4anti-CD4 antibodies;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 CD4HIV only replicates in CD4++ cells, but most human cells cells, but most human cells support replication if transfected with provirus;support replication if transfected with provirus;

• CD4 transfer to CD4CD4 transfer to CD4-- cells makes them HIV susceptible. cells makes them HIV susceptible.(This does not work for CD4(This does not work for CD4-- mouse cells.) mouse cells.)

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How CXCR4 and CCR5 were discoveredHow CXCR4 and CCR5 were discovered

2 sets of mouse cells were set up, to act as:2 sets of mouse cells were set up, to act as:

mock human CD4mock human CD4++ cells cells mock HIV virionsmock HIV virions

cells contain vector cells contain vector cells contain vector cells contain vector with CD4 gene with CD4 gene with gp120/gp41 genes with gp120/gp41 genes

They do not fuse.They do not fuse.

Selections from human cDNA library introduced into cells on the left.Selections from human cDNA library introduced into cells on the left.

Some now fuse with cells on the right.Some now fuse with cells on the right.

The cDNA in these cases contains gene for CXCR4 or CCR5.The cDNA in these cases contains gene for CXCR4 or CCR5.

How was the fusion detected?How was the fusion detected?

Cells on left Cells on left contain T7 RNA polymerase gene.contain T7 RNA polymerase gene.Cells on right Cells on right contain contain galactosidase gene behind T7 promoter. galactosidase gene behind T7 promoter.

Only when cells fuse is Only when cells fuse is galactosidase detected. galactosidase detected.

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gp41 at the cell surfacegp41 at the cell surface(just one gp41 molecule shown)(just one gp41 molecule shown)

cell membranecell membrane N N

virion envelopevirion envelope CC

‘‘pre-hairpin intermediate’pre-hairpin intermediate’ ‘triple hairpin’‘triple hairpin’

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HIV entry into the body: HIV entry into the body: dendritic cells as Trojan horsesdendritic cells as Trojan horses

Dendritic cells occur at vulnerable mucosal surfaces.Dendritic cells occur at vulnerable mucosal surfaces.

They detect pathogens, internalise and degrade them, and display They detect pathogens, internalise and degrade them, and display fragments on their surface.fragments on their surface.

They migrate to lymph nodes and present the fragments to CD4They migrate to lymph nodes and present the fragments to CD4++ T cells, which initiate an immune response to the pathogen.T cells, which initiate an immune response to the pathogen.

gp120 non-specifically binds DC-SIGN gp120 non-specifically binds DC-SIGN (‘dendritic cell-specific ICAM grabbing non-integrin’).(‘dendritic cell-specific ICAM grabbing non-integrin’).

HIV is internalised, carried to lymph nodes HIV is internalised, carried to lymph nodes and, displayed intact on the dendritic cell surface, presented to CD4and, displayed intact on the dendritic cell surface, presented to CD4++ T cells. T cells.

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Virion +RNAVirion +RNA

5’ 5’ 3’3’

capcap____________________________________________________________________________________________poly A poly A tailtail

1 92131 9213

R U5 PBS U3 RR U5 PBS U3 R

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The product of reverse transcriptionThe product of reverse transcription

Virion +RNAVirion +RNA

5’ 3’ 5’ 3’ cap______________________________________________poly A tailcap______________________________________________poly A tail

1 92131 9213

R U5 PBS U3 RR U5 PBS U3 R

Double-stranded DNADouble-stranded DNA

--__________________________________________________________________________________________________________________

+_________________________________________________________+_________________________________________________________

11 9213 9213 U3U3 R U5 PBS U3 R R U5 PBS U3 R U5U5

5’-LTR 3’-LTR5’-LTR 3’-LTR

-455 -455 92989298

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The product of integrationThe product of integration

The product of reverse transcriptionThe product of reverse transcription

- _________________________________________________________- _________________________________________________________

+_________________________________________________________+_________________________________________________________ 11 9213 9213

U3 R U5 PBS U3 R U5U3 R U5 PBS U3 R U5

-455 -455 5’-LTR 3’-LTR 5’-LTR 3’-LTR 92989298

The product of integrationThe product of integration

--------------------------__________________________________________________________________________________________________________________ ----------------------------

--------------------------__________________________________________________________________________________________________________________ ----------------------------

cell DNA cell DNA cell DNA cell DNA

U3 R U5 PBS U3 R U5U3 R U5 PBS U3 R U5

-453 9296-453 9296

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Cell activationCell activation

For T cells, this involves presentation of foreign antigens, interaction with For T cells, this involves presentation of foreign antigens, interaction with cytokines, infection with particular viruses, oxidant stress or DNA damage.cytokines, infection with particular viruses, oxidant stress or DNA damage.

stimulates cell protein kinase C,stimulates cell protein kinase C,which catalyses phosphorylation which catalyses phosphorylation

and inactivation of Iand inactivation of IB.B.

IIB is normally complexed to NFB is normally complexed to NFB, B, a constitutive cytoplasmic protein.a constitutive cytoplasmic protein.

When IWhen IB is inactivated, NFB is inactivated, NFB is able to move to nucleus B is able to move to nucleus (it has a nuclear signal sequence, like those in p17, Vpr, Tat and Rev (it has a nuclear signal sequence, like those in p17, Vpr, Tat and Rev

Sections III, VII).Sections III, VII).

In the nucleus, NFIn the nucleus, NFB binds to enhancers B binds to enhancers at the start of different genes in different tissues.at the start of different genes in different tissues.

For T cells, these include genes for cytokines and cell-surface adhesion molecules.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.Their expression allows T cells to influence other cells of the immune system.

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Positions of TATA box, enhancer Positions of TATA box, enhancer and NRE at the 5’LTRand NRE at the 5’LTR

Only +DNA shownOnly +DNA shown

cell DNA U3 R U5cell DNA U3 R U5

-453 1 98-453 1 98

NRE enhancer TATANRE enhancer TATA

transcription of -DNAtranscription of -DNA

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HIV has additional genes, shown below, in 3 frames, HIV has additional genes, shown below, in 3 frames, on the +DNAon the +DNA

-453 1 -453 1 vifvif rev2 tat2rev2 tat2 9213 9296 9213 9296

gag tat1 vpugag tat1 vpu

pol env nefpol env nef

U3 R U5 U3 R U5U3 R U5 U3 R U5

vpr rev1vpr rev1

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Current model of Tat activityCurrent model of Tat activityTat Tat recruits histone acetyl transferases recruits histone acetyl transferases that remodel chromatin around the provirus that remodel chromatin around the provirus and initiate transcription; and initiate transcription; this leads to TAR formation.this leads to TAR formation.

TatTat-TAR interaction recruits transcription factors -TAR interaction recruits transcription factors that covalently modify RNA pol II, making it more processive.that covalently modify RNA pol II, making it more processive.

Independently of TAR, cytoplasmic Independently of TAR, cytoplasmic TatTat aids aids NFNFB migration to the nucleus.B migration to the nucleus.

These 3 activities account for These 3 activities account for TatTat transcriptional activation. transcriptional activation.

Further, secreted Further, secreted TatTat remains attached to the cell surface, remains attached to the cell surface, and, by interacting with receptors on the same or other cells, and, by interacting with receptors on the same or other cells, affects signalling pathways.affects signalling pathways.

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The function of NefThe function of Nef

4 activities have been defined (in cultured cells). 4 activities have been defined (in cultured cells).

Nef:Nef:

• decreases cell surface [CD4], thus stopping superinfection of decreases cell surface [CD4], thus stopping superinfection of cells;cells;

• inhibits HLA I expression, making the cell resistant to inhibits HLA I expression, making the cell resistant to cytotoxic T cell attack;cytotoxic T cell attack;

• assembles a cell-surface signalling complex that primes the assembles a cell-surface signalling complex that primes the cell for fast HIV replication when the cell is activated;cell for fast HIV replication when the cell is activated;

• activates expression of cytokines that cause apoptosis in activates expression of cytokines that cause apoptosis in nearby cytotoxic T cells.nearby cytotoxic T cells.

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In the In the absenceabsence of of RevRev, ,

transcripts are eventually spliced. transcripts are eventually spliced. This removes CRS’s, and RNA exits the nucleus, This removes CRS’s, and RNA exits the nucleus, becoming early viral mRNAs that encode Tat, Nef and Rev.becoming early viral mRNAs that encode Tat, Nef and Rev.

In the In the presencepresence of of RevRev, ,

RevRev-RRE interaction prevents activity of CRS’s.-RRE interaction prevents activity of CRS’s.Complexed with Rev, unspliced or singly spliced transcripts Complexed with Rev, unspliced or singly spliced transcripts move to the cytoplasm, move to the cytoplasm, becoming late viral mRNAs (Section X).becoming late viral mRNAs (Section X).

RevRev, prompted by its nuclear signal sequence, , prompted by its nuclear signal sequence, moves back to repeat the process. moves back to repeat the process.

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gaggag

11 92139213

1629 16351629 1635

UUUUUUUUUUAUUA

Gag polyprotein N aa Gag polyprotein N aa PhePhe LeuLeu aa C aa C

Occasionally the loaded leucyl tRNA slips back, from UUA to UUU:Occasionally the loaded leucyl tRNA slips back, from UUA to UUU:

UUUUUUUUUUUUAA

Gag-Pol polyprotein N aa Gag-Pol polyprotein N aa PhePhe

LeuLeu aa new C aa new C

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4 Inhibitors of gp41-mediated membrane fusion (Section II)4 Inhibitors of gp41-mediated membrane fusion (Section II)

NN

enfuvirtideenfuvirtide cell membranecell membrane36-mer peptide;36-mer peptide;licensed 2003;licensed 2003;homologous to homologous to gp41 C-terminal region; gp41 C-terminal region; competes for N terminal bindingcompetes for N terminal binding

virion envelopevirion envelope CC

‘ ‘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 6Acquires 6thth helix from gp41 C region, helix from gp41 C region,

thus preventing the C region from interacting with the N thus preventing the C region from interacting with the N region.region.

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HOHHOH22C thymineC thymine

N= N= NN= N= N

AZTAZT is triphosphorylated by cell is triphosphorylated by cell kinases kinases and then inhibits and then inhibits reverse transcription by:reverse transcription by:

• competing with dXTPs for competing with dXTPs for rtase;rtase;

• chain terminating.chain terminating.

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Inhibitors of the viral proteinase Inhibitors of the viral proteinase (p10; Section XI)(p10; Section XI)

Inhibitors of cell aspartyl proteinases, like renin, Inhibitors of cell aspartyl proteinases, like renin, are well explored as drugs for lowering blood pressure.are well explored as drugs for lowering blood pressure.

p10 cleaves bonds (p10 cleaves bonds (e.g.e.g. X-Pro) resistant to cell proteinases. X-Pro) resistant to cell proteinases.

X-ray pictures of p10 have enabled transition state X-ray pictures of p10 have enabled transition state analogues to be developed.analogues to be developed.

Saquinavir was licensed in 1995.Saquinavir was licensed in 1995.There are now 10 such drugs in use, often in There are now 10 such drugs in use, often in HAART combination with rtase inhibitors. HAART combination with rtase inhibitors.

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XX HOW DO SOME PEOPLE ‘ESCAPE’ XX HOW DO SOME PEOPLE ‘ESCAPE’ AIDS?AIDS?

Some people, repeatedly exposed to HIV, fail to progress to AIDS Some people, repeatedly exposed to HIV, fail to progress to AIDS (‘long-term non-progressors’) or even to register HIV(‘long-term non-progressors’) or even to register HIV++ (‘elite controllers’). (‘elite controllers’).

It is not clear what allows such ‘protection’, but two probable factors have emerged.It is not clear what allows such ‘protection’, but two probable factors have emerged.

1 Often, such individuals mount a very potent CD81 Often, such individuals mount a very potent CD8++ cytotoxic T cell response to HIV. cytotoxic T cell response to HIV.

This has focussed attention on vaccines that promote this response (Section XVII).This has focussed attention on vaccines that promote this response (Section XVII).

As regards the origin of the natural ‘protection’, As regards the origin of the natural ‘protection’, mutations in HLA I of cells that present (HIV) antigens to CD8mutations in HLA I of cells that present (HIV) antigens to CD8++ cells seems to be a cells seems to be a factor. factor.

The mutations variously:The mutations variously:lead to enhanced presentation;lead to enhanced presentation;more complicatedly, discourage HIV escape, more complicatedly, discourage HIV escape,

by by gaggag mutation, from the CD8 mutation, from the CD8++ cell response, cell response, because, in such HLA I mutants, because, in such HLA I mutants, two successivetwo successive gaggag mutations are needed for escape. mutations are needed for escape.

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2 Some individuals are homozygous for a natural 32bp 2 Some individuals are homozygous for a natural 32bp deletion in the CCR5 (Section II) gene.deletion in the CCR5 (Section II) gene.

They are largely resistant to infection They are largely resistant to infection by CCR5-binding HIV strains (Section II), by CCR5-binding HIV strains (Section II), and have no detrimental phenotype.and have no detrimental phenotype.

The condition is present in 1-2% Caucasians.The condition is present in 1-2% Caucasians.

Heterozygotes (~20% Caucasians) Heterozygotes (~20% Caucasians) show some delayed progression to AIDS.show some delayed progression to AIDS.

Other mutations affecting expression of CCR5 Other mutations affecting expression of CCR5 and/or of its natural ligands and/or of its natural ligands are associated with accelerated or delayed progression. are associated with accelerated or delayed progression.