Andrew Lever

University of Cambridge

BHIVA 2010

The talk with no nameGetting intimate with HIV

- the virus in the cell

Lohse, N. et. al. Ann Intern Med 2007;146:87-95

Antiretroviral therapy: >3 million years of life saved

Predicted Survival if HIV+ at age 25 years

Current issues

- Search for new therapeutic targets

- Viral latency

- Lack of a vaccine

Current issues

- Search for new therapeutic targets- Viral RNA

- Viral latency

- Lack of a vaccine

Envelope glycoproteins

Lipid envelope

Dimeric RNA genome

2 x 10,000 nucleotides

MatrixCapsid

Nucleocapsid

Enzymes: RT IN etc

HIV virion

Transcription

Translation

A CellCell surfacereceptors

Cell membrane

Nucleus

DNA

Messenger RNA

RibosomeProtein secreted

Budding

Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

Retrovirus life cycle

Provirus

Envelope glycoproteins

Dimeric RNA genome

Lipid envelope

Uncleaved Gagand Gag/Pol polyproteins

MatrixCapsid

Nucleocapsid

Enzymes: RT IN etc

Gag Pol EnvLTR LTR

Simple retrovirus

Complex retrovirus

Gag Pol EnvLTR LTR

LTR Pol

Tat

Rev

NefVif

Vpu

Gag Pol EnvLTR LTR

Budding

Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

Retrovirus life cycle

Nucleus

HIV

CD4

Chemokine receptorCCR5/CXCR4

Binding to receptor

Cell membrane

Nucleus

HIV

Envelope fusion

Virus entry

HIV TM protein forms coiled coil

Enfuvirtide disrupts coiled coil, blocks fusion

Drug resistant viruses already identified

Cell membrane

Nucleus

Entry and uncoating

Owl monkey

Virus entry – no productive infection

Nucleus

Entry and uncoating

Cellular restriction factorTRIM5α

Overcome by HIV Capsid interaction with cell Cyclophilin protein

Cell membrane

Nucleus

Reverse transcription

APOBEC3G cytidine deaminase, overcome by HIV Vif protein

Cell membrane

Nucleus

Entry and Reverse transcription

Reverse transcriptase enzyme

Highly error prone - 1 in 104

Virus RNA 10,000 nucleotides long Increases diversity of virusDiversity increased further by recombination

• Budding

• Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

HIV

• Budding

• Maturation

Ψ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

HIV

Recombination

• Budding

• Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

HIV

Nucleus

Integration

LEDGF +

Rev -

• Budding

• Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

Packaging in retroviruses

ΨTranscription

Translation

Ψ

Ψ

Packaging signal/Ψinteraction

Budding and protease cleavage

• Budding• Maturation

Ψ

Ψ

Transcription

Ψ

Translation

Packaging

ESCRTs

Packaging in retroviruses

ΨTranscription

Translation

Ψ

Ψ

Packaging signal/Ψinteraction

Secondary Structure of the HIV-1 Packaging Signal Region

5'- GTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAG

CAG - 3’

G U G C

U G C G

GAG

A

StemLoop 1

AUG

GAGAG

CUCUC

A G G A

U U U U G A

A

C U A G CG A U C G

GG

AG

A

AA

G C G

C G C

G G

A AG G

G C U C

C G A G

G

A GG

C G G C A A G

G U C G U U C

C

G A A

G C A

CA

GC U

GC

PutativeDimerisationInitiation Site

Major Splice Donor

Gag Initiation Codon

G

UCA

GCG

AGUA

CGC

G UG G

Principal Packaging Signal

Primer Binding Site

AA

StemLoop 2

StemLoop 3

StemLoop 4

- Phylogenetic comparison- Free energy minimisation- Biochemical probing Geoff Harrison

RNA Packaging in HIV

Ψ

ΨΨ

Ψ

Ψ

Ψ

Ψ

Ψ

Structure of SL3 region of psi

Stuart Hassard

Peter Varnai

Packaging inhibition as a therapeutic target

Oligonucleotide Binding to packaging signal RNA

5`

3`

UG

CU

UG

GG G

G

G

AA

AA ACCU U U

A

A

G

G

G AA G G

AA

M

500250125

62.531.25-O R M

1000

Oligo nM 15.63

100RNA+oligo

RNA

SL3 oligo

Doug BrownMike Gait

G

G G

C

A

U AC

CC

C U

U CC U

Oligonucleotide inhibition of packaging in retroviruses

• Budding• Maturation

Ψ

Ψ

Ψ

Ψ

Transcription

Transport

Ψ

Translation

Packaging

Ψ

Oligonucleotide protection against viral challenge

2`OMe/LNA Scr 2`OMe/LNA SL3125nM

2`OMe/LNA SL31000nM

Doug BrownMike Gait

Oligonucleotide inhibition of packaging in retroviruses

• Budding• Maturation

Ψ

Ψ

Ψ

Ψ

Transcription

Transport

Ψ

Translation

Packaging

Ψ

Therapeutic vaccination

Budding

Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

HIV

Tat/TAR

Rev/RRE

Gag/Ψ

Critical RNA structures

Lentivirus RNA packaging

Multiple structural changes in RNA during progress through cell

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Translation atpolysome

Rev-dependentexport

Gag binding

Membrane targeting

Capsid assemblyand budding

Immatureparticle

Maturevirion

Tat/TAR dependenttranscription

Cellular chaperones?

Brass et al siRNA screen

siRNA library screens and HIV-1

313 18

9270 281

MED6

MED7

RELA

Brass et al.

Zhou et al.König et al.

239

313 18

9270 281

MED6

MED7

RELA

Lentivirus RNA packaging

Multiple structural changes in RNA during progress through cell

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Translation atpolysome

Rev-dependentexport

Gag binding

Membrane targeting

Capsid assemblyand budding

Immatureparticle

Maturevirion

Tat/TAR dependenttranscription

Cellular RNA chaperones – RNA helicases

5 helicases of interest

DDX5

DDX10

DDX17

DDX28

DDX52

Claire Williams

RNA as a target?

Conclusions

RNA structures are potential antiviral targets

Oligonucleotides can block Gag/RNA interactionand inhibit viral spread

Packaging inhibition would lead to autovaccination

Current issues

- Search for new therapeutic targets

- Viral latency

- Lack of a vaccine

• Budding

• Maturation

Ψ

ΨΨ

Ψ

Ψ

Ψ

ΨΨ

Transcription

TranslationPackaging

Cell membrane

Nucleus

Prevalence of viral latency

- PCR studies of CD4+ lymphocytes

- In situ studies of lymph node biopsies of cells + for viral nucleic acid

DNA positive 10 fold commoner than RNA positive

90% of proviruses latent

CD4+ T cells

Majority quiescent, in G0~50% naïve cells t1/2 unknown ? Very long~50% memory cells t1/2 6-44 months

Naive

Memory

ActivatedCytopathic

Latency

106

104

Virus load

CD4+Lymphocytest1/2 1 day

Macrophagest1/2 2 weeks

Memory TLymphocytest1/2 44 months

2-3 years

Effect of HAART on viral load

106 cells 73 years to decay

106

104

Virus load

Reactivation of latent virus

Stop HAART

HAART stopped - new viruses identified including drug sensitive variants

Maldarelli et al. PLoS Path 2007

100 copies

10 copies

1 copy

Persistent HIV Production despite ART

Using vectors to study latency

• Budding• Maturation

Ψ

HIV vector withTransgene

Translation

Packaging

Ψ

Ψ

Viral structural genes

HIV-1 vector

HIV-1 gag/pol expressor

VSV-G envelope

Transient co-transfection

Pseudotyped vector

Infect target cells

HIV-1 based Pseudotyped Vector System

Assay for transgene

Transcription

Translation

Nucleus

Co-transfect plasmids for structural genesand vector containing transgene

Transgeneexpression

Jing ZhaoPadraig Strappe

Efficiency of infection/transduction and gene expression of HIV-1 vector

Time of examination (days post transduction)

Percentage of cells containing

the vector

Percentage of puromycin

resistant cells

Percentage of active vectors

1 7 0.292 0.0171 5.92 7 0.258 0.0595 23

14 0.726 0.0843 123 35 0.214 0.0110 5.14 40 0.187 0.00747 4.05 Long term 0.952 0.0274 2.9

Mixed cultures lose vector activityClones still have vector present

Table 3. Stability of vector gene expression in selected transduced cell clones

Time post transduction

(months)

Number of puromycin resistant

cell clones

Number of puromycin sensitive

cell clones

Percentage of clones that are puromycin

resistant

3 31 0 1006 31 0 10011 28 2 9318 22 5 81

Resistant clones express stably long term

Transcriptional activity of transgene associated with active chromatin

At least two distinct mechanisms of viral silencing

Mok 2006, 2007

Latent virusCell membrane

Nucleus

Retrovirus life cycle

• Integration site • Resting state of memory T cells, low NFAT, NF-kB (HMBA, Prostratin)

• Host miRNA: HIV RNA translational effect, HIV LTR heterochromatin effect

• Resting cell deficient in RNA export factor: PTB

• Epigenetics of viral promoter: acetylation, methylation, etc.

“Open” Histones•Acetylated Histone tails

•Reduced Higher Order Structure•Access to Transcription Factors

•Transcription Active

“Closed” Nucleosome•Hypo-Acetylated Histone tails

•Stable, Compact Chromatin•Less accessible to Transcription Factors

•Transcription Repressed

deacetylated

acetylated

HIVlives

withinchromatin

LTR

Margolis et al., J Virol 1994Romerio et al., J Virol 1997Coull et al., J Virol 2000He & Margolis, MCB 2002Ylisastigui et al. JID 2002

Histonedeacetylases

can shut downHIV expression

LSF LSFYY1

HDAC1

Better targeting of HDAC:Which of the 11 HDACs matter in patient’s resting CD4+ T cells

Class I Class II Class IV

Nancy Archin

Karen Keedy

Margolis lab

10% Input

IP:

TSA: - + - + - + - +

IP:10% Input IgG α-AcH4 α-HDAC4

IP:10% Input IgG α-AcH4 α-HDAC6

IP:10% Input IgG α-AcH4 α-HDAC7

HDACs 1, 2 & 3found at

the HIV LTR

HDACs4, 6 & 7

notfound

Keedy J Virol 2009

Infe

cted

uni

ts p

er b

illion

rest

ing

CD

4+ T

cel

ls

PHA

10

100

1000

10000

Class IIinhibitor

Infe

cted

uni

ts p

er b

illion

rest

ing

CD

4+ T

cel

ls

PHA Class Iinhibitor

10

100

1000

10000

Infe

cted

uni

ts p

er b

illion

rest

ing

CD

4+ T

cel

ls

PHA VPAglobal HDAC

inhibitor

10

100

1000

10000

100000

Infe

cted

uni

ts p

er b

illion

rest

ing

CD

4+ T

cel

ls

PHA

10

100

1000

Class IIinhibitor

Limit ofdetection

Archin AIDS 2009

Vorinostat:

Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor with nanomolar potency licensed for the treatment of

cutaneous T cell lymphoma

Inhibits HDACs 1, 2, 3, and 8 (class I)and HDAC 6 (class II)

Archin ARHR 2009Contreras JBC 2009

Start ART

Target residual viraemia(?)

Anti-latencytherapy

…..eradicationor remission

Future Paradigm

Conclusions

Several mechanisms of viral latency

Selective targeting and inhibition of proteins known to repress HIV may be possible

Latency inhibitors may prevent or reverse latencyand leave the virus susceptible to antiviral agents, and thecell to immune clearance

AcknowledgementsPast

Jenny RichardsonGeoff HarrisonGino MieleLisa ChildFrederique GuesdonPreetha BalanEamonn McCannDorte HaselhorstStuart HassardDavid ChadwickRos FisherNijsje DormanJoan ThomasSteve GriffinDebbie PattisonTom MonieNikki RoseJane AllenBushra Jamil

Present

Jing ZhaoAnne L’HernaultJulia KenyonTruus AbbinkUlrich DesselbergerWilson LiWinsome CheungClaire WilliamsJames StephensonJames RichardsNatasha LeeLiam PrestwoodNeil Bell

Collaborators

Eric HunterGabriele VaraniMartin CranageJames FawcettRobin FranklinMichael LairmoreMarie-Christine DokhelarDavid GraingerAshley MoffettJose GallegoMike GaitSusan LeaMartin ZachariasRay HicksRoger PomerantzPeter LukavskyTahir RizviPeter VarnaiShankar BalasubramanianDavid KlenermanKuan-Teh Jeang

Naomi BishopAlison MaguireNick BennettKate NashErin MyersPadraig StrappeCatherime MurfittJohn SeamonsAlison MuirDoug BrownEmma PooleHoi Ping MokEmma AndersonMatt WheelerHarriet GroomJane GreatorexEmily Manktelow

FundingMRCWellcome TrustBHF

Deaths from HIV/AIDS globally