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University College London

Professor Greg Towers

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Thursday 29 September 2011, Royal College of Physicians, London

Does HIV-1 uncoat at the nuclear pore as part of nuclear entry?

Greg TowersInfection and Immunity

University College London

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Also anti-viral activities called restriction factors

Viruses are obligate Intracellular Parasites

TRIM5α

APOBEC3G/F

Tetherin

Brass, A. L. et al. 2008. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319:921-6.

HIVHIV--1 travels light with only 9 genes1 travels light with only 9 genes

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HIV-1 does not induce type 1 interferon responses in monocyte-derived macrophages

Tsang, J., B. M. Chain, R. F. Miller, B. L. Webb, W . Barclay, G. J. Towers, D. R. Katz, and M. Noursade ghi. 2009. HIV-1 infection of macrophages is dependent on evasion of innate immune cellular activation. AIDS 23:2255-2263.

HIV-1 Goes Under the Innate Radar

0.010.11

0

20

40

60

80

100IFN ββββ pre-treated

Untreated

IFN ββββ post-treated

5.0Innoculum (MOI)

p24

+ve

cel

ls (

%)

HIV-1 replication (infection) in MDM is IFN sensitive

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Tetherin causes Retention of Virions on the Plasma Membrane which are then Degraded

Modified from Neil, S. J., Zang, T., &Bieniasz, P. D. (2008) Nature 451, 425-430. Degradation in lysosomes

Vpu and Nef antagoniseTetherin

HIV uses its accessory genes to antagonise some interferoninduced restriction factorsAccessory genes may make excellent drug targets

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“Here, you see” , said the Red Queen to Alice “ it takes all the running you can do to stay in the same place”.Leigh Van Valen 1973

The Red Queen Hypothesis

The Red Queen Hypothesis

Advantage

Advantage

For an evolutionary system, continuing development is needed just in order to maintain its fitness relative to the systems it is co-evolving with."

Leigh Van Valen 1973

Viruschanges

Hostchanges

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The Red Queen Hypothesis

Advantage

Advantage

For an evolutionary system, continuing development is needed just in order to maintain its fitness relative to the systems it is co-evolving with."

Leigh Van Valen 1973

Viruschanges

Evolve Vpu

HostChanges

Evolve THN

The Red Queen Hypothesis

Advantage

Advantage

For an evolutionary system, continuing development is needed just in order to maintain its fitness relative to the systems it is co-evolving with."

Leigh Van Valen 1973

Viruschanges

Evolve Vpu

HostChanges

Evolve THNDrug Vpu

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Proteasome

Ubi

How Does TRIM5α Work?

Hope LabSodroski LabAiken LabTowers Lab

Proteasome

Ubi

How Does TRIM5α Work?

?

Nature. 2011 Apr 21;472(7343):361-5.

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Nup358 is the Biggest Cyclophilin in the Cell

Splinter D et al. Bicaudal D2, Dynein, and kinesin-1 associate with NPCs and regulate centrosome and nuclear positioning during mitotic entry (PLOS Biology2010)

SUMO E3 ligase activity (not RING-like, not HECT-like); Interaction with Ubc9 � SUMO1-RanGAP1

Interaction with BICD2 � Dynein-Dynactin recruitment at G2 (positioning of nucleus relative to centrosomes)

Nuclear import

Alpha/beta importin dependent

Transportin dependent

Proposed Functions:

Gammaretroviruses (MoMLV)Require Cell Division toAccess The Nucleus

Lentiviruses (HIV-1) can enter the nucleus of non-dividing cells via the nuclear pore

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A Hypothetical Model of the HIV-1 Post Entry Journe y

Nup358

TNPO3

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

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Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

Nup358

TNPO3

CypA

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

Nup358

TNPO3

CypA

11

Chromatin

Nuclear Membrane

Plasma Membrane

HIV-1 PIC

The HIV-1 Post Fusion Journey

Nup358

TNPO3

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

HIV-1 PIC

Nup358

TNPO3

12

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

HIV-1 PIC

Nup358

TNPO3

PRR

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

HIV-1 PIC

Nup358

TNPO3

13

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

Provirus

HIV-1 PIC

Nup358

TNPO3

Chromatin

Nuclear Membrane

Plasma Membrane

The HIV-1 Post Fusion Journey

Nup358

TNPO3

P90A

14

Chromatin

Nuclear Membrane

Plasma Membrane

HIV-1 PIC

The HIV-1 Post Fusion Journey

Nup358

TNPO3

P90A

Chromatin

Nuclear Membrane

Plasma Membrane

HIV-1 PIC

The HIV-1 Post Fusion Journey

Nup358

TNPO3

AlternateRoute to

P90A

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Summary

HIV-1 is an obligate intracellular pathogen that uses its accessory genes to antagoniserestriction factors (Nef/Vpu/Vif antagonise Tetherin/APOBEC3G

We hypothesise that primate lentiviruses such as HIV-1 are locked in evolutionary conflict with their hosts

We propose that drugs that target HIV accessory gene will switch the advantage back to the host mimicking host evolution

HIV-1 does not activate interferon. We hypothesise it must avoid triggering innate responses as it is sensitive to inhibition by interferon

Summary

Nup358 and Transportin 3 are cofactors for HIV-1 nuclear entry

The C terminal Nup358 Cyp domain directly binds the HIV-1 capsid protein

Nup358 contributes to a pathway of nuclear entry that influences integration targeting

Substitutions in the capsid protein can shift HIV-1 to genes in high density regions or to random genes (low density)

Redirecting HIV-1 integration targeting away from its favored sites leads to reduced LTR directed gene expression and reduced replication in primary human macrophages

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Hypotheses

Nup358 destabilises the intact core on arrival at the nuclear pore

The nuclear pore associated uncoating step reveals the PIC to Transportin3 which assures integration into preferred sites

We propose that late uncoating contributes to avoidance of pattern recognition

We speculate that early uncoating allows promicuouskaryopherin use leading tointegration targeting into different sets of genes

We hypothesise that the use of these proteins keeps HIV in a pathway that allows avoidance of innate responses. We are seeking to push HIV off this pathway to activate immune defenses

We propose that future HIV therapy will seek to disturb HIV’s ability to evaderestriction and pattern recognition and will switch on the innate response to HIV thereby appropriately activating adaptive responses.

Outstanding Questions

The restriction factors so far defined are the tip of the iceberg How many are there out there?

Do Restriction factors have broad antiviral activit y? (tetherin)

Are viral countermeasures or host cofactors druggabl e

Can we drug HIV-1 off its “safe” pathway activating better innate and adaptive responses

Will understanding the details of these host virus Interactions may help us: Make Better Vectors for Gene DeliveryEnhance/Adjuvant Vaccination and Design Potent, Broa dly Specific Antiviral Therapeutics?

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But Interferon is strongly induced during HIV -1seroconversion

Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections.Stacey AR, Norris PJ, Qin L, Haygreen EA, Taylor E, Heitman J, Lebedeva M, DeCamp A, Li D, Grove D, Self SG, Borrow P.J Virol. 2009 Apr;83(8):3719-33.

IP10

IFNα

Viral Load

University PennsylvaniaRick BushmanTroy BradyKaren Ocwieja, Keshet Ronen

MRC Laboratory of Molecular Biology, CambridgeLeo JamesAmanda Price

Towers LabTorsten SchallerLaura HilditchAdam FletcherChoon Ping TanSarah PetitCaroline BlondeauJane RasaiyaahJoanne RowleyChris Van Tulleken

National Cancer Institute, FrederickVineetKewalRamaniKyeungeon Lee

UCL Infection and ImmunityMahdadNoursadeghiJhen Tsang