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LEDGIN (ALLINI) Disrup0on of HIV-‐1 Assembly Does Not Involve LEDGF/p75 Hind J. Fadel1, James H. Morrison1, Dyana T. Saenz1, James R. Fuchs2, Mamuka Kvaratskhelia3, Stephen C. Ekker4 and Eric M. Poeschla1 1Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester MN, USA, 2Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA, 3Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA, 4Department of Biochemistry & Molecular Biology, Mayo Clinic College of Medicine, Rochester MN, USA. Abstract LEDGIN (ALLINI) Disrup0on of HIV-‐1 Assembly Does Not Involve LEDGF/p75 Hind J. Fadel, James Morrison, Dyana Saenz, Stephen C. Ekker, James R. Fuchs, Mamuka Kvaratskhelia and Eric M. Poeschla Background: LEDGF/p75, the product of the PSIP1 gene, is an HIV-‐1 integraYon cofactor that tethers the viral pre-‐integraYon complex to the site of its subsequent integraYon. Allosteric integrase inhibitors or ALLINIs (also known as LEDGINs) disrupt the interacYon of LEDGF/p75 with the HIV-‐1 IN dimer and impair integraYon. However, a considerably more potent (and apparently main) mechanism of ALLINI acYon was recently idenYfied: disrupYng proper parYcle assembly. It is not clear whether this anYviral effect involves LEDGF/p75. Some studies suggest that it is LEDGF/p75-‐independent but others suggest LEDGF/p75-‐dependence and, consistent with this, that LEDGF/p75 incorporaYon into HIV-‐1 parYcles occurs and is necessary for normal HIV-‐1 infecYvity. RNAi-‐ depleted cells sYll contain some LEDGF/p75 protein, and a fracYonally minute LEDGF/p75 residuum can maintain integraYon cofactor funcYon. Mouse PSIP1 gene knockout cell lines exist but cannot be used for HIV assembly studies. Here we definiYvely answer two quesYons: does LEDGF/p75 play a role in HIV-‐1 assembly and does the ALLINI anYviral mechanism involve LEDGF/p75? To do so, we used TAL effector nucleases (TALENs) to delete the PSIP1 gene from informaYve human cell lines. Methodology: We designed and constructed mulYple TALEN pairs that site-‐specifically target PSIP1. We used them to generate clonal Jurkat and 293T cell lines that are -‐/-‐ for the enYre 42 kb gene, and separately, -‐/-‐ for the only the exons that encode the LEDGF/p75 integrase binding domain (IBD). Correct deleYon of all PSIP1 alleles was verified at the DNA and protein levels, precise chromosomal deleYon juncYons were determined, and comprehensive viral life cycle analyses were conducted in the presence and absence of drug. Results: HIV-‐1 integraYon is impaired, and spreading viral replicaYon is blocked, but HIV-‐1 producYon and infecYous parYcle assembly are normal in PSIP1 -‐/-‐ cells. This is the case whether the enYre gene is deleted or only the IBD-‐encoding gene segment is deleted. We further determined that ALLINI potency for blocking HIV-‐1 infecYous parYcle formaYon is unaffected by total removal of LEDGF/p75-‐encoding capacity from the cell. TALEN gene KO was efficient. For example, a third of single cell clones generated from a single transfecYon were found to contain bi-‐allelic gene disrupYons due to NHEJ-‐generated indels at the specific target site, suggesYng efficacy for gene targeYng cure strategies aimed at this and other HIV dependency factor genes such as CCR5. Conclusions: The main ALLINI mechanism is independent of LEDGF/p75. This protein also does not play a detectable role in parYcle assembly. The LEDGF/p75 cofactor role is confined to the integraYon step of the viral life cycle. Background TAL Effector Nucleases • • • • • Bacterial plant pathogens of the genus Xanthomonas inject TAL effector proteins that bind specific DNA sequences and alter plant host cell transcripYon. They contain a conserved tandem of 33-‐35 amino acid repeats that differ mainly at posiYons 12 and 13. These are termed repeat variable diresidues (RVDs). Different RVDs recognize different DNA base pairs, which creates a simple code: the order of RVD repeats in the TALE corresponds to the nucleoYde sequence of the target DNA. Customized TALENs are designable with TALEs that recognize a specific DNA sequence at the N-‐terminus; these are fused to an NLS and FokI at the C terminus. A pair of TALENs are built to bind sequences to the right and the leh of the target DNA, causing a DSB when the two FokI domains dimerize. Error-‐prone NHEJ leads to gene-‐disrupYng indels at the target. Use of two TALEN pairs can produce full deleYon of the intervening chromosome DNA and provision of a template in trans can lead, via HR, to gene replacement, ediYng or knock-‐in. In vivo TALEN-‐induced genome edi0ng outcomes TALEN TALEN A very small frac0onal chroma0n-‐bound residuum of LEDGF can maintain cofactor func0on: strip it from chroma0n in CD4+ T cells and HIV-‐1 is impaired Llano et al., Science, 2006 GFP U6 Pol III LEDGF mRNA HIV-1 reporter virus infection shRNA 12% 2% TC2 TL1 TL2 TC2 TL1 TL2 Results Efficient TALEN mediated gene targe0ng of PSIP1 gene Uncut Cut Cut Cut Uncut Cut PCR primers: 1 and 3 PCR primers: 2 and 3 Jurkat cells (CD4+ T-‐cells) PCR primers: 1 and 3 PCR primers: 2 and 3 293T cells S2 frac0on Cell lysate 293T cells IBD -‐/-‐ (B) S2 frac0on LEDGF(∆IBD) 50 IBD -‐/-‐ (A) PCR primers: 2 and 3 LEDGF 75 Parent IBD -‐/-‐ Parent Water PCR primers: 1 and 3 Cell lysate Jurkat 293T LEDGF -‐/-‐ Parent Jurkat LEDGF -‐/-‐ Parent TALEN mediated dele0on of PSIP1 or IBD exons in 293T and Jurkat cell lines Water 293T Jurkat cells Clones indels sequencing TALEN pair X and B Gene sequence TALEN X spacer TALEN B spacer gactcgcgajtcaaACCTGGAGACCTCAT-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐/ 42Kb /-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐AAGTCTACAATGTTGtataacaagjtaag Jurkat PSIP1 -‐/-‐ (X to A): gactcgcgajtcaaACCAGGjaag gactcgcgajtAACTAGgjaag 293T PSIP1 -‐/-‐ (X to A): gactcgcgajtcaaACCTGGAGGAGAATGTTGtataacaagjtaag TALEN pair A and B Gene sequence TALEN A spacer TALEN B spacer caatggajctcgacTTCAAAGGATACATGC-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐/ 0.5 kb /-‐-‐-‐-‐-‐-‐-‐-‐-‐AAGTCTACAATGTTGtataacaagjtaag Jurkat IBD -‐/-‐ Clone A: caatggajctcgacTTTTGtataacaagjtaag catggajctcgaccTTCAAAGAAGTCTACAATGTTGtataacaagjtaag Jurkat IBD -‐/-‐ Clone B: caatggajctcgacTTCAAAGGATTTACCACAATGTTGtataacaagjtaag aggaacaaatggaaactgagcaCTTAAATGagjtaag 293T IBD -‐/-‐: caatggajctcgacTTCAAGTTGtataacaagjtaag caatggajctcgacTTCAGTGTATAACAAGTTTGagaacatgjcjggtt Len0viral infec0on is impaired in LEDGF KO cells 160000 250000 HIV-‐luc 200000 293T 80000 293T LEDGF-‐/-‐ 40000 293T IBD -‐/-‐ RLU 120000 RLU HIV-‐luc Jurkat 150000 Jurkat LEDGF -‐/-‐ 100000 Jurkat IBD -‐/-‐ clone A 50000 Jurkat IBD -‐/-‐ Clone B 0 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 0 1.E+03 100 12000 FIV-‐GFP 60 293T 40 293T LEDGF -‐/-‐ 293T IBD -‐/-‐ 20 Jurkat 6000 Jurkat LEDGF -‐/-‐ 4000 Jurkat IBD -‐/-‐ Clone A Jurkat IBD -‐/-‐ Clone B 2000 0.1 1 0 10 0.1 60 MLV-‐GFP 50 40 293T 30 293T LEDGF -‐/-‐ 20 293T IBD -‐/-‐ 10 0.1 1 Input (ul) 10 1 10 Input (ul) 100 % GFP posi0ve cells % GFP posi0ve cells 1.E+07 8000 Input (ul) 0 0.01 1.E+06 FIV-‐luc 10000 80 0 0.01 1.E+05 Input (RT unit) RLU % GFP posi0ve cells Input (RT units) 1.E+04 35 MLV-‐GFP 30 25 Jurkat 20 Jurkat LEDGF -‐/-‐ 15 Jurkat IBD -‐/-‐ Clone A 10 Jurkat IBD -‐/-‐ Clone B 5 0 0.1 1 10 Input (ul) 100 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 HIV-‐luc HIV-‐luc Jurkat Jurkat LEDGF -‐/-‐ Jurkat LEDGF -‐/-‐ BC 0.1 1 10 Input (MOI) RLU RLU HIV-‐1 infec0vity is rescued in T-‐cells by re-‐expression of LEDGF or expression of an alterna0vely tethered IBD (LANA31-‐GFP-‐IBD) 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 0.01 Jurkat Jurkat LEDGF -‐/-‐ Jurkat LEDGF -‐/-‐ LANA-‐ GFP-‐IBD Jurkat LEDGF -‐/-‐ LANA-‐ GFP-‐IBD D366N 0.1 1 Input (MOI) 10 BC= back-‐complemented LEDGF LEDGF GFP Tubulin Tubulin Jurkat cells HIV-‐1 produc0on and infec0ous par0cle assembly are normal in PSIP1 -‐/-‐ cells Reporter virus produc0on Titra0on of produced virus on target cell Measure GFP by flow cytometry to calculate Yters 90 80 70 60 50 40 30 20 10 0 1.E+04 VIrus produced in 293T Virus produced in 293T IBD KO 1.E+05 1.E+06 1.E+07 Input (RT units) 1.E+08 Infect target cells with normalized viruses % GFP posi0ve cells % GFP posi0ve cells Produce virus by transfecYon in 6 well plate • 293T cells • 293T LEDGF -‐/-‐ cells • 293T IBD -‐/-‐ cells 100 100 90 80 70 60 50 40 30 20 10 0 1.E+03 Virus produced in 293T Virus produced in 293T LEDGF KO 1.E+04 1.E+05 1.E+06 Input (RT units) 1.E+07 Titer (TU/p24) 1.E+03 1.E+02 1.E+01 1.E+00 0.01 Titer (TU/p24) 293T 1.E+04 0.1 1 10 Concentra0on (uM) 293T LEDFG -‐/-‐ 1.E+04 1.E+02 1.E+00 0.01 0.1 1 Concentra0on (uM) 10 100 293T LEDGF shRNA 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 0.01 100 Titer (TU/p24) Titer (TU/p24) ALLINI potency for blocking HIV-‐1 infec0ous par0cle forma0on is unaffected by total removal of LEDGF/p75-‐encoding capacity from the cell 0.1 1 Concentra0on (uM) 10 100 10 100 293T IBD -‐/-‐ 1.E+04 1.E+02 1.E+00 0.01 0.1 1 Concentra0on (uM) p24 (ng/ml) HIV-‐1 NL4-‐3 challenge of PSIP1 -‐/-‐ cells 180 160 140 120 100 80 60 40 20 0 MOI 0.1 Jurkat Jurkat LEDGF -‐/-‐ Jurkat IBD -‐/-‐ Jurkat LEDGF -‐/-‐ LANA-‐GFP-‐ IBD 0 10 20 30 40 50 60 Jurkat LEDGF -‐/-‐ LANA-‐GFP-‐ IBD D366N p24 (ng/ml) Days post-‐infec0on MOI 1 180 160 140 120 100 80 60 40 20 0 Jurkat Jurkat LEDGF -‐/-‐ Jurkat IBD -‐/-‐ Jurkat LEDGF -‐/-‐ LANA-‐GFP-‐ IBD 0 10 20 30 40 50 Days post-‐infec0on 60 Jurkat LEDGF -‐/-‐ LANA-‐GFP-‐ IBD D366N Conclusions • PSIP1 (LEDGF) gene deleYons and PSIP1 IBD exon segment deleYons were achieved in human diploid and polyploid cell lines with TALENs. • Single gene disrupYons occurred with high efficiency, with virtually all TALENS we constructed being effecYve. The predictable cipher provides an advantage over zinc finger nuclease technology. • Large chromosome segments > 40 kb were excised with two TALEN pairs. • Both T cells and adherent cells were targetable. • HIV-‐1 integraYon is inhibited in PSIP1 -‐/-‐ cells. • We find that HIV-‐1 parYcle producYon levels and infecYous HIV-‐1 parYcle assembly are normal in PSIP1 -‐/-‐ cells. LEDGF does not play a detectable role in parYcle assembly and its cofactor role depends on its role in early events. • We also find that the potency of a representaYve allosteric inhibitor of HIV-‐1 integrase (ALLINI), which binds to the LEDGF IBD binding site on IN, is unaffected by total removal of LEDGF from the cell. • We conclude that the dominant ALLINI mechanism is enYrely independent of LEDGF. References 1. 2. 3. 4. 5. 6. Cermak, T., E.L. Doyle, M. ChrisYan, L. Wang, Y. Zhang, C. Schmidt, J.A. Baller, N.V. Somia, A.J. Bogdanove, and D.F. Voytas, Efficient design and assembly of custom TALEN and other TAL effector-‐based constructs for DNAtargeYng. Nucleic Acids Res, 2011. 39 (12): p. e82. Bedell, V.M., Y. Wang, J.M. Campbell, T.L. Poshusta, C.G. Starker, R.G. Krug, 2nd, W. Tan, S.G. Penheiter, A.C. Ma, A.Y. Leung, S.C. Fahrenkrug, D.F. Carlson, D.F. Voytas, K.J. Clark, J.J. Essner, and S.C. Ekker, In vivo genome ediYng using a high-‐efficiency TALEN system. Nature, 2012. 491 (7422): p. 114-‐8. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, ChrisYan M, Voytas DF, Long CR, Whitelaw CB, Fahrenkrug SC. Efficient TALEN-‐mediated gene knockout in livestock.Proc Natl Acad Sci U S A. 2012 Oct 23;109(43):17382-‐7 Bogdanove, A.J., S. Schornack, and T. Lahaye, TAL effectors: finding plant genes for disease and defense. Curr Opin Plant Biol, 2011. 13 (4): p. 394-‐401. Llano, M., D.T. Saenz, A. Meehan, P. Wongthida, M. Peretz, W.H. Walker, W. Teo, and E.M. Poeschla, An EssenYal Role for LEDGF/p75 in HIV IntegraYon. Science, 2006. 314 (5798): p. 461-‐4. Llano, M., M. Vanegas, N. Hutchins, D. Thompson, S. Delgado, and E.M. Poeschla, IdenYficaYon and CharacterizaYon of the ChromaYn Binding Domains of the HIV-‐1 Integrase Interactor LEDGF/ p75. Journal of Molecular Biology, 2006. 360 : p. 760-‐73. Future Direc0ons • IntegraYon site analysis • Knock out HRP2 also. • CCR5 and other dependency factor KO. • Determine the importance of LEDGF in vivo. A germline knockout of PSIP1 (LEDGF) is in progress in the domesYc cat. This integraYon cofactor is used by all lenYviruses, including FIV, yet by no other retroviruses. Therefore, gene disrupYon in the cat will be informaYve. Acknowledgments • Poeschla Lab Ø Eric Poeschla Ø Dyana Saenz Ø Anne Meehan • Ekker lab Ø Stephen Ekker Ø Jarryd Campbell Ø Alvin MA Ø Mary Peretz Ø Tommy Rinkoski Ø Dan Rogstad Ø SwaY Kumar • Mamuka Kvaratskhelia (Ohio State Center for Retrovirus Research) Ø Manita Wijayarat • Funding: R01 AI77344
