retrovirology journal club presentation at ucsd by erick tatro

7/27/2019 Retrovirology Journal Club Presentation at UCSD by Erick Tatro

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HIV-1 causes CD4 cell death

through DNA-dependent proteinkinase during viral integration

Nature, 2013Cooper, Garcia, Petrovas, Yamamoto, Koup, Nabel

Virology Lab & Immunology Lab; Vaccine Research Center at NIAID

Erick Tatrofor

Retrovirology Journal Club at UCSD, August 2, 2013

1Monday, September 30, 13


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CD4 Cell Death in HIV

CD4 T cell death, which seems to coincide withpeak viremia during acute HIV

Nature Medicine12, 289 - 295 (2006)Published online: 6 March 2006; | doi:10.1038/nm1380


This study focuses on attempting to understand mechanisms of early HIV dynamics.


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CD4 Cell Death in HIV

CD4 T cell death, which seems to coincide withpeak viremia during acute HIV


here is a similar graph, but which takes into account HAART initiation.


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Lifespan of plasma virus and virus-producing

cells is remarkably short

t1/2 = 2 0.9 days

Nature363, 117-122 (1995)

Nature363, 123-126 (1995)4Monday, September 30, 13

Early on, we understood that plasma virus is short lived; meaning new virus must be producedto maintain high viremia; also infected cells are short-lived; so new virus must come from fairly

recently infected cells.


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Infected Macrophages & Resting CD4

Cells S ared From Cell Death

Even after total CD4 T cell depletion in SIV model,lymph node macrophages maintain SIV infectionwhile CD3 T cells do not.

PNAS (2001)

98:2

:658-662

Macrophage

T - Cells

HIV RNA

HIV RNA

Overlap

No Overlap


Some infected cells; like macrophages and >>resting


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Infected Macrophages & Resting CD4

Cells Spared From Cell Death

Infected memory, resting, T-cells

survive and maintain infection through

aviremia in treated patients.

Nature Medicine 15, 893 - 900 (2009)


even in aviremic patients; "resting" CD4 cells survive as HIV+.


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Why would mechanism

of cell death matter?

Figure out how to stop it if newly Dxpatient to preserve T cells

Whats different about the infected cells

that die -- cause death of reservoir?



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Proposed Mechanisms of

CD4 Cell Death in HIVSyncitia

Autologous mechanisms (e.g.,Tc,

macrophage, FasL)

Apoptosis

Bystander effect

Signal transduction from viral

proteins


cells nearby to dying & infected cells

induced to die through paracrine signallingof inflammatory factors

binding of HIV proteins (e.g., Tat or gp120) induce intracellulsignaling cascade (e.g., p38 or STATs) which lead to death

natural immune response to infected cellsactively kill them to prevent spread

many cells fusing into a mass and dying


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Essential Aims

Investigate molecular mechanism

underlying HIV1 induced cell deathduring acute infection

Why do infected CD4 cells die, but

infected resting CD4 cells, macrophagessurvive?



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In vitro infection dynamics

CEMXT174


CD4 T cell line

by Day 2; 20% of cellsproducing viral protein

of those, only 15% are dead;whereas 60% of thosenot producing viralproteins die.


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In vitro infection dynamicsCEMXT174 - VSVG


vsvg - model of HIV infection where the cells produce a greenfluorescent protein; another method of counting how many

cells go through: entry->reverse transcription -> dnaintegration- viral protein transcription -> translation

40% of cells producing viral protein

. Few of them die

But those that do not produceviral protein; 50% die


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In vitro infection dynamicsPrimary CD4 T cells


From a living human donor (not infected); cells exposed to HIV in the lab, "ex vivo"

by day 10, 20% of cells producingHIV proteins

of that, few die, but up to 70% othose not producing

viral proteins die


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In vitro viability after

infectionPrimary Lymphocytes - various HIV isolates



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In vitro viability after

infectionPrimary Lymphocytes - various HIV isolatesCD4+ T cells die



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Viral cDNA detected in dying

GFP negative cells


However; in thosecells that died,

detected ~5 copiesof viral cDNA

Meaning Entry -> ReverseTranscription occured; but notnecessarily integration


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Dying cells lacking viral expression had beenproductively infected prior to cell death

17Monday, September 30, 13appears as though cells that are infected and produced viral proteins but thenstop producing go on to die

I h f P I


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In the presence of P.I.,Ral (I.I.) & Efv (R.T.I)

prevented cell death



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NRTI

NNRTIII

II

Preventing cell death with ...



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Death decreased without integrase;

2LTRs accumulate


mutation at catalytic site


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Even if transcripts dont

circularize, cells still survive



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Even if transcripts dont

circularize, cells still survive

EMBO J (2001) 20:12:327222Monday, September 30, 13


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Integrated virus, lacking gene

expression ... cells still die


implies that there's something about the integration process that is lethal


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Cells from infected

donors



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... 3 days of activation in vitro,

non-expressing cells die


Th ll did h


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Those cells did have

HIV cDNA



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Raltegravir treatment

in ex vivo culture


DNA D R


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DNA Dmg Response

Following proviralintegration-p53-DNA-PKamage H2AX death

NA


phosphorylated DNA protein kinase phosphorylated p53 gammaH2AX


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Nuclear Translocation ofDNA PK


DNA Dmg Response


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DNA Dmg ResponseFollowing proviral integration

-p53-DNA-PKamage H2AX deathNA

NU7026


The lack of p-DNA-PK doesn't make much sense;unless the inhibitor blocks DNA-PK activation;

DNA Dmg leads to cell death


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DNA Dmg leads to cell death

-p53-DNA-PKamage H2AX deathNA

NU7026 &

NU7441

(Actively infected)

Gated for p24-


li t ifi


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... ruling out non-specific

protection


(toxin)

C l li k 53 & ll d th


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Causal link p53 & cell death

in infection

-p53-DNA-PKDamage H2AX deathDNA

Pifithrin


ATM lt ti t


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ATM - alternative to

DNA-PK

-p53-DNA-PKamage

H2AX deathNA

-ATM

KU55933


Testing whether an alternative pathway is involved

C l i


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Conclusion

Integration triggers DNA Dependent Kinase signaltransduction leading to cell death.

It is associated with productive, not abortive,

infection

See 2010 Cellpaper by

Greene.

Notably, killing of abortiveinfected cells required fusion withHIV from nearby infected cells.


N H th


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New H otheses

Infected Macrophages and Resting CD4 cells thatsurvive to maintain reservoir lack DNA-PKresponse.

Inducing DNA-PK response in cells would kill themduring acute infection.

Inhibiting DNA-PK response in CD4 T cells would

rescue them during acute infection.

Implications for Raltegravir.


How is this paper like The Iliad?


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How is this paper like The Iliad?

Achilles -> CD4 T cell

Hector -> HIV

DNA PK presence / priming -> Achilles prophesy (hell die if isparticipates in siege of Troy)

Hectors killing of Patroclus & wearing Achilles armor -> HIV

cDNA & Integrase inducing DNA damage

Achilles going to battle -> DNA-PK-

Achilles being killed -> CD4 cell death by !H2AX and apoptosis


i like to draw from analogies to explain biology; so if you know the story of Achilles,this is kinda how the process is proposed to work, based on this report.

tragic protagonist (his own actions, presumably carriedout b/c of good qualities, lead to his death). CD4 cellsready to respond to DNA dmg.

antagonist

pre-destiny; pre-existing condition

catalytic event triggeringresponse by the hero

DNA damage leads to phosphorylatiof DNA-PK and events ensue ...

retrovirology journal club presentation at ucsd by erick tatro

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