Foreword

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HIV has a very complex system of transcription regulation and mRNA splicing. This produces something that approximates "life-cycle" stages. Expression from the promoter always occurs but at first it is at a reduced level and at first the transcripts are very short. The shortness is because the pre-initiation complex that forms is of low quality. Polymerase begins transcription in a low processivity state. This early stage is critical for HIV. Later on the transcription complexes become better quality and polymerase can make full length transcripts.

We have noted before that many transcription factors have multiple functions - kind of like Swiss Army Knifes. HIV amplifies this to the point of absurdity.

5' LTR provides acts as a very weak promoter.3' LTR provides a polyadenylation signal.This is very clever considering that they are the same sequences!

PBS

PBSR U5U3

R U5U3 R U5U3

R U5U3

5' LTR : weak promoter [Inr, TATA-box, and three Sp1 sites] + enhancer elements.

One enhancer binds a host transcription factor called NF-kappa-B. This transcription factor is typically activated when cells of the immune system respond to an infection (cellular response). The immune response involves proliferation of immune cells and NF-kappa-B is turned on when these cells make the decision to divide.

The HIV promoter is also activated by NF-kappa-B. Therefore, when the immune system is stimulated the expression of HIV is also stimulated!! HIV has subverted a defensive host response system for its own use.

LTR

Taube  R, Fujinaga  K, Wimmer  J, Barboric  M, Peterlin  BM. 1999. Tat transactivation: a model for the regulation of eukaryotic transcriptional elongation. Virology. Nov;264(2):245-53

PBS

PBSR U5U3

R U5U3 R U5U3

R U5U3

One enhancer binds a host transcription factor called NF kappaB (nuclear factor [kappa]B) AND NFAT (nuclear factor of activated T cells) and Ets family members. These transcription factors are typically activated when cells of the immune system respond to an infection (cellular response). The immune response involves proliferation of immune cells and NF-kappa-B is turned on when these cells make the decision to divide.

The HIV promoter is also activated by NF-kappa-B. Therefore, when the immune system is stimulated the expression of HIV is also stimulated!! HIV has subverted a defensive host response system for its own use.

NF [kappa]B and NFAT relocalize to the nucleus after lymphocyte activation. In the cytoplasm NF-[kappa]B is associated with a cytoplasmic inhibitor, I[kappa]B. Lymphocyte activation causes the phosphorylation, ubiquitination, and proteosomal degradation of the I[kappa]B. NFAT is dephosphorylated by calcineurin (a reaction inhibited by cyclosporin A) and, after its nuclear import, assembles with AP1 to form the fully active transcriptional complex.

LTR -new

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Figure 1. StructureoftheHIVgenomeandtheviralpromoterelements.The5 0 LTRpromoterconsistsofthreestructuralsequenceelements,U3 (nt!453 to!1),R (nt"1to"98),andU5(nt"99 to"180)with"1asthestart siteoftranscriptionandfourmainfunctionaldomains: aregulatory region (nt!454 to!104), anenhancerelement (nt!105 to!79), acorepromoter (nt!78 to!1), andaTARelement (nt"1to"60).Within theTARelement two initiatorelements (InR) are located from!3 to"8 and"35 to"59, andan inducerof short transcripts (IST) spans the initiation regionfrompositions!5 through"26 and"40 through"59.Regardless the site of integration, the 5 0 LTRpromoterof HIV ispackagedby twonucleosomes, nuc-0, which is locatedbetweennucleo-tides!415 and!255, andnuc-1, which is locatedbetweennucleotides"10 and"155.The 5 0 LTRpromoter incorporates several cis-acting DNA sequences for binding cellular transcriptionfactors including,COUP, chickenovalbuminupstreampromoter transcriptionfactor; RAR, retinoicacidreceptor; Myb,Mybbindingprotein; NFAT, nuclear factorofactivatedTcells; USF, upstreamstimulating factor; Ets-1, Ets binding factor; LEF-1, lymphocyte enhancer factor; C/EBP,CCAAT/enhancer binding protein; NF-kB, nuclear factor kappa B; AP-2, activator protein 2; SP-1, SP1bindingprotein;TFIID, transcriptionfactor IID; LSF, late SV40 transcriptionfactor;TDP-43,TARDNA-bindingprotein; FBI-1, factor thatbinds to IST; CTF/NF1,CAAT-box transcriptionfactor/nuclearfactor1; AP-1, activator protein1; IRF, interferon regulatory factors.

HIV-1

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SCRIPTIO

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TARGET

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ERAPY

*597

Miguel Stevens, Erik De Clercq, Jan Balzarini. 2006. Medicinal Research Reviews, 26:595-625

NFK-b

• In cytoplasm bound to I-kappa-B alpha• Phosphor of IkBa causes its destruction• NFK-b goes into nucleus• NFK-b gets phosphorylated• It can recruit CBP or HDAC which means

that it can stimulate or repress.

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Figure 2.

HIV-1 TRANSCRIPTION AS TARGET FOR CHEMOTHERAPY * 601

How NF-kappa-b is activated.

p50/p65 dimers are NFkB

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7Figure 3.

HIV-1 TRANSCRIPTION AS TARGET FOR CHEMOTHERAPY * 603

p50 dimers are NFkB

p50/p65 dimers are NFkB

Nucleosome 0 and 1 are reproducibly positioned (remodeled).

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Figure 3.

HIV-1 TRANSCRIPTION AS TARGET FOR CHEMOTHERAPY * 603

p50 dimers are NFkB

p50/p65 dimers are NFkB

TFIIH - ATP-dep helicaseTFIIH - kinase

nuc-1 has to be moved (remodeled)

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Stages of HIV replication

Why are the transcripts stuck in the nucleus during the rev minus stage?

intron

RRE+ / -

RRE

exon 1 exon 2

splice factors

RRE

RRE

RRE

REV

+ / -

-

REV

REV

RRE

+REV

nuclear mem

brane

B-globin transcripts are very efficiently spliced.

Experiment which first shows a function of rev.

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Rev

• Rev/RRE binds host cell factor called CRM-1. This guy causes nuclear export of the Rev/RRE complex.

• CRM-1 exports it using the exportin 1 pathway.

• Rev then goes back into the nucleus.

Tat

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Tat does more than you think• 101 amino acids in clinical isolates• In addition to regulating transcription it can

– leave the cell and enter uninfected cells! It remains able to regulate transcription in these cells.

– Extracellular Tat can stimulate production of cytokines and expression of cytokine receptors, modulate survival, proliferation and migration of different cell types and has angiogenic effects.

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Miguel Stevens, Erik De Clercq, Jan Balzarini. 2006. Medicinal Research Reviews, 26:595-625

Kaposi’s sarcoma

• Human herpesvirus 8 HHV8• Found in all KS• Much more commonly found in males• Becoming less common• Also seen in those at risk for but without HIV• Old paper showed that Tat expression in mouse epithelia

would induce something that looked like Kaposi’s sarcoma. Cells that are in the tumor do not express Tat.

Hengge et al. 2002. The Lancet Infectious Diseases 2:344

Accessory Proteins

• So called because they are not required for replication in culture. BUT they are required for successful persistence in a real host.

• Nef• Vpu• Vif• Vpr

NefHelps keep the infected cell alive.

• blocks multiple apoptosis signals that would cause the infected cell to die.

• down regulates CD4 from cell surface, reduces chance of autofusion or syncytial cell fusion

• down regulates surface levels of MHCI proteins. This prevents cell from telling other immune cells that it is infected.

• activates the intercellular signaling system that causes bystander apoptosis.

• activates the infected T cell which helps get HIV genome expressed.

• expressed before integration.

Nef deletionA cohort of HIV positive men who were infected with a virus with a deletion in the Nef gene were identified in Australia. They have been mostly asymptomatic since 1985.

They seem to be able to live with the virus - very, slow progression of the disease.

Learmont JC, et al. 1999. N Engl J Med. Jun 3;340(22):1715-22. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort.

Birch MR, et al. 2001 J Clin Virol. Oct;22(3):263-70. An examination of signs of disease progression in survivors of the Sydney Blood Bank Cohort (SBBC).

Vif, Vpu and Vpr

• Vif– Cell puts CEM15, a cytidine deaminase (cell defense

system), into the maturing virus particle to tries to damage the viral genome. HIV puts Vif into the particle and these inhibits the activity of CEM15.

• Vpu– Stimulates degradation of CD4 within the endoplasmic

reticulum. This prevents the Env polyprotein from being bound up in the endoplasmic reticulum.

• Vpr– gives HIV the ability to enter nuclei of non-dividing

cells. Connects the pre-initiation complex to the importin 7 nuclear import pathway.

99 bp DNA flap common for LentivirusesImportant for entering non-dividing cells

RTC = reverse transcription complex. PIC = preintegration complex.

Zennou et al. 2000. HIV-1 Genome nuclear import is miediated by a central DNA flap Cell 101:173-185

• Vpr gives HIV the ability to enter nuclei of non-dividing cells. Connects the pre-initiation complex to the importin 7 nuclear import pathway.

What possible advantage to the virus is a 2 stage life cycle?

Rev- -----> Rev+

Provides time for Nef to down regulate CD4 and to adjust MHCI so that the cell does not appear to be infected.

Other reasons may exist that await discovery.

LTR polgag

gag polmRNA

HIV gag and pol regions shown.

The unspliced mRNA contains the coding regions for boththe gag and pol genes. However, they overlap one another andare in different reading frames.

Translation in the gag reading frame produces a 55 kDa protein.

Pr55

HIV protease cleaves Pr55 to produce:

p17p24

p7p6

a polyprotein

Polyproteins

gag polmRNA

The polycistronic mRNA can also be translated in another way.frame shift region

Translation begins in the gag reading frame but then a ribosome frame shift occurs!This now causes the pol gene to be read in the correct reading frame. Now a biggerprotein is made:

pr160

First part is in the gag reading frame. Then a translational frameshift occursand the second part is in the pol reading frame.

polyprotein

p11

p66/51p32

this is the protease itself

this is reverse transcriptase

this is the integrase

Proteolysis produces the followingfrom the polyprotein.

Quasi-species

• Reverse transcription error rate is 10-4 to 10-5.

• Daily in vivo virus production rate is > 1010 per day• Viral recombination

– Copy-choice recombination --> Strand switching– Sources of diversity when 2 variants are in same cell.

Inherent in mechanism of reverse transcription to allow this.

– Superinfection oddly enough appears common.

Malim & Emerman. 2001. Cell 104: 469–472

Ideas have been prev covered. Read for clarification if you wish.

Quasispecies

Quasispecies

Quasispecies

So what?

• The diversity generated in a single infected individual can be greater than the worldwide diversity of influenza A virus during an epidemic

• 36.1 million people are currently estimated to be infected.

Weiss RA 2001. NATURE 410:963-967

This has been discussed.

What is possible with such diversity?

• Many of the variants are produced invisible to some or ALL components of the immune system - true.

• Restriction - Genetic bottleneck associated with the mode of transmission. <-- Helps restrict things a bit.

• What if mode of transmission changed!!!!????

Weiss RA 2001. Nature 410:963-967Rambaut, A., Posada, D., Crandall, K. A. & Holmes, E. C. (2004) Nat Rev Genet 5, 52-61.

HIV2 differs from HIV 1.

• A large number of small changes• HIV 2 lacks vpu but has vpx (dupl of vpr).

Not presented in class. Read for clarification if you wish.

Origin• HIV 1 is related to SIVcpz

– simian immunodeficiency virus, cpz = chimpanzees• HIV 2 is related to SIVsmI

– smI = mangabeys• HIV1 and 2 entered humans at different times and probably

multiple time.• Humans hunt primates for food. It is believed that during the

hunt or during preparation of the meat that humans were contaminated by blood transfer.

• When? A number of early samples from infected humans have been found from the 1959-the mid 1960’s. These do not represent the original entry. Evolutionary studies suggest that the original tntry time was between 1915 to 1941.

Current therapies• Only 16 drugs in use.• Nucleoside analogs

(Nucleoside Reverse Transcriptase Inhibitor - NRTI)

• Non-nucleoside Reverse Transcriptase Inhibitors (NNRTI)

• HIV protease inhibitors• Fusion inhibitors such as Enfuvirtide these

inhibit gp41 <-- a viricide.• HAART - highly active antiviral therapy

(used to be called Combination Therapy).

Inhibitors of reverse transcriptasenucleoside analogs that work just like the dideoxynucleotides used to sequence DNA. Basically, the trick with these is to find one that the reverse transcriptase accepts readily but which the host's DNA polymerases tend to reject.

AZT or azidothymidine has been used since 1987.N3 is an azido group which is attached to the 3' position of the deoxyribose. Reverse transcriptases use AZT preferentially over TTP bout our cellular DNA polymerases prefer TTP. It takes an additonal 10 to 20 times more TTP to block DNA synthesis by DNA polymerases. The use of AZT along prolongs life by about 2 years. Unfortunately, AZT resistant mutants appear after about 6 months of treatment. Fortunately, these mutants are still sensitive to other analogs (ie. ddC, ddI & 3TC).

Reverse Transcription Inhibitors

Combination therapy ... Now called HAART

$15,000 / year for drugs alone

Other ideasParts of life cycle that differ most greatly from how cells do

things are the good candidates for targets.

• RNAs that look like TAR or RRE can soak up all of the TAT and REV.

• RNAi• Ribozymes• Vaccines• small molecule inhibitors• Transgenically modify person’s immune cells in

culture and reintroduce them– ARGs, make them more likely to kill themselves if

infected, express anti-HIV ribozymes, 39

Viricidesgp41• When it promotes fusion, gp41 exposes a

hydrophobic peptide.• A peptide matching another part of gp41 can

block fusion. This one is called T20 and it may represent a part of gp41 that normally binds the hydrophobic peptide.

• Approved for use in March 2003.

gp41

41

E. De Clercq / The International Journal of Biochemistry & Cell Biology 36 (2004) 1800–1822 1805

(Maeda et al., 2001). From the spirodiketopiperazine

class of compounds, AK602 was recently quoted

as binding only partially to CCR5, while exhibit-

ing much greater anti-HIV activity: this compound

was also reported to block R5 HIV-1 replication in

HIV-1 (JRFL)-infected hu-PBM NOD-SCID mice

and to possess favorable oral bioavailability in rodents

(Maeda et al., 2002).

N

NH

N

O

O

O

O

E913

8

In addition to the afore-mentioned CCR5 antag-

onists, a wealth of, primarily, 1,3,4-trisubstituted

pyrrolidine CCR5 receptor antagonists have been de-

scribed which possess oral bioavailability and/or po-

tent anti-HIV activity (Hale et al., 2001, 2002; Lynch,

& Gentry et al., 2002; Lynch, & Hale et al., 2002;

Willoughby et al., 2003). The general structure of this

1,3,4-trisubstituted pyrrolidine series is depicted in 9.

The compound’s site of interaction with CCR5 has

been mapped to a cavity, near the extracellular surface,

formed by the TM helices 2, 3, 6 and 7 (Castonguay

et al., 2003). This site is overlapping yet distinct from

that reported for TAK-779 (Dragic et al., 2000).

N

N

O

1

2

3

4

5

1,3,4-Trisubstituted pyrrolidine

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5. Virus–cell fusion inhibitors

The interaction of the X4 or R5 HIV-1 envelope

gp120 with the co-receptors CXCR4 or CCR5, re-

spectively, is followed by a spring-loaded action of

gp41 that then anchors through its amino terminus

into the target cell membrane and, in doing so, initi-

ates the fusion of the viral envelope with the cellular

plasma membrane. At the onset of the fusion process,

hydrophobic grooves on the surface of the coiled

coil gp41 ectodomain become available for binding

with extraneous inhibitors, such as enfuvirtide (T-20,

DP-178, pentafuside, FuzeonTM) (10), a synthetic,

36-amino acid peptide corresponding to residues

127–162 of gp41 or residues 643–678 of the gp160

precursor.

In an initial clinical trial carried out with T-20 at

four doses (3, 10, 30 and 100mg twice daily, intra-

venously, for 14 days) in HIV-infected adults, at the

highest dose (100mg, twice daily) T-20 achieved by

the fifteenth day a 1.5–2.0-fold reduction in plasma

HIV RNA, thus providing proof-of-concept that HIV

fusion inhibitors are able to reduce virus replication

Reservoirs• Quiescent T-cells - remember past infections. Half

live 4 years• Monocytes CD14&CD16 + CCR5 bind HIV. Can go

into the brain & other places. Wait months or decades. Drugs may not get there. Mature into macrophages - turn up anywhere. Macrophages make a lot of virus probably more on a per cell basis than a T-cell.

• Monocytes also make dendritic cells. Show up at every entry point into the body where they interact with T-cells and B-cells.

• Fat cells !

May, M. Playing Hide and seek the deadly way. 2004 The Scientist vol 18: 16-19

Shock & Kill

http://www.cdc.gov/hiv/dhap.htm

http://hivinsite.ucsf.edu/InSite.jsp?page=KB

http://www.ama-assn.org/special/hiv/hivhome.htm

http://www.niaid.nih.gov/default.htm

http://www.aafp.org/afp/980600ap/maenza.html

http://www.thebody.com/treat/expdrugs.html

http://www.fda.gov/oashi/aids/hiv.html

CDC site

Aids Knowledge Base

Jama HIV/AIDS Information Center

National Institute of Allergy and Infectious Diseases

Combination Antiretroviral Therapy June 1998 Am.Acad.Family Physicians

The Body: Gene Therapy for HIV/AIDS

HIV Infection and AIDS, FDA related treatment, testing, fraud, prevention,clinical trials