Alternative RNA splicing in latently infected T cells generates chimeric cellular:HIV

mRNAs with the potential to generate Tat and reactivate infection

Con Sonza, Talia Mota, Jonathan Jacobson, Michelle Lee, Giovana Bernardi, Jane Howard, Damian Purcell

The University of Melbourne, Department of Microbiology and Immunologyat the Peter Doherty Institute

cART is able to suppress plasma viraemia below detectable levels, however the reservoir of latently infected cells persists. Interruption or discontinuation of cART is followed by rebound of viraemia and progression to AIDS.

The current need for Latency targeting therapy:

Interruption of cART

(weeks)

cART

Adapted from: Eisele And Siliciano.

Immunity (2012) HIV-1 infected cell

CD4+ T cell

Latency Rebound - AIDSEarly infection

Adapted from: Han and Siliciano, Nat Med., 2007

Cellular factors-Limited transcriptional

activators

Viral factors-Site of integration (into active gene)-Transcriptional interference*-Low acetylation-High methylation

Impaired RNA export from nucleus

HIV specific host microRNA

HIV gene expression during latency in resting memory T-cells

Cell-activation /senescence

RNA splicing

RNA transcription

Chromatin remodellin

g(epigenetic

s)

microRNA expression

m7G-capping

Tat

Adapted from Siliciano RF, Greene WC. 2011

Mechanisms that establish and/or maintain latency: Transcriptional Interference

CENTRAL HYPOTHESIS: cART selects HIV provirus integrated into the introns of transcriptionally active genes where read-through transcription includes HIV RNA

A7

Cap-dependent Tat vs IRES-Tat

pcDNA3.1-

100200

300400

500600

200400

600800

10001200

14000

0.20.40.60.8

11.21.41.61.8 Transfection of TZM-bl reporter cells

Rela

tive

Luci

fera

se A

ctivi

ty

Cap-Tat (ng) IRES-Tat (ng)

Michelle Lee, 2014

ACH2 latent T cell line

1. Uninfected Jurkat cells 2. Unstimulated ACH2 cells 3. PMA-stimulated ACH2 cells

Ex2 Ex6Ex1intronsA

UG

Ex3 Ex4 Ex5 Ex7 Ex8 Ex9

2595PCR primers

32P-probes

1 2 3

Odp2603tat exon 2

2603

A2 A3

2603

1 2 3

Odp2601gag-5’

2601

D1

2601

1 2 3

Odp2604env/vpu

2604

D4

2604

1 2 3

Odp2605HIV-U3

2605

Ex5 U3

2605

- p

A

- p

A

- p

A

A3

D1 D4

Read-through transcription splices HIV tat exon2 onto cell NT5C3 mRNA in the ACH-2 cell line model of HIV-1 latency

Jane Howard, 2013

Latently infected J-Lat6.3 cells produce chimeric cell:tat RNA by read-through transcription and splicing

Michelle Lee, 2013

Functional Tat protein is expressed from spliced cell:tat mRNA using an Internal Ribosome Entry Site (IRES) underlying the Tat coding sequence

Transduction of the latently infected ACH2 and J-Lat6.3 cell lines with a Tat responsive LTR-Luciferase reporter pseudovirus

A3.01 Unstimulated PMA stimulated0

2000

4000

6000

8000

ACH2

Luci

fera

se a

ctivi

ty

FlucNef2 5∆Ga

g∆Env

U3 R U5Nef U3 R U5

Pseudovirus (after RT):

A3.01 Unstimulated TNF stimulated0

5000

10000

15000

20000

25000J-Lat

Luci

fera

se a

ctivi

ty

Jurkat

Giovana Bernardi, 2013

Alu-tat PCRs of cDNA from CCL19-treated,memory CD4 T cells infected with NL4.3

Latently-infected primary CD4 T cells express chimeric cellular:tat mRNAs

tat exon 2

MW + + + - DNAseMW + + - + RT

MW + - :RT

Nested tat PCR

Uni

nfec

ted

cont

rol

Don

or 1

Don

or 2

Uni

nfec

ted

cont

rol

Don

or 1

Don

or 2

Southern blotprobed with tat exon 2 probe

Cloned, sequenced Talia Mota

Distribution of integration sites in five patients. A total of 2410 integration sites were obtained from PBMCs or negatively selected

CD4+ cells from the five patients.

F Maldarelli et al. Science 2014;345:179-183

Detection of chimeric cell:tat RNA in primary resting CD4 T cell latency model

PCR of cDNA from CCL19 chemokine-induced resting CD4 T cells infected with HIV-1NL4.3

(Saleh et al, Blood, 2007) using various cellular gene exon forward primers andtat exon2 reverse primer

STAT5BExon 5

Exon 8

DDX6Exon 2

Exon 5

HORMAD2Exon 2

Exon 9

ExonExon

- pA

- pAA3

D1 D4

Read-through transcription and splicing generate chimeric cell:tat RNAs in latently infected primary CD4 T cells

1 2 3 4 5 1 2 3 4 5STAT5B HORMAD2

Reverse primers1. LTR-U32. gag3. tat exon 2-ls4. tat exon 2-cs5. SD1/SA3

Forward Primers

Exon Exon

- pA

- pA

A3

D1 D4

Conclusions • During read-through transcription in latently infected T cell

lines and primary resting CD4 T cells, chimeric cell:tat RNAs are generated by the usual cellular mechanisms of alternative RNA splicing

• An IRES-like element in tat leads to translation of this mRNA in a cap-independent manner and expression of functional Tat protein (POSTER THPE006: G. Khoury)

• Because of the central role of Tat in the establishment and maintenance of latency, factors affecting transcription, splicing, cytoplasmic localization or translation of Tat from chimeric RNAs will impact on HIV latency

• Such factors could be targeted to develop novel, more specific, strategies to assist in the activation and clearance of the latent reservoir or prevent viral rebound upon cessation of cART (POSTER THPE016: J. Jacobson)

Acknowledgments

Sharon LewinPaul CameronFiona WhitemanSuha SalehVanessa Evans

Alfred Hospital / Burnet institute / Monash University

Damian PurcellTalia MotaJonathan Jacobson Michelle LeeGiovana BernardiJane HowardLeanne Ng

University of Melbourne

NHMRC and ACH2 for funding.