Single-Cell Analysis Shows Molecular Signatures of HIV Latency In

CROI 2020 PP E-01 Mechanisms of Latency 306 7-11 March 2020 Contact Single-Cell Analysis Shows Molecular Signatures of HIV Latency in Primary Cell Models Name: Sushama Telwatte Address: 4150 Clement St San Francisco, CA 94121 1,2 3 4 5 1,2 6 6 5 3 Email: Sushama Telwatte , Mauricio Montano , Rachel S. Resop , Emilie Battivelli , Sara Morón-López , Douglas Arneson , Atul Butte , Eric Verdin , Warner C. Greene , [email protected] Alberto Bosque4, Joseph K. Wong1,2, Steven A. Yukl1,2 1. San Francisco VA Medical Center (SFVAMC), San Francisco, CA, USA 2. University of California, San Francisco (UCSF), San Francisco, CA, USA 3. Gladstone Institutes, San Francisco, CA, USA 4. George Washington University, Washington, DC, USA 5. The Buck Institute for Research on Aging, Novato, CA, USA 6. Bakar Computational Health Sciences Institute, UCSF, San Francisco, CA, USA

Introduction Results • Primary cell models have greatly advanced our understanding of HIV latency. HIV expression drives the differences between populations across donors in the all of the primary cell model tested However, it is unclear which mechanisms underlie latency in these primary cell models. Dataset Group1 Group2 Differentially expressed genes (FDR p<0.05) Dataset Group1 Group2 Differentially expressed genes (FDR p<0.05) WT Virus HIV+ HIV- (exposed but UI) Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), Nef, PolyA Resting Cell (blood CD4) Latent HIV+ HIV- (exposed but UI) Gag, Env (IPDA), PolyA Resting Cell Model (Blood CD4) HIV+ Uninfected (HIV-unexposed) Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), Nef, PolyA Latent HIV+ UI (unexposed/uninfected) Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), PolyA Hypothesis: Molecular signatures can distinguish uninfected, latently- Dual Productive Latent Gag, Pol, Env (IPDA), PolyA, Tat-Rev CDK13 is differentially-expressed between latently-infected, HIV-exposed but uninfected, and HIV- and productively-infected populations in these models Reporter Productive Uninfected Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), PolyA, Tat-Rev Resting Cell (tonsil CD4) Latent (mCh-) UI (unexposed/uninfected) Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), unexposed cells Latent Uninfected Long LTR, Gag, Gag (IPDA), Pol, Env (IPDA), PolyA B Methods A Donor 11 Latent (HIV+) Donor 13 Latent (HIV+) • We assessed 4 primary cell models [blood CD4T cells: models from labs of Alberto Donor 11 HIV- (HIV-exposed) Donor 13 HIV- (HIV-exposed) Bosque1 (WT Virus Model), Eric Verdin2 (Dual Reporter Virus Model), and Warner Wild Type Virus Model Donor 11 UI (HIV-unexposed) Greene3,4 (Resting Cell Model); tissue (tonsillar) CD4+ T cells: model from Warner Upregulation of BCL6 and HLA-DR and of STAT1 distinguish latent HIV+ cells from Donor 13 UI (HIV-unexposed) Greene]. Single cells from each model (2 donors) were FACS-sorted into 96-well downregulation plates and multiplex RT-qPCR (Biomark HD) was used to quantify 88 human RNAs uninfected populations previously implicated in HIV infection/latency and 8 HIV targets (5'LTR, Gag, Pol, 5 Nef, MS Tat-Rev, U3-PolyA, and the IPDA assays for Env and Gag). We compared sampleID 30 sampleID MAIN FINDINGS HIV-unexposed, HIV-exposed but uninfected, and latently- +/- productively-infected A B BD77HIV+Donor 77 Latent (HIV+) Long LTR 25 *No Tat-Rev populations from each model to identify genes with ≥2-fold difference in median BD79HIV+Donor 79 Latent (HIV+) expression BD77HIVDonor 77 HIV− - (HIV-exposed) expression levels and P<0.05 or FDR-corrected P<0.05. Gag 20 ●Donor 11 HIV+●Donor 11 HIV−●Donor 11 UI detected BD79HIVDonor 79 HIV− - (HIV-exposed) ●Donor 13 HIV+●Donor 13 HIV−●Donor 13 UI Gag (IPDA) BD77UIDonor 77 UI (HIV-unexposed) C 15 WT Virus Model: All Genes No HIV Genes BD79UIDonor 79 UI (HIV-unexposed) ● Primary Cell Models ● ● Pol 2 ●● ● ● ● ● 10 ● ●● ● ● ● D ● ● ● ● ● • ● ● ● ● • Each model differed in the virus employed: BCL-6 and HLA-DR are ● 1 ● ● ● ● ● ● Env (IPDA) ●● ● ● 5 ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PolyA sampleID upregulated and STAT1 is 0 ● ● ● ● ● ●●● Nef ● ● ● ● ● PolyA 0.6 GD11HIV+ 0 ● ● ● ● ● ● ● ● sampleID APOBEC3G PTB/PTBP1 IFI16 STAT1 SF2/ASF (SRSF1) GAPDH IRF9 G9a/EHMT2 OX40/TNFRSF4 NFKB1 PBAF/SMARCA4 BCL11B/CTIP2 PAPOLA POLR2A CDK9 beta7 integrin/ITGB7 KAT2B/PCAF BCL6 NELFA/WHS2 PRDM1/Blimp − 1 CD4 TCRA ELL SF3B2 BCL2 HLA − DR alpha CASP3 PAF − 1 TGFB1 FAS STING/TMEM173 SLFN11 FoxP3 TBX21/t − bet TRIM5 CD28 CD3 delta CD44 C − GAS/MB21D1 RIG − I/DDX58 CTLA4 GATA3 HTATSF1 MATR3 PPIA SAMHD1 CD69 NFATC1 p53 CDK11a CCR5 CREBBP/CBP PRMT6 PSIP1 MDA5/IFIH1 BST2/Tetherin IFNA1 NFKBIA CCNL2/cyclin L2 CXCR4 CCR7 CEBPB TCF7 CDK7 CDK13 RPL13a CD25/IL2RA LCK HDAC5 Sp1 EP300 EZH2 CCNT1 YY1 ● ● ● ● ● ● ● ● ● ● ● ● ● ●● 0.4 GD13HIV+ Wild Type Virus UMAP_2 ● ● −1 ● ● ● ● WT HIV-NL4.3 PolyA ● ● ● ● ● ● ● ● ● 0.2 downregulated in latent HIV+ ● ● ● ● ● ●● ●● 0 model Competent ● ● ● ● −2 ● −0.2 Tat−Rev ● ● −0.4 cells vs. uninfected cells. ● −2 −1 0 1 2 −1 0 1 2 UMAP_1 C D Fig. 4(A) Schematic of Resting Cell model (blood and tonsillar CD4 T cells). Resting Cell ●Donor 77 HIV+●Donor 77 HIV−●Donor 77 UI ●Donor 79 HIV+●Donor 79 HIV−●Donor 79 UI Single-cell data generated (blood CD4 T cells) are represented by (B) heat HIVmCherry-LUC All Genes No HIV Genes 0.3 sampleID model 4 Dual Reporter Virus Model: map for HIV transcript expression (C) UMAP projections with/without HIV ● PolyA Table 4. Differentially expressed genes between Resting Cell Model (blood CD4 T) populations ●● ●● ● BD77HIV+,BD79HIV+ ● ●●● Competent* ● ●●●● ● ● 0.2 ● ●● ● ● ● ●● ● targets, and (D) correlation matrix showing degree of correlation between ●● ● ●● ● ● Ave. Log ●● ●● ●● ● ●● ● ● 2 ● ●● ● ● ● ● ● ● ● ●● Group1 Group2 gene p_val N1 N2 FDR ● ● ●●● ●● ● ● ● ● ●● ● Tat−Rev • ● ● ● ● ●● ●● ● CDK7, CEBPB and Cyclin L2 expression of each cellular gene (columns) and HIV RNA-specific PolyA ● ● ● ● ●● ● ● ● ● 0.1 Fold Change ● ● ●● ● ● ● ●● ● ●● ●● ● ● ● ●●● ● ● ●● sampleID NFKB1 CDKN1A ELL HTATSF1 CCR7 CDK13 BST2/Tetherin CTLA4 HDAC5 PAF − 1 APOBEC3G ATF − 3 BCL6 IFI16 CREBBP/CBP RIG − I/DDX58 beta7 integrin/ITGB7 G9a/EHMT2 PRDM1/Blimp − 1 TRIM5 NFATC1 TNFRSF8 CD38 CDK9 GATA3 FAS Sp1 PTB/PTBP1 HLA − DR alpha LCK TCF7 CCNL2/cyclin L2 CXCR4 STAT1 SUV39H1 EP300 OX40/TNFRSF4 MDA5/IFIH1 p53 CCNT1 TBX21/t − bet YY1 ZNF431 BCL11B/CTIP2 CD69 STING/TMEM173 KAT2B/PCAF CD44 EZH2 CEBPB MATR3 C − GAS/MB21D1 CD4 GAPDH CASP3 CDK7 EGR1 POLR2A TCRA CD28 BCL2 SAMHD1 FoxP3 TRBP/TARBP RPL13a SF2/ASF (SRSF1) PRMT6 IFNA1 CDK11a PPIA CCR5 CD3 delta Ki67/MKI67 SLFN11 IRF9 NELFA/WHS2 NFKBIA CD25/IL2RA PAPOLA PSIP1 TGFB1 PBAF/SMARCA4 SF3B2 Greene’s model ●● ●● ● ● ● ● ● ●● ●● ●●● ● ● ● ●●●●●● ● ● ● ● ● ● ● Latent (HIV+) HIV- (HIV-exposed) CDK13 0.001 -1.266 29 6 0.022 (rows). PolyA is grouped by donor (Donor 11 and 13). Color scale (right) ● ●● ● ● ● 0 ●● ● ● ● ● ●● ● ● ● ●● 0 ●●● ●● ●● ● ●● ● ● ● Dual Reporter ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● distinguish latent HIV+ cells HIV- (HIV-exposed) UI (HIV-unexposed) CDK13 7.3E-05 1.795 6 30 0.005 indicates Spearman r values. UMAP_2 ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● HIV-GKO ● ● ● ● ●● ● −0.1 −2 ●● ●● ● ● ● ● ● Virus model ● ●● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ●●●● ●● ●● ●● ●● ● ●●●● vs. uninfected cells Incompetent ● ●● ● −0.2 ● ● ● ●●● −4 ●● ● −2 −1 0 1 2 −2 −1 0 1 2 −0.3 Resting Cell Model (Tonsil CD4) Assay Panel UMAP_1 Table 1. Cellular and HIV targets in single-cell multiplex qPCR panel Upregulation of transcriptional factors, immune checkpoint markers, T cell phenotypic/function Fig. 2 (A) Schematic of Wild Type (WT) Virus model. Sorted single cells from the WT Virus model populations were analyzed for HIV expression by a qPCR-based pre-screen Resting Cell Model: followed by a multiplex qPCR for 96 HIV and cellular targets (Biomark HD platform)[Table 1]. Single-cell data generated are represented by (B) heat map to demonstrate cell- associated genes in productive tonsillar CD4 T cells compared to latent (mCherry-) and HIV-unexposed to-cell variation in levels of each HIV target (rows), where each vertical line represents a single cell, (C) UMAP projections with/without HIV targets, and (D) correlation matrix Blood CD4 T cells: cells●GTD1SmCh+●GTD1SmCh−●GTD1mCh−●GTD1UI●GTD1mCh+ showing degree of correlation between expression of each cellular gene (columns) and HIV RNA-specific target (rows) (n=2 donors). Color scale (right) indicates Spearman r ●GTD3SmCh+●GTD3SmCh−●GTD3mCh−●GTD3UI values. • CDK13 is downregulated in 35 Table 2. Differentially expressed genes between WT Virus primary cell model populations sampleID sampleID All Genes No HIV Genes Donor 1 Productive (mCh+)[Stim.] GTD1SmCh+ ●● ● 30 Donor 1 Productive (mCh+)[Stim.] ● ●●● ● Long LTR 2 ●● ● ●●● ● Donor 3 Productive (mCh+)[Stim.] Ave. Log Fold GTD3SmCh+ ● ● ● ●● ● latent cells compared to A Donor 3 Productive (mCh+)[Stim.] B ●● ● ● Group1 Group2 gene p_val N1 N2 FDR ● 25 ●● ● ● ●● Donor 1 Productive (mCh+) Change GTD1mCh+Donor 1 Productive (mCh+) ● ● ● ● Gag 1 ● ● ● ● ● ●● ● ● ● ●● ● ● GTD1SmCh− ● ●● ● ● ● ● ●● Donor 1 Latent (mCh-) [Stim.] Latent (HIV+) HIV- (HIV-exposed) BCL6 0.001 1.598 102 15 0.016 20 Donor 1 Latent (mCh-) [Stim.] ● ● ● ● ● ● ● ● ● ● ● Gag (IPDA) GTD3SmCh− ● ● ● ● ● ● ● ●● uninfected populations Donor 3 Latent (mCh-) [Stim.] ● ● ● ● ● Donor 3 Latent (mCh-) [Stim.] 0 ● ● ●● ● Latent (HIV+) UI (HIV unexposed) HLA-DR alpha 0.004 1.319 102 37 0.036 ●● ●● ● ● ●● 15 GTD1mCh− ●● ● ● ● ●●● ● ● ● Donor 1 Latent (mCh-) UMAP_2 ● ● ● ● ● ● ● ● ● ● Donor 1 Latent (mCh-) Latent (HIV+) UI (HIV unexposed) STAT1 2.19E-04 -0.050* 102 37 0.002 Pol ● ● ● ●● ●● 10 GTD3mChDonor 3 Latent (mCh− -) −1 ● ●● ● ● ●● ● ● ● Donor 3 Latent (mCh-) *Median difference >1 CT ● ● ● ● ● Env (IPDA) GTD1UIDonor 1 UI ● ● ● ●● ●● ● ●●● ● 5 ●● ●● ●● Donor 1 UI GTD3UI −2 ●●● ● ●● Donor 3 UI ● ● ● Nef ● Donor 3 UI Tonsil CD4 T cells: 0 −2.5 0.0 2.5 −4 −2 0 2 UMAP_1 Dual-Reporter Virus Model PolyA • Latent cells are distinguished Downregulation of CDK7, CEBPb, and Cyclin L2 distinguish latent cells from uninfected Tat−Rev 1 PolyA sampleID C Tat−Rev populations from productive and HIV- GTD1SmCh+,GTD3SmCh+Productive (mCh+)[Stim.] PolyA GTD1mCh+Productive (mCh+) Tat−Rev 0.5 GTD1SmChLatent (mCh−,GTD3SmCh-) [Stim.] − PolyA unexposed cells by * Tat−Rev GTD1mChLatent (mCh−,GTD3mCh-) − PolyA 0 Tat−Rev A B sampleID IFNA1 SF3B2 SUV39H1 BCL11B/CTIP2 STING/TMEM173 Sp1 TRIM5 MDA5/IFIH1 CDKN1A RIG − I/DDX58 G9a/EHMT2 IFI16 CDK11a TBX21/t − bet BST2/Tetherin ELL EGR1 CDK7 CASP3 PRMT6 APOBEC3G EZH2 PAPOLA BCL2 NFATC1 TGFB1 TRBP/TARBP MATR3 NFKB1 CD25/IL2RA CEBPB HTATSF1 OX40/TNFRSF4 TCRA p53 PSIP1 CCNT1 CDK9 YY1 CD44 TCF7 GAPDH PTB/PTBP1 BCL6 PPIA SF2/ASF (SRSF1) PAF − 1 SAMHD1 CTLA4 EP300 NFKBIA NELFA/WHS2 POLR2A HDAC5 CXCR4 IRF9 C − GAS/MB21D1 FAS KAT2B/PCAF CCNL2/cyclin L2 CDK13 HLA − DR alpha CD38 PBAF/SMARCA4 CREBBP/CBP LCK FoxP3 SLFN11 ATF − 3 PD − 1 (PDCD1) CD69 CD28 PRDM1/Blimp − 1 CD103/ITGAE RPL13a beta7 integrin/ITGB7 RORC CCR7 CD3 delta GATA3 Ki67/MKI67 FosB TNFRSF8 STAT1 CCR5 CD4 Donor 1 Productive differential expression of −0.5 Donor 2 Productive Donor 1 Latent transcriptional factors, Donor 2 Latent Fig. 5. Single-cell data generated (tonsil CD4 T cells) are represented by (A) heat map for HIV transcript expression (B) UMAP projections with/without HIV targets, and (C) Donor 1 Dbl Negative immune checkpoint markers, correlation matrix showing degree of correlation between expression of each cellular gene (columns) and HIV RNA-specific PolyA (rows). Color scale (right) indicates Spearman Donor 2 Dbl Negative r values. Donor 1 UI and T cell phenotypic/ Donor 2 UI Table 5. Differentially expressed genes between Resting Cell Model (tonsil CD4 T) populations function associated genes Group1 Group2 Differentially expressed genes (FDR p<0.05) BCL6↑, CD28↑, CD3 delta↑, CD4↑, CTLA4↑, G9a↑, GAPDH↑, LCK↑, PD-1↑, PPIA↑, PSIP1↑, SF2/ASF↑, TCRA↑, YY1↑; LongLTR↑, Productive (mCh+ ) Latent (mCh-) Gag↑, Gag (IPDA)↑, Pol↑, Env (IPDA)↑, Nef↑, Tat-Rev↑ Productive (mCh+ ) Stimulated Latent (mCh-) LCK↑; Gag↑ BCL6↑, CASP3↑, CD38↑, CD4↑, CD44↑, GAPDH↑, HTATSF1↑, LCK↑, MATR3↑, NFKB1↑, NFKBIA↑, PD-1↑, PPIA↑, PSIP1↑, SP1↑, Productive (mCh+ ) UI (unexposed/uninfected) TBX21↑, TCRA↑, YY1↑; LongLTR↑, Gag↑, Gag (IPDA)↑, Pol↑, Env (IPDA)↑, Nef↑, PolyA↑, Tat-Rev↑ Latent (mCh-) UI (unexposed/uninfected) Long LTR↑, Gag↑, Gag (IPDA) ↑, Pol↑, Env (IPDA) ↑ Stimulated mCh- (Latent) UI (unexposed/uninfected) IFI16↑, STAT1↑, TBX21↑; Long LTR↑, Gag↑, Gag (IPDA) ↑, Pol↑ Env (IPDA) ↑ Stimulated Productive (mCh+ ) BST2↑, C-GAS↑, CCR7↑, CD25↑, EGR1↑, FAS↑, HLA-DR↑, IFI16↑, IRF9↑, MDA5↑, OX40↑, PD-1↓, PRDM1↑, STAT1↑, STING↑, Productive (mCh+ ) TCF7↓ Stimulated Productive (mCh+ ) C D Stimulated Latent (mCh-) CD25↑, CD3 delta↑, STING↑; LongLTR↑, Gag↑, Gag (IPDA)↑, Pol↑, Env (IPDA)↑, Tat-Rev↑ ●Donor 1 Productive●Donor 1 Latent●Donor 1 Dbl Negative●Donor 1 UI ●Donor 2 Productive●Donor 2 Latent●Donor 2 Dbl Negative●Donor 2 UI * * * * * * * * * * * * * * * * * * * * * * * * PolyA sampleID * * * * * * * * * * * * * * * * * * * * * * * * * * * * Tat−Rev 0.6 VD1Pro,VD2Pro PolyA All Genes No HIV Genes Tat−Rev VD1Lat,VD2Lat 0.4 ● ● PolyA VD1DbN,VD2DbN ● Tat−Rev Discussion & Conclusions ● ●● ●● ● ● sampleID RPL13a CASP3 STING/TMEM173 CREBBP/CBP PAF − 1 C − GAS/MB21D1 NELFA/WHS2 HTATSF1 PAPOLA BCL2 SF3B2 CXCR4 CD28 CD44 GAPDH IFI16 CD25/IL2RA CTLA4 IFNG CDKN1A KAT2B/PCAF MDA5/IFIH1 PRDM1/Blimp − 1 PD − 1 (PDCD1) RORC ZNF431 BCL6 NFKBIA FAS CD69 OX40/TNFRSF4 EP300 Sp1 TCRA CDK7 CCNT1 MATR3 PPIA SUV39H1 BST2/Tetherin SF2/ASF (SRSF1) TGFB1 HLA − DR alpha LCK CD4 TBX21/t − bet CDK9 IFNA1 CD3 delta NFKB1 POLR2A YY1 PRMT6 Ki67/MKI67 PSIP1 G9a/EHMT2 IRF9 PBAF/SMARCA4 RIG − I/DDX58 ATF − 3 NFATC1 GATA3 CCR7 HDAC5 CCNL2/cyclin L2 ELL CDK13 TNFRSF8 CD38 STAT1 FoxP3 TRIM5 EGR1 beta7 integrin/ITGB7 CCR5 TCF7 CEBPB BCL11B/CTIP2 SAMHD1 PTB/PTBP1 SLFN11 EZH2 TRBP/TARBP CDK11a p53 ●●● ● ● ● ●● ● ● 2 ●● ● ● ● ●● ●● 0.2 ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ● ● ●●●●● ● ● ● ● ● ● 0 ● ●● ● ●● ● ● ● ● ● ●●● 1 ● ● ●● ● ● ● ●● 1. Multiple HIV targets distinguished uninfected, latently-infected, and productively-infected cells in all 5. Resting (blood) CD4 T cell model: Lower CDK13 expression in latent CD4 T cells relative to ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −0.2 ●● ● ●●●● ● ● ● ● ● ● ● ● ● 12 0 ● ● ● ● ● ● ●● ●● ● primary cell models uninfected (HIV-exposed) cells may be associated with block to HIV multiple-splicing observed UMAP ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● Fig. 1. Schematic of HIV targets in multiplex qPCR panel and the HIV RNA/DNA regions that they target. Included ● ● ● ● ●●● ●● ● ● 11 ● ● ● ● ● ●● ● ● ● ● ● ● ● 5 ●● ● ● ● ● ●● ● ● ● ● ● ●● ●● • Our bulk cell data suggest that a primary block to HIV transcription in latent cells in this model occurs at −1 ● ● ● ●● ● ●●● ● ●● ●●● ● ● 2. Each primary cell model differed in the cellular factors that distinguished populations, with some are two additional assays that distinguish intact proviral DNA (‘Gag [IPDA]’ and ‘Env [IPDA]’), that can also ●●●● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● Fig. 3(A) Schematic of Dual-Reporter Virus model. Single-cell data generated are represented by (B) heat map to ● ● ● ●● ● ●● ● ●● ●● ●●● ● ● ●● ● ●●● ●● multiple-splicing ● ● ● ● ● detect HIV RNA. ●● ● ●● ●●● ● ● demonstrate cell-to-cell variation in levels of each HIV target (rows), where each vertical line represents a single cell, −2 ●●●● ● ●● ●● differences between donors ● (C) UMAP projections with/without HIV targets, and (D) correlation matrix showing degree of correlation between 6. Resting cell (tonsil) CD4 T cell model: Upregulation of multiple transcriptional factors, immune −2 0 2 −3 −2 −1 0 1 2 3. Wild Type Virus model: Upregulation of BCL6 in latent cells relative to uninfected (HIV-exposed) UMAP expression of each cellular gene (columns) and HIV RNA-specific target (rows). HIV targets are grouped by population checkpoint markers, and interferon stimulated genes distinguish productively-infected cells from References cells and upregulation of HLA-DR in latent cells relative to uninfected (HIV-unexposed) cells may 1. Macedo et al. AIDS Human and Retroviruses 34(9):769-777 (2018) 2. Battivelli et al., eLife 7:e34655 (2018) 3. Lassen (n=2 donors). Color scale (right) indicates Spearman r values. latent and uninfected cells. et al., PLoS One 7(1): e30176.(2012) 4. Doitsh et al., Cell 143(5) :789-801 (2010). 5. Bruner et al., Nature be associated with T cell activation6 and inhibition of antiviral defenses7 566(7742):120-125 (2019) 6. Cai et al., J Virol. 93(2). pii: e01073-18 (2019) 7. Amet et al., J Leukoc Biol. 102(2): 527– • Nef and Vpu reportedly counteract STAT1 phosphorylation to also evade antiviral defenses8 7. Though each primary cell model differs in the virus used and methods employed to establish 536 (2017) 8. Nguyen et al., Innate Immun. 24(8): 490–500.(2018) 9. Kim et al., EMBO J. 9;25(15):3596–3604 (2006) Table 3. Differentially expressed genes between Dual Reporter Virus primary cell model populations 10. Mameli et al., J Gen Virol. 88(Pt 2):631-40 (2007) 11. Moron-Lopez et al., Poster: WEPEA041, 10th International Group1 Group2 Differentially expressed genes (FDR p<0.05) 4. Dual Reporter Virus model: Downregulation of CDK7 and CEBPb in latent cells relative to latent infection, our data suggest that these models could be useful for identifying molecular AIDS Society Conference (IAS) 2019, Mexico City, Mexico. 12. Berro et al., J Virol. 82(14): 7155–7166 (2008) BCL11B/CTIP2↓, BCL2↓, CASP3↓, EGR1↑, EZH2↓, GAPDH↓, Ki67↓, p53↓, PBAF↓, TNFRSF8↓; LongLTR↑, Gag↑, Pol↑, Env(IPDA),↑PolyA↑, uninfected populations may contribute to transcriptional silencing at initiation9,10 signatures of HIV latency in vivo. Productive Latent Tat-Rev↑ Acknowledgments 9 ATF3↓, BCL2↓, beta7 integrin↑, C-GAS↓, CCNL2↓, CD44↓, CDK7↓, CEBPB↓, FoxP3↓, MDA5↓, NFKBIA↓, OX40↓, p53↓, PAPOLA↓, PBAF↓, • CDK7 phosphorylates the C-terminal domain of RNA polymerase II to mediate HIV transcription initiation This project was supported by the NIDDK (R01DK108349-01 to SY), NIAID (1R01AI132128 to SY), 11 Productive Uninfected PPIA↓, PRMT6↓, PSIP1↓, RPL13A↓, TRBP↓, YY1↓; LongLTR↑, Gag↑, Gag (IPDA)↑, Env (IPDA)↑, Tat-Rev↑ • Our bulk cell data suggest that a primary block to HIV transcription in latent cells relative to productive the amfAR Institute for HIV Cure Research (109301). ST is supported by a CFAR Mentored Scientist Latent Uninfected CCNL2↓, CDK7↓, CEBPB↓; LongLTR↑, Gag↑, Gag (IPDA)↑, Pol↑, Env (IPDA)↑, PolyA↑ cells occurs at transcriptional initiation in HIV Award (Grant# P30 AI027763, Award # A120163, PI: Paul Volberding). We thank Fluidigm specialists Drs. Stacey Meyers, Katy Richards-Hrdlicka and Sam Lasse for their technical assistance. Double Negative Uninfected beta7 integrin↑, CD44↓, CEBPB↓, OX40↓; Long LTR↑, Gag↑, Pol↑, PolyA↑