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)