HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration

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

Virology Lab & Immunology Lab; Vaccine Research Center at NIAID

Erick Tatro for

Retrovirology Journal Club at UCSD, August 2, 2013

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CD4 Cell Death in HIV

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

Nature Medicine 12, 289 - 295 (2006) Published online: 6 March 2006; | doi:10.1038/nm1380
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This study focuses on attempting to understand mechanisms of early HIV dynamics.

CD4 Cell Death in HIV

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

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

Lifespan of plasma virus and virus-producing

cells is remarkably short
1/2

= 2 0.9 days

Nature 363, 117-122 (1995)

Early on, we understood that plasma virus is short lived; meaning new virus must be produced to maintain high viremia; also infected cells are short-lived; so new virus must come from fairly recently infected cells. Nature 363, 123-126 (1995)

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Infected Macrophages & Resting CD4 Cells Spared From Cell Death

Even after total CD4 T cell depletion in SIV model,

lymph node macrophages maintain SIV infection while CD3 T cells do not.

PNAS (2001) 98:2:658-662

HIV RNA Macrophage Overlap

HIV RNA
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T - Cells

No Overlap
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Some infected cells; like macrophages and >>resting<< CD4 cells; do not die.

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)

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

Why would mechanism of cell death matter?

Figure out how to stop it if newly Dx
patient 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 HIV Syncitia Autologous mechanisms (e.g.,T ,
many cells fusing into a mass and dying

macrophage, FasL)

natural immune response to infected cells actively kill them to prevent spread

Apoptosis Bystander effect Signal transduction from viral

proteins
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cells nearby to dying & infected cells induced to die through paracrine signalling of inflammatory factors

binding of HIV proteins (e.g., Tat or gp120) induce intracellula signaling cascade (e.g., p38 or STATs) which lead to death
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Role of DNA PK in Cell Death is a Controversy

DNA PK has a role in retroviral integration

Science (1999)

DNA PK not required for lentiviral integration J Virol (2000)

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces efciency of stable transduction Mol Cell Biol (2001)

DNA damage sensors ATM, ATR, DNA PK, and PARP1 are dispensable for HIV Integration J Virol (2005)
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various studies have looked at DNA Protein Kinase and viral integration; it looks like when DNA-PK is present, is involved, but when not present, integration still happens.

Essential Aims
Investigate molecular mechanism
underlying HIV1 induced cell death during acute infection infected resting CD4 cells, macrophages survive?

Why do infected CD4 cells die, but

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In vitro infection dynamics EMENTARY INFORMATION

SUPPLEMENTARY FIGURES CEMXT174
doi:10.1038/nature12274

CD4 T cell line

by Day 2; 20% of cells producing viral protein

Supplementary Figure 1. Time course analysis of viral replication (a) and cell viability primary CD4+ T cells infected with replication-competent HIV-1. Viral replication was monitored using intracellular p24 staining and cell viability analysis was simultaneously

of those, only 15% are dead; whereas 60% of those not producing viral proteins die.

performed on the indicated, gated p24- and p24+ populations using the permeability dye V and AnnexinV. c, Viability of cell subsets in PBMCs from a healthy donor infected with

indicated viruses or uninfected for 6 days. Data are representative of two independent tary Figure 1. Time course analysis of viral replication (a) and cell viability (b) in

experiments performed with activated primary PBMCs from at least two different donors 4+ T cells infected with replication-competent HIV-1. Viral replication was
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done in triplicate. using intracellular p24 Monday, September 30, 13 staining and cell viability analysis was simultaneously

on the indicated, gated p24- and p24+ populations using the permeability dye Vivid

In vitro infection dynamics

CEMXT174 - VSVG vsvg - model of HIV infection where the cells produce a green
fluorescent protein; another method of counting how many cells go through: entry->reverse transcription -> dna integration- viral protein transcription -> translation
40% of cells producing viral protein

But those that do not produce viral protein; 50% die

. Few of them die

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In vitro infection dynamics

Primary 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 producing HIV proteins

of that, few die, but up to 70% of those not producing viral proteins die

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Primary Lymphocytes - various HIV isolates

In vitro viability after infection

ementary Figure 1. Time course analysis of viral replication (a) and cell viability (b) i
+

y CD4 T cells infected with replication-competent HIV-1. Viral replication was

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red using intracellular p24 staining and cell viability analysis was simultaneously

Primary Lymphocytes - various HIV isolates CD4+ T cells die

In vitro viability after infection

ementary Figure 1. Time course analysis of viral replication (a) and cell viability (b) i
+

y CD4 T cells infected with replication-competent HIV-1. Viral replication was

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red using intracellular p24 staining and cell viability analysis was simultaneously

Viral cDNA detected in dying GFP negative cells

However; in those cells that died, detected ~5 copies of viral cDNA

Meaning Entry -> Reverse Transcription occured; but not necessarily integration

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Dying cells lacking viral expression had been productively infected prior to cell death

ORMATION

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appears as though cells that are infected and produced viral proteins but then stop producing go on to die

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

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EMENTARY INFORMATION

Preventing cell death with ...

NRTI NNRTI II II

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ORMATION

Death decreased without integrase; 2LTRs accumulate

mutation at catalytic site

2. a, Quantitative real-time PCR analysis for late reverse transcripts

d populations originating from pre-sorted GFP+ cells shown in Fig. 2c, at

Cell death analysis gated on GFP-negative populations of CEMX174 cells

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oding, VSVG-pseudotyped HIV-1 in the absence or presence of the

Even if transcripts dont circularize, cells still survive

RESEARCH SUPPLEMENTARY INFORMATION

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Even if transcripts dont circularize, cells still survive

EMBO J (2001) 20:12:3272

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Integrated virus, lacking gene expression ... cells still die

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

Cells from infected donors

SUPPLEMENTARY IN

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SUPPLEMENT

... 3 days of activation in vitro, non-expressing cells die

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Those cells did have HIV cDNA

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Raltegravir treatment in ex vivo culture

Supplementary Figure 3. Flow cytometry analyses of viral replication (a) and cell death (b)

CD3 high, CD8 negative cells in PBMCs from a healthy donor (subject # 1) and three infected

patients (subjects # 2, 3 and 4) that had been activated for 3 days (see Fig. 3e). Viral replicatio
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was monitored using intracellular p24 gag staining and cell viability analysis was simultaneou

DNA Dmg Response Following proviral integration

-DNA-PK

DNA Damage

-p53

H2AX

death

phosphorylated DNA protein kinase phosphorylated p53 gammaH2AX

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ESEARCH SUPPLEMENTARY INFORMATION

Nuclear Translocation of DNA PK

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DNA Dmg Response Following proviral integration

NU7026 H2AX death

DNA DamageRESEARCH -DNA-PK -p53 SUPPLEMENTARY INFORMATION

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

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RMATION

DNA Dmg leads to cell death

NU7026 & NU7441 -DNA-PK

DNA Damage

-p53

H2AX
Gated for p24-

death

(Actively infected)
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... ruling out non-specic protection

(toxin)

Supplementary Figure 4. a, Western blot analysis for DNA-PK, Matrin

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and cytoplasmic fractions from primary CD4 lymphocytes uninfected or

Causal link p53 & cell death in infection

Pithrin DNA Damage -DNA-PK

-p53

H2AX

death

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ATM - alternative to DNA-PK

-DNA-PK -ATM
Testing whether an alternative pathway is involved

DNA Damage

-p53

H2AX

death

KU55933

Figure 4. a, Western blot analysis for DNA-PK, Matrin3 and

entary Figure 4. a, Western blot analysis for DNA-PK, Matrin3 and eIF4e
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Conclusion
Integration triggers DNA Dependent Kinase signal
transduction leading to cell death. infection

It is associated with productive, not abortive,

See 2010 Cell paper by Greene.
Notably, killing of abortive infected cells required fusion with HIV from nearby infected cells.
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New Hypotheses
Infected Macrophages and Resting CD4 cells that
survive to maintain reservoir lack DNA-PK response. during acute infection.

Inducing DNA-PK response in cells would kill them Inhibiting DNA-PK response in CD4 T cells would
rescue them during acute infection.

Implications for Raltegravir.

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How is this paper like The Iliad?

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.

Achilles -> CD4 T cell Hector -> HIVantagonist DNA PK presence / priming -> Achilles prophesy (hell die if is
participates in siege of Troy) pre-destiny; pre-existing condition cDNA & Integrase inducing DNA damage
response by the hero

tragic protagonist (his own actions, presumably carried out b/c of good qualities, lead to his death). CD4 cells ready to respond to DNA dmg.

armor -> HIV Hectors killing of Patroclus & wearing Achilles catalytic event triggering

damage leads to phosphorylatio Achilles going to battle -> DNA-PK- DNA of DNA-PK and events ensue ... Achilles being killed -> CD4 cell death by !H2AX and apoptosis

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