dahabieh et al. 2013 1 a double-fluorescent hiv-1 reporter shows

Dahabieh et al. 2013

1

A double-fluorescent HIV-1 reporter shows that the majority of integrated 1 HIV-1 is latent shortly after infection. 2 3 Running Title: Double-labeled HIV-1 model 4 5 Matthew S. Dahabieh1, Marcel Ooms2, *, Viviana Simon2, 3, Ivan Sadowski1, * 6 7 1 Department of Biochemistry and Molecular Biology; University of British 8 Columbia; Vancouver, BC, V6T1Z3; Canada 9 2 Department of Microbiology, The Global Health and Emerging Pathogens 10 Institute; Icahn School of Medicine at Mount Sinai; New York, NY, 10029; USA 11 3 Division of Infectious Diseases, Department of Medicine, Icahn School of 12 Medicine at Mount Sinai; New York, NY, 10029; USA 13 14 Abstract: 215 words 15 Text: 7880 words 16 Figures: 6 17 18 * Corresponding Authors: 19 Dr. Ivan Sadowski, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T1Z3 20 Phone: 6048224524; Fax: 6048225227; email: [email protected] 21 22 Dr. Marcel Ooms, One Gustave Levy Place, Box 1124, New York, NY, USA 23 10029 24 Phone: 2122418388; Fax: 2125343240; email: [email protected] 25 26 Keywords: HIV-1, latency, LTR, CMV, subtype, promoter, eGFP, mCherry, 27 double-label, silent-infection28

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.03478-12 JVI Accepts, published online ahead of print on 13 February 2013

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Abstract 29 30 HIV-1 latency poses a major barrier to viral eradication. Canonically, 31 latency is thought to arise from progressive epigenetic silencing of active 32 infections. However, little is known about when and how LTR-silent infections 33 arise since the majority of the current latency models cannot differentiate 34 between initial (LTR-silent) and secondary latency (progressive silencing). 35 In this study, we constructed and characterized a novel, double-labeled 36 HIV-1 vector (Red-Green-HIV-1, RGH) that allows for detection of infected cells 37 independently of LTR activity. Infection of Jurkat T cells and other cell lines with 38 RGH suggests that the majority of integrated proviruses were LTR-silent early 39 post infection. Furthermore, the LTR-silent infections were transcriptionally 40 competent, as the proviruses could be reactivated by a variety of T cell signaling 41 agonists. Moreover, we used the double-labeled vector system to compare LTRs 42 from seven different subtypes with respect to LTR silencing and re-activation. 43 These experiments indicated that subtypes D and F LTRs were more sensitive to 44 silencing, whereas subtype AE LTR was largely insensitive. Lastly, infection of 45 activated human primary CD4+ T cells yielded LTR-silent as well as productive 46 infections. 47 Taken together, our data, generated using the newly developed RGH 48 vector as a sensitive tool to analyze HIV-1 latency on a single cell level, shows 49 show that the majority of HIV-1 infections are latent early post infection. 50 51

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Introduction 52 Upon infection of target cells, HIV-1 integrates into the host genome and 53

the subsequent transcription of the provirus by the cellular machinery leads to an 54 accumulation of genomic and spliced viral RNAs. However, the HIV-1 LTR can 55 be transcriptionally inactive and, as a result, no viral proteins are produced. 56 Transcriptionally inactive, latent HIV-1 proviruses can form as a result of multiple 57 and progressive epigenetic mechanisms that silence otherwise productive 58 infections (reviewed in (10, 37). These mechanisms include, but are not limited 59 to: a) host cell activation state and transcription factor pools, b) HIV-1 LTR 60 chromatin structure and modifications (e.g., nucleosome positioning, histone tail 61 modification, DNA methylation), c) proviral genomic location (e.g., host chromatin 62 environment, provirus orientation relative to host genes), d) threshold levels of 63 the viral transactivator Tat, and e) upstream control of HIV-1 activation by a 64 currently unknown protein kinase (52). Cumulatively, these molecular 65 mechanisms have lead to the prevailing view that HIV-1 latency is largely the 66 product of progressive epigenetic silencing of active infections. 67

Post-integration HIV-1 latency is one of the main obstacles to viral 68 eradication, since latently infected cells are not detected by the immune system 69 and are not affected by highly active anti-retroviral therapy (HAART) (19, 40, 44). 70 The latent reservoir is formed within days of initial infection (4, 11, 12, 53), and is 71 primarily composed of long lived resting memory CD4+ T-cells, although other 72 cell types may exist (10). Latency makes lifelong HAART a requirement for HIV-1 73 infected individuals (9), as cessation of therapy results in viral rebound within 74 weeks (25). 75

Eradication of HIV-1 requires novel pharmacotherapies directed at 76 modulating the latent reservoir. Such strategies will necessitate compounds that 77 are able to potently and specifically induce latent HIV-1 expression from the viral 78 long terminal repeat (LTR) promoter in a broad range of epigenetic environments 79 and cellular states. To date, efforts to purge the latent reservoir have focused on 80 the usage of histone deacetylase inhibitors (HDACi) to induce LTR transcription. 81 While able to induce HIV-1 expression ex vivo, early efforts with these drugs 82

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showed little impact on the latent reservoir in vivo (1, 41, 45). A recent study 83 demonstrated, however, the induction of cell-associated HIV-1 RNA in patients 84 treated with the HDACi suberoylanilide hydroxamic acid (SAHA) (3). A more 85 comprehensive understanding of the processes determining the establishment of 86 HIV-1 latency is essential to discovering efficacious new therapies. 87

Several model systems have been developed to study latency in cell 88 culture, since studies in vivo are complicated because latently infected cells from 89 patients are extremely rare (less than ~1x106 per patient) and phenotypically 90 indistinguishable from uninfected cells (44). Typically, HIV-1 latency models 91 utilize a single marker of active viral infection, such as an LTR driven fluorescent 92 protein like green fluorescent protein (GFP), followed by selection and long-term 93 cell culture to establish latency (reviewed in (13, 24, 36). However, this design 94 inherently limits the ability to study early latency establishment because of the 95 absence of a positive marker for latent infection. Additionally, the cell selection 96 steps employed may polarize the model towards specific latent populations i.e. 97 either initial or secondary latency. Some evidence suggesting that ‘silent 98 infections’ may play a role in HIV latency (17); however, the frequency and 99 regulation of these infections remains largely unknown. 100

In order to better study HIV-1 early latency establishment, we constructed 101 a ‘double-labeled’, single-cycle HIV-1 vector suitable for studying HIV-1 102 transcription at the single cell level (Red-Green HIV-1, RGH). We show that in 103 infected Jurkat cells, a substantial proportion of infections (approximately 65%) 104 are LTR-silent very early after infection. This LTR-silent population is 105 transcriptionally competent and can be re-activated by several HIV-1 activating 106 compounds. In addition, we show that the frequency of early silent infections is 107 variable with respect to specific HIV-1 subtype LTRs. Finally, we show that both 108 LTR-silent and productive infections are also represented in activated primary 109 human CD4+ T cells. In summary, the series of RGH vectors described here 110 provides a new model system to effectively study different aspects of HIV-1 111 latency at the single cell level without the need for selection steps or extensive 112 culturing. 113 114

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Materials and Methods 115 Construction of a panel of Red-Green-HIV-1 (RGH) vectors 116

To construct the RGH molecular clone, an ApaI/BssHII fragment 117 containing gag-enhanced Green Fluorescent Protein (eGFP) was cloned from 118 the Gag-iGFP NL4-3 clone (27) into pLAI (38) to create pLAI-Gag-iGFP. Of note, 119 the Envelope open reading frame was disrupted by the introduction of a 120 frameshift at position 7136 by digestion with KpnI, blunting, and re-ligation. The 121 CMV-mCherry (43) cassette was PCR amplified from pcDNA3.1+-mCherry and 122 cloned into pLAI-Gag-iGFP using the BlpI/XhoI sites to create the final double-123 labeled construct. The ΔU3 RGH clone was created by cloning a ΔU3 linker from 124 pTY-EFeGFP (7, 15, 29, 54) into the KpnI/SacI sites of the 3’ LTR in the double-125 labeled clone. The ΔCMV RGH clone was created by removing a BsmBI/PmeI 126 fragment containing the CMV promoter from the double-labeled construct. 127

The Integrase mutant D116A was created by two-step PCR mediated site 128 directed mutagenesis and cloning the final amplicon into the ApaI/SalI sites of the 129 RGH construct. The group M subtype promoters (N = 7; B, A, C, D, AE, F, G) 130 were cloned into the RGH construct by transferring a HindIII/XhoI fragment from 131 subtype constructs (30) to the 3’ LTR of the RGH clone. 132

133 Cell culture, virion production, and transduction 134

Jurkat E6-1 (51), SupT1 (49), U937 (ATCC), HeLa (ATCC), and HEK293T 135 (ATCC) cells were cultured according to standard conditions as previously 136 described (16). 137

VSV-G pseudotyped viral stocks were created by transfecting HEK293T 138 cells with viral molecular clones and pHEF-VSVg (7) in a 10:1 ratio as previously 139 described (16). Purified, concentrated viral stocks were prepared by 140 centrifugation of clarified supernatants from transfected HEK293T cells through a 141 6% Iodixanol/Optiprep (Sigma) cushion. Concentrated virions were resuspended 142 in complete media. 143

Jurkat E6-1 cells were infected by spinoculation as previously described 144 (35), except that 5x105 cells were spinoculated for 1.5 hours in 12 well plates in 1 145

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mL complete media supplemented with 4 μg/mL polybrene (500 x g, room 146 temperature). 25 μL of viral supernatant was added to the cells to yield an 147 average infection rate of 10%, thereby ensuring single copy integrations. Unless 148 otherwise indicated, infected cells were utilized for experiments at four days post 149 infection. 150 151 Primary T cell infections 152 Primary CD4+ T cells from three healthy donors HC-8537, HC-8536, and 153 HC-4142 were obtained from Sanguine Biosciences and thawed according to the 154 company’s recommendations. Cells were cultured for 48 hours in a humidified 37 155 °C, 5% CO2 atmosphere in RPMI 1640 + 10% (v/v) fetal bovine serum, 100 156 U/mL penicillin, 100 mg/mL streptomycin, 20 U/mL IL-2 (33). 2.5 x 106 cells from 157 each donor were either incubated with IL-2 alone (non-activated) or with IL-2 and 158 Phytohaemagglutinin (PHA-P, 2 μg/mL, activated) for 48 hours. 159 Activated and non-activated primary CD4+ T cells were spinoculated with 160 purified RGH viral stocks as described for Jurkat cells, except that 2.5x105 cells 161 were spinoculated in 24 well plates in 0.5 mL of complete media supplemented 162 with 2 μg/ml polybrene. 10 or 100 μL of 25x concentrated viral supernatant was 163 added to the activated or non-activated cells, respectively. For the remainder of 164 the experiment, cells were maintained in complete RPMI media supplemented 165 with IL-2 (20 U/mL) and media was replenished every two days. Cells were 166 analyzed by flow cytometry six days post infection. 167 168 Flow cytometry and drug treatments 169

Prior to FACS analysis, cells were fixed in 1% (v/v) formaldehyde for 10 170 minutes at room temperature. 10000-20000 live cells events (as based on 171 FSC/SSC profiles) were analyzed on a Becton Dickinson LSRII flow cytometer 172 and the data was analyzed using FlowJo. Statistical tests (Student’s T-test) were 173 performed using R 2.15.1. 174

Cells were treated with various drugs for the times and durations indicated 175 in the individual experiments. Drugs were added at the listed concentrations to 176

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complete media. Unless otherwise stated, drugs were used at the following 177 concentrations: Raltegravir, 10 μM; TNFα, 10 ng/mL, SAHA, 1 μM (34); TSA, 50 178 ng/mL; PMA, 4 ng/mL; Ionomycin, 1 μM; 5-aza-dC, 5 μM. 179 180

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Results 181 Characterization of the double-labeled HIV-1 molecular clone RGH 182

We constructed a HIV-1 LAI molecular clone containing two fluorescent 183 markers to identify both silent and productive HIV-1 infections (Figure 1A). 184

The eGFP protein is flanked by HIV-1 protease cleavage sites and placed 185 in gag between matrix and capsid (27, 28). Each Gag protein contains eGFP 186 resulting in green virions, which upon fusion with the target cell, release eGFP 187 into the target cell (Figure 1B). Upon integration, Gag-eGFP expression is under 188 control of the HIV-1 promoter and may serve as a quantitative marker for HIV-1 189 LTR gene activity. Moreover, Gag-eGFP is a late gene product and, hence, is 190 likely indicative of HIV-1 virus production. Of note, low-level Gag expression in 191 primary cells can occur in the absence of virion production (37), suggesting that 192 certain Gag expression levels are required for efficient production of new virions. 193

The second reporter, inserted in the viral nef position, consists of a 194 cassette containing the CMV immediate-early promoter expressing mCherry 195 fluorescent protein (43). mCherry is constitutively expressed and allows for 196 detection of integrated virus regardless of the transcriptional state of the HIV-1 197 LTR. 198

The combination of the two fluorescent proteins under the HIV-1 LTR and 199 an internal CMV promoter (Red-Green-HIV-1, RGH) allows for quantitative 200 detection of active virus production (eGFP+ and mCherry+, yellow), but more 201 importantly, also detects non-productive HIV-1 (eGFP- and mCherry+, red, 202 Figure 1C). In addition, the HIV-1 envelope is inactivated, which prevents 203 spreading viral replication, super-infection, as well as envelope 204 induced/associated cell toxicity. The deletion in envelope also allows us to 205 circumvent the use of antiretroviral drugs in cultures, which would be required to 206 prevent multiple rounds of infections. 207

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Red-Green-HIV-1 identifies LTR-silent infections, which are established early 212 after infection and require integration 213

To test the double-labeled system (Figure 1), we produced VSV-G 214 pseudotyped RGH viral stocks in HEK293T cells and infected Jurkat T-cells at 215 low MOI (~0.2). At one-day post infection distinct green, yellow and red 216 populations were observed, but only the yellow and red populations, representing 217 integrated proviruses, persisted through later time points (Figure 2A and B). The 218 number of LTR-silent infected cells (red) consistently outnumbered the double 219 positive HIV-1 producing cells (yellow), indicating that the majority of HIV-1 220 infections are silent early post infection. Interestingly, day one post infection was 221 characterized by a high number of GFP+ mCherry- cells, which were no longer 222 observed at later time points (Figure 2A and 2B). This transient eGFP+ cell 223 population is the result of the Gag-eGFP fusion proteins incorporated in the virus 224 used to infect the cells, and hence, rapidly disappears after infection (Figures 1B 225 and references 24, 25). Calculation of the ratio of red to yellow cells for all the 226 time points of the infection (Figure 2C), showed a transient increase of red cells 227 (mCherry+) over yellow cells (eGFP+/mCherry+) two days post infection. This 228 spike could reflect delayed late Gag-eGFP expression as compared to more 229 rapid mCherry accumulation and/or the observed drop in mCherry+ cells could 230 result from CMV promoter silencing. Importantly however, the red-yellow cell 231 ratio stabilized approximately four to five days after infection (Figure 2C). 232 Non-integrated 2-LTR circles have been described to express several 233 HIV-1 non-structural proteins, such as Tat, Rev and Nef (22, 23). To exclude that 234 the higher mCherry signal results from expression from 2-LTR circles, we 235 infected Jurkat cells with RGH virus in the presence of integrase inhibitor 236 raltegravir (Figure 2D) or with an RGH virus bearing a D116A inactivating 237 mutation in Integrase (Figure 2E, reference 4). Both raltegravir and the Integrase 238 mutant prevented the formation of red and yellow cells (Figure 2D and 2E), 239 indicating that the red and yellow signals observed in RGH infected cells 240 represent integrated proviruses and not 2-LTR circles. The eGFP expression at 241 day one post-infection, in contrast, was not affected by raltegravir or the 242

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integrase mutation underscoring that the initially observed eGFP peak is caused 243 by the virally packaged eGFP and not from newly expressed eGFP in the target 244 cells. Thus, the RGH vector can efficiently discriminate between active and silent 245 HIV-1 expression. Moreover, infection of Jurkat cells with RGH suggests that the 246 majority of infections are latent early post infection. 247 248 Identification of LTR-silent infections is dependent on the presence of both LTR 249 and CMV promoters 250

Infection of Jurkat cells with RGH indicates that the majority of integrated 251 proviruses are LTR-silent four to five days post infection (Figure 2). To 252 characterize the effect of both the LTR and CMV promoters on this infection 253 profile, we constructed a number of control RGH variant viruses that lack either 254 CMV-mCherry, the CMV promoter, or the HIV-1 promoter (Figure 3A). As 255 expected, CMV-mCherry deleted virus only produced green cells (Figure 3B). 256 RGH lacking the U3 promoter region of the 3’ LTR results in an integrated virus 257 devoid of the 5’ HIV-1 promoter, and results in red only cells (Figure 3B). Finally, 258 the deletion of the CMV promoter in RGH renders mCherry dependent on the 259 HIV-1 promoter and results in the simultaneous expression of eGFP and 260 mCherry, thereby producing exclusively eGFP/mCherry double positive (‘yellow’) 261 cells (Figure 3B). 262

Inspection of Jurkat cells infected with wild-type RGH by fluorescence 263 microscopy showed two distinct cell populations: cells expressing both 264 fluorescent proteins (eGFP+/mCherry+, ‘yellow’) and cells expressing only 265 mCherry (eGFP-/mCherry+, ‘red’) (Figure 3C). Taken together, these data 266 confirm that having a virus with independent expression of both fluorescent 267 reporters is necessary to detect early LTR-silent infections. 268

We next wanted to determine if the LTR-silent infections were specific for 269 Jurkat T-cells or a bona fide property of HIV-1 infection. To do this, we infected 270 different human cell lines such as the T-cell line SupT1, the monocyte line U937, 271 and the non-immune cell lines HEK293T and HeLa, with the RGH viral stocks. As 272 shown in Figure 3D, cell lines differed in susceptibility to infection (between five 273

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and 37% infection rate, Figure 3D) but most infected cell lines yielded at least 274 twice as many eGFP-/mCherry+ (‘red’) cells as eGFP+/mCherry+ (‘yellow’) 275 double positive cells (SupT1: red only: 24.5%, yellow: 12.3%, ratio: 1.99; U937: 276 red only: 5.5%, yellow: 0.7%, ratio: 7.85; HEK293T: red only: 6.8%, yellow: 1.9%, 277 ratio: 3.58; HeLa red only: 2.8%, yellow: 2.5%, ratio: 1.12). These results indicate 278 that LTR-silent infections are common and evident in many different cell types 279 (Figure 3D). Collectively, these data suggest that the independent expression of 280 both fluorescent reporters is necessary to detect early LTR-silent infections, and 281 that these infections are a bona fide feature of HIV-1 infection. 282 283 Silent LTRs in infected cells are transcriptionally competent 284

The HIV-1 LTR contains binding sites for a multitude of transcription 285 factors (such as NFκB, NFAT, and AP-1) that act downstream of the T-cell 286 signaling pathway, and hence, can be activated by a variety of signaling agonists 287 (reviewed in (13, 32). For example, Tumor necrosis factor alpha (TNFα), and 288 phorbol 12-myristate 13-acetate (PMA) / Ionomycin (Iono) are both potent 289 activators of HIV-1 (TNFα in transformed T cells only) by stimulating the protein 290 kinase C (PKC), and MAP kinase / Calcineurin pathways, respectively (20, 21). 291 Additionally, general epigenetic modifying drugs like the HDAC inhibitors SAHA 292 and trichostatin A (TSA) cause hyperacetylation of LTR nucleosomes and 293 activation of LTR-driven transcription (2, 14). The DNA methyltransferase 294 inhibitor 5-aza-2′deoxycytidine (5-aza-dC) has been shown to activate HIV-1 295 transcription, although its effects depend on the model system used (18). 296 We next determined whether the majority of RGH infected Jurkat cells, 297 which are LTR-silent four days after infection (Figure 2), could be reactivated. 298 We treated RGH infected Jurkat cells four days post infection with TNFα, PMA, 299 Iono, PMA/Iono, 5-aza-dC, SAHA, TSA, TNFα/SAHA or DMSO (negative control) 300 for 24 hours followed by FACS (Figures 4A and 4B). TNFα, PMA, PMA/Iono, 301 SAHA, TSA and TNFα/SAHA activated silent LTRs as compared to the DMSO 302 control (approx. 3-fold decrease in red-yellow ratio, Figures 4A and 4B). 303 Interestingly, 5-aza-dC failed to activate silent LTRs (Figures 4A and 4B). It has 304

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been proposed that DNA methylation is a late mark in HIV-1 proviral silencing 305 and, thus, 5-aza-dC would not be expected to play a role at an early time point 306 after integration (32). Additionally, the combination of TNFα and SAHA did not 307 have an additive effect on LTR activation, suggesting that maximal effect had 308 been reached with either drug alone (Figures 4A and 4B). 309

Interestingly, we consistently noticed a decrease in the double negative 310 cell population upon re-activation of RGH (eGFP- mCherry-, lower left quadrant, 311 Figures 4A and 4B), which suggests that the double-negative cells may harbor 312 infected cells with inactivated LTR and CMV promoters. To better understand the 313 re-activation features of the different RGH infected cell populations, we sorted 314 RGH infected Jurkat cells into their constituent populations (3 days post-315 infection). Each of the three sorted cell populations as well as unsorted infected 316 Jurkat cells were activated with TNFα (Figures 4C and 4D). The pre-sorted 317 infected Jurkat cells showed the typical infection profile for both DMSO and TNFα 318 treatment with ‘red’ cells outnumbering the ‘yellow’ cells for DMSO and a reversal 319 of this distribution for TNFα (Figure 4D). TNFα treatment of the sorted double 320 negative cells resulted in both red and yellow cells (4.5% ‘red’, 8.2% ‘yellow’, red-321 yellow ratio: 0.55, Figure 4D), indicating that a portion of the “uninfected” cell 322 population, indeed, harbors fully competent proviruses with both promoters 323 silenced. The sorted mCherry single positive population remained predominantly 324 mCherry positive and showed an increase of yellow cells upon TNFα treatment 325 compared to the DMSO treatment (Figure 4D). The sorted double positive, yellow 326 cell population comprises primarily LTR-active proviruses, and TNFα activation 327 resulted in only a minor increase in yellow cells, thus confirming that the HIV-1 328 LTR is already fully activated in these cells (Figure 4D). 329 Taken together, these data indicate that LTR-silent RGH proviruses in 330 Jurkat cells are transcriptionally competent, and can be reactivated by a variety 331 of T-cell signaling agonists and HDAC inhibitors. 332 333 334 335

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Group M subtype LTRs differ in their sensitivity to latency 336 Given that the RGH vector can detect LTR-silent infections in Jurkat cells 337

(Figures 2, 3, and 4), we reasoned that the ratio of LTR-silent red cells to LTR-338 active yellow cells would likely be affected by intrinsic determinants within the 339 HIV-1 LTR, such as transcription factor binding sites. Therefore, we cloned 340 natural subtype LTR variants (30) into the RGH vector and assessed the effect of 341 LTR variation on silent-infections and reactivation potential. Jurkat cells were 342 infected with each of the RGH subtype LTR viruses (subtypes B, A, C, D, AE, F 343 and G, Figure 5A) at low MOI and treated with PMA/Iono or DMSO four days 344 post infection to activate LTR-silent RGH (Figures 5B and 5C). After PMA/Iono 345 activation, the red-yellow ratio dropped substantially (approx. 2-4 fold) for most 346 subtypes compared to control DMSO treatment. However, subtype AE exhibited 347 the lowest red-yellow ratio difference between DMSO and PMA/Iono treatment, 348 suggesting that the subtype AE promoter is constitutively expressed and, thus, 349 cannot be further activated by PMA/Iono (Figures 5C and 5D). Since the subtype 350 AE promoter only contains one canonical NFκB binding site (30, 50), limited 351 activation by the PMA/Iono treatment could be the underlying reason (Figures 352 5C and 5D). 353

Interestingly, the fold change in latency ratios is significantly higher for 354 subtype D and F LTRs suggesting that these promoters are more prone to 355 transcriptional silencing in Jurkat T cells (Figure 5D). Of note, for subtype AE the 356 fold difference in latency ratios between DMSO and PMO/Iono treatment is 357 substantially lower compared to B-LAI (Figure 5D). 358 Our results suggest that different subtypes display different levels of LTR 359 silencing. Subtype AE, contrary to the other subtypes, is less likely to become 360 silenced, which is in full agreement with previous reports (30, 50) while subtypes 361 D and F are more prone to silencing. Using the RGH Jurkat cell model, we 362 observed differences in the occurrence of LTR-silent infections between 363 subtypes, which recapitulated well the results observed in earlier studies using 364 different models to study HIV-1 latency. 365 366

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LTR-silent RGH infections occur in primary CD4+ T cells 367 The majority of RGH infections in Jurkat cells result in direct LTR-silencing 368 (Figures 2, 3, 4, and 5). Although Jurkat cells are widely used to study HIV-1 369 latency, they may not accurately represent the memory CD4+ T cells that make 370 up the latent HIV-1 reservoir in vivo. To study RGH infection in a more relevant 371 context, we infected human primary CD4+ T cells obtained from three healthy 372 donors. We spinoculated activated (IL-2+PHA treated) and non-activated (IL-2 373 alone) cells from the different donors with RGH and cultured the infected cells for 374 six days prior to flow cytometry analysis (Figure 6A and 6B). 375 The activated cells (IL-2+PHA) showed both red and yellow cells indicative 376 of successful infections. Red cells outnumbered yellow cells in all donors 377 (Figures 6A and 6C), demonstrating the presence of LTR-silent infections in 378 primary cells, similar to Jurkat cells (Figures 2, 3, 4, and 5). However, many cells 379 were eGFP+ positive only (Figure 6A, compare RGH infected to mock infected) 380 and microscopy inspection of the cells shortly after infection revealed the 381 presence of large intracellular eGFP aggregates that persisted for more than six 382 days (data not shown). This eGFP signal represents the initial, virally derived 383 eGFP signal that, contrary to Jurkat cells (Figure 2B), persists for a long time in 384 primary CD4+ T cells. 385 Despite using ten-fold more virus to infect non-activated CD4+ T cells (IL-386 2 alone), we could not detect red or yellow infected cells (Figure 6B), indicating 387 that the RGH virus is unable to productively infect non-activated cells. Of note, 388 non-activated CD4+ T cells are difficult to infect which may be overcome in some 389 models by using a very high MOI (reviewed in (24, 37). Despite the absence of 390 productive infections, we detected eGFP+ only cells similar to the levels 391 observed in activated cells from the same donor (Figure 6B). 392 Collectively, these data suggests that both LTR-silent and productive 393 infections occur in primary CD4+ T cells. 394 395

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Discussion 396 A better understanding of the dynamics of HIV-1 latency and the cellular 397

mechanisms controlling these processes is instrumental to developing HIV-1 398 eradication strategies. In this study, we construct and characterize a novel 399 double-labeled HIV-1 vector (Red-Green-HIV-1, RGH) (Figure 1), which reduces 400 the selection bias inherent to traditional single-label models of latency. 401 Additionally, this model allows for examination of several phases of HIV-1 latency 402 and reactivation including establishment, maintenance, and expression. By 403 incorporating the CMV-mCherry reporter, the RGH model can positively identify 404 latently infected cells rather than utilizing excluding selection steps, which is a 405 common denominator for many other latency systems. Moreover, utilization of 406 CMV-mCherry allows for the distinction of latently infected cells from uninfected 407 ones, thereby allowing transcriptome and proteome comparisons, without the 408 need for viral reactivation by T-cell activation. Reactivation likely destroys the 409 unique transcriptional properties of latently infected cells, thus preventing 410 meaningful comparisons of the viral and cellular states of both latently and 411 actively infected cells. Thus with the RGH vector, all populations of infected cells, 412 including those with initially suppressed LTRs, can be studied and quantified at 413 both the single-cell and population levels. Additionally, the RGH vector may also 414 be used to study canonical mechanisms of HIV-1 latency by continual passaging. 415 These mechanisms, which contribute to the progressive epigenetic silencing of 416 active infections, include both cis-acting factors (e.g. HIV-1 integration site and 417 chromatin environment) and trans- acting factors (e.g. cellular activation state 418 and transcription factor pools, reviewed in 10, 37). 419

The major advantage of the RGH vector system is the LTR-independent 420 marker of infection (mCherry), which requires a separate, HIV-1 independent 421 promoter to drive its expression. We choose the CMV promoter for this purpose 422 since it is widely used by the research community. Of note the CMV promoter 423 may also have its limitations, especially in primary cells (26, 39, 42). Activation of 424 RGH infected Jurkat cells (Figures 4A and 4B, whole cell population and Figure 425 4D, double negative sorted cells) revealed that the eGFP-/mCherry- cell 426

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population also contains LTR and CMV silent proviruses, which can be activated 427 by TNFα treatment (Figure 4). Inactive/silenced CMV promoters would, therefore, 428 underestimate the true infection rate in our system. Moreover, the CMV promoter 429 has been shown to be responsive to T cell activation signals and, consequently, 430 is expressed at much lower levels in non-activated cells (26, 39, 42). Thus, 431 alternative constitutive non-viral promoters like EF1α, PGK, or UbC may be 432 useful alternatives to the CMV promoter used in the current RGH vector. Despite 433 these CMV promoter associated potential limitations, we observed that the 434 majority of RGH CMV-mCherry+ infections were LTR-silent (Figures 2, 3 and 4) 435 in different cell lines (T cell lines: Jurkat, Sup-T1; myeloid, epithelial etc.). In this 436 light, utilizing a stronger mCherry promoter would lower the frequency of double-437 silent promoter proviruses thereby ensuring that the detectable cells (eGFP- and 438 mCherry+) better represent the silenced HIV-1 provirus. 439

Importantly, the RGH infection experiments suggest that transcriptionally 440 competent, LTR-silent proviruses are common in T cell lines, as well as other 441 transformed cell types (Figure 2). Moreover, we also observed LTR-silent 442 infections in primary CD4+ T cells, suggesting that RGH vector may also be 443 suitable for studying direct-silent infections in primary cells (Figure 6). However, 444 some shortcomings of the RGH vector became more apparent in primary CD4+ T 445 cell infections. First, a substantial and persistent eGFP+ only signal reflecting 446 eGFP labeled incoming particles, was present in both resting and activated CD4+ 447 T cells (Figure 6A and 6B). This was also observed for RGH infected Jurkat cells, 448 but this virally packaged eGFP signal disappeared rapidly in the Jurkat cells 449 (Figure 2). Apparently, the virally packaged eGFP is much more stable in primary 450 cells, possibly because the CD4+ T cells divide less and, thus, eGFP would not 451 be lost by dilution. Importantly, the presence of the eGFP+ only signal may 452 complicate the distinction of the red and yellow infected cell populations, as 453 virally derived eGFP may cause red cells to shift into the yellow gate, thereby 454 underestimating the LTR-silent population. In addition, some of the cells in the 455 eGFP+ population could be infected, with only the LTR transcriptionally active 456 (eGFP+ only). Indeed, the CMV promoter has been observed to be expressed 457

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with lower efficiency in some primary lymphocyte systems (6, 39, 47, 48). 458 Moreover, the low infectivity in CD4+ T cells is likely caused by the insertion of 459 eGFP into Gag, which was reported to have cell type specific effects on 460 replication (27). Future versions of the vector could address these CD4+ T cell 461 specific limitations by using other constitutive promoters driving mCherry (EF1α, 462 PGK, or UbC) and/or by replacing certain HIV-1 genes, such as Vif or Vpr with 463 eGFP, so that the fluorescent reporter would no longer be packaged into virions, 464 and may be less detrimental to viral infectivity. 465

Despite these shortcomings, the RGH vector data suggest that direct 466 silent-infections have been underappreciated so far and, thus, it is likely that 467 these cells contribute to HIV-1 latency. To our knowledge, only one other study 468 has identified silent-infection as a contributor to HIV-1 latency. In this study, 469 which used a singly-labeled vector to infect a cell line, silent infections were 470 correlated with NFκB levels in cells at the time of infection, with transcriptional 471 interference from host genes being the actual silencing mechanism (17). Further 472 studies are needed to definitely address whether control of latency by NFκB is 473 indeed causative, and if this mechanism applies to all sites of viral integration. 474

Differential mechanisms that lead to silent-infection or silencing from 475 canonically formed latency may require unique pharmaco-therapeutics to 476 reactivate latent proviruses. One of the most commonly proposed eradication 477 strategies, called “shock and kill”, would utilize small molecules to induce HIV-1 478 expression from latently infected cells, thus allowing induced cells to be cleared 479 by the immune system or killed by viral cytopathic effects (reviewed in (8). 480 However, any efficacious drug suitable to purge HIV-1 latency would have to 481 target both forms of latent infection, as excluding any part of the latent reservoir 482 would render such therapy less effective. Given its ability the minimize selection 483 bias and include all populations of infected cells, the RGH vector model will be 484 very useful tool to screen for latency modulating drugs that fulfill these criteria. 485

In conclusion, we generated and characterized a comprehensive panel of 486 single cycle vectors to study the very early events involved in establishment of 487 HIV-1 latency. Our results suggest that the proviral LTR is silenced immediately 488

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upon integration in a high proportion of infections. HIV-1 latency, thus, may not 489 simply the result of progressive epigenetic silencing of active infections but rather 490 represents a multi-faceted process that can form very early post integration and 491 in the absence of previous LTR activity. This underappreciated and previously 492 hard to measure population of initially latent cells may have profound 493 consequences for the establishment and treatment of latency. A better 494 understanding of the mechanisms controlling the fate of integrated proviruses will 495 inform on improved treatment strategies. 496 497

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Acknowledgements 498 We thank Andy Johnson and Justin Wong of the UBC Flow Cytometry 499

Facility for performing FACS, as well as assistance with flow cytometry. We also 500 gratefully acknowledge Ben Berkhout for providing the molecular clone pLAI and 501 HIV-1 subtype constructs, and Benjamin Chen for providing the molecular clone 502 Gag-iGFP. We thank Vicente Planelles for critical review of this manuscript and 503 helpful suggestions. We also thank Jacob Hodgson, Adam Chruscicki, Kevin 504 Eade, Benjamin Martin, and Nicolas Coutin for helpful discussion throughout this 505 project. 506

The following reagents were obtained through the AIDS Research and 507 Reference Reagent Program, Division of AIDS, NIAID, NIH: Jurkat Clone E6-1 508 from Dr. Arthur Weiss, Sup-T1 from Dr. James Hoxie, Raltegravir (Cat # 11680) 509 from Merck & Company, Inc, SAHA (Vorinostat), pHEF-VSVG from Dr. Lung-Ji 510 Chan, pTY-EFeGFP from Dr. Lung-Ji Chang, and Human rIL-2 from Dr. Maurice 511 Gately, Hoffmann - La Roche Inc. 512

This work was supported by Canadian Institute of Health Research (CIHR) 513 grants to I.S. (MOP-77807, HOP-120237), and NIH/NIAID grants to V.S. 514 (AI064001, AI089246, AI90935). M.S.D is supported by a CIHR fellowship (CGD-515 96495). 516 517

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Figure Legends 709 Figure 1. Schematic representation of the Red-Green-HIV-1 (RGH) vector. 710 A: HIV-1 B-LAI Δenv is labeled with eGFP as an in-frame gag fusion flanked by 711 HIV-1 protease cleavage sites (inverted triangles), and by a CMVIE-driven 712 mCherry cassette located in place of nef. 713 B: Schematic depiction of RGH infection of target cells and the resultant 714 fluorescent protein profiles over time. 715 C: Schematic depiction of HIV-1 RGH infected cell populations detected by 716 FACS. 717 718 Figure 2. LTR-silencing in RGH infected Jurkat cells occurs early and 719 requires integration. 720 A: Flow cytometry time course of RGH infected Jurkat cells. Plots shown are 721 representative of triplicate infections. 722 B: Plot of positive cells from each colored fraction (eGFP+ mCherry-, eGFP- 723 mCherry+, eGFP+ mCherry+) over the course of infection. Error bars represent 724 standard deviations of triplicate experiments. 725 C: Data from the experiment shown in panel B is enumerated as ratio of red to 726 yellow cells (proportion of eGFP- mCherry+ to eGFP+ mCherry+). Error bars 727 represent standard deviations of triplicate experiments. ‘ns’ non-significant, * 728 p<0.05 (Student’s T-test). 729 D: Jurkat cells were infected with RGH in the presence of the integrase inhibitor 730 raltegravir (10 μM). Cells were analyzed by flow cytometry over a period of seven 731 days. Error bars represent standard deviations of triplicate experiments. 732 E: Jurkat cells were infected as in panel D, except that an RGH variant bearing a 733 catalytically defective Integrase variant (D116A) was used. Additionally, 734 raltegravir was excluded. 735 736 737 738

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Figure 3. Identification of LTR-silent RGH infections is dependent on both 739 the LTR and CMV promoters, and is not specific for Jurkat T cells specific. 740 A: Schematic representation of RGH variant viruses. Gag-eGFP lacks the CMV-741 mCherry construct, while ΔU3 and ΔCMV both contain both fluorescent coding 742 sequences (eGFP and mCherry) but lack U3 in the 3’ LTR, and the CMV 743 promoter, respectively. Variants are otherwise isogenic. 744 B: Jurkat cells were infected with WT and variant RGH and analyzed by flow 745 cytometry four days post infection Plots shown are representative of triplicate 746 experiments. 747 C: Fluorescence microscopy (100x) of RGH infected Jurkat cells (4 days post 748 infection). eGFP- mCherry+ and eGFP+ mCherry+ cells are indicated in the 749 merge panel by red and yellow arrows, respectively. Images shown are 750 representative of triplicate experiments. 751 D: SupT1, U937, HEK293T, and HeLa cells were infected with RGH and 752 analyzed by flow cytometry four days post infection Plots are representative of 753 duplicate experiments. 754 755 Figure 4. Silent LTRs in RGH infected cells are transcriptionally competent. 756 A: RGH infected Jurkat cells (4 days post infection) were treated with DMSO, 757 TNFα, PMA, PMA/Iono, 5-aza-dC, SAHA, TSA, or TNFα/SAHA for 24 hours prior 758 to analysis by flow cytometry. Plots shown are representative of triplicate 759 experiments. 760 B: Data from panel A is enumerated as the red-yellow ratio of the infected 761 population. Error bars represent standard deviations of triplicate experiments. ‘ns’ 762 non-significant, ** p<0.01, *** p<0.001 (Student’s T-test). 763 C: RGH infected Jurkat cells were sorted to greater than 90% purity three days 764 post infection. The left panel depicts the Jurkat cells infected with RGH at day 3 765 post infection and the right panel shows the purity of the individual cell 766 populations after sorting (composite of three individual FACS plots). Cells were 767 left to recover for 24 hours prior to treatment with either DMSO or TNFα for a 768 further 24 hours, followed by analysis by flow cytometry. 769

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D: Data from the experimental outline in panel B. Cells were analyzed by flow 770 cytometry. Error bars represent standard deviations of duplicate experiments. ** 771 p<0.01, *** p<0.001 (Student’s T-test). 772 773 Figure 5. RGH recapitulates differences in latency observed between HIV-1 774 Group M subtypes. 775 A: Schematic representation of RGH variants containing promoter sequences 776 from the major group M viral subtypes. Viruses are isogenic except for a 209 bp 777 BseAI/AflII fragment containing the core promoter region of the LTR that 778 stretches from position -147 to +68. 779 B: Jurkat cells were infected with the subtype RGH variants and analyzed by flow 780 cytometry four days post infection Plots shown are representative of triplicate 781 experiments. C: Jurkat cells were infected with the subtype RGH variants. Four 782 days post infection cells were treated with either DMSO or PMA/Ionomycin for 24 783 hours, and then analyzed by flow cytometry. Error bars represent standard 784 deviations of triplicate experiments. 785 D: Data from panel C is enumerated as fold change between the PMA and 786 DMSO treatments. Error bars represent standard deviations of triplicate 787 experiments. ‘ns’ non-significant, *** p<0.001 (Student’s T-test). 788 789 Figure 6. LTR-silent RGH infections occur in primary CD4+ T cells. 790 A: Activated (IL-2 + PHA) primary CD4+ T cells from three healthy donors were 791 infected with purified RGH. Infected cells were cultured in IL-2 containing media 792 for six days prior to FACS analysis. 793 B: Non-activated (IL-2) primary CD4+ T cells from three healthy donors were 794 infected with ten-fold more virus than in panel A. Infected cells were cultured in 795 IL-2 containing media for six days prior to FACS analysis. 796 C: Data from panel A is displayed graphically with the ratio of red to yellow cells 797 indicated below. 798

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Figure 1

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102

103

104

0.24284.40 102 103 104 105

0

102

103

104

0.36882.30 102 103 104 105

0

102

103

104

0.10192.40 102 103 104 105

0

102

103

104

0.49979

LTR - eGFP

B

Red

1

1.5

2

ow

Rat

io

ns

****

Infection(Day 0)

Drug treatment

Yel

low

0

0.5

1

Red

-Yel

l

*** ****** **

FACS(Day 4)

g(Day 3)

100

eGFP - mCherry+ eGFP+ mCherry+DC

Live sorting(Day 3)

DMSO / TNFα(Day 4)

FACS(Day 5) *** **

******

20

40

60

80

% P

osi

tive

cel

ls

***

Pre-sorting

103

104

105 6.88 3.49

mC

her

ry

mC

her

ry

92.2% 91.1%

Post-sorting purity

( ay 3) ( ay ) ( ay 5)

**

*** **

**

0

20

- + - + - + - +

%

NegativeeGFP-mCherry+

eGFP+mCherry+Total inf.

Pre-sorting Post-sorting

0 102 103 104 105

0

102

0.17289.5

LTR - eGFP

CM

V -

m

LTR - eGFP

CM

V -

m

99%TNFα:

on Novem


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Figure 5A

U3 R U5 U3 R U5MA CA NC

polvifvpr Δenv

vpurev

tat

gag-eGFP5’ LTR 3’ LTR

CMV-mCherry

TTCAA NFκB NFκB SP1x3 TATAATTCAA NFκB NFκB SP1x3 TATAAB-LAI

TACAA AP1 AP1NFκB NFκB SP1x3 TATAATACAA AP1 AP1NFκB NFκB SP1x3 TATAAA

TACAA AP1 NFκB NFκB NFκB SP1x3 TATAATACAA AP1 NFκB NFκB NFκB SP1x3 TATAACTACAA NFκB NFκB SP1x3

TATAATACAA NFκB NFκB SP1x3

TATAADGroup Msubtypes

Core promoter elementsTATAATATAA

TATAA AP1 GABP NFκB SP1x3 TAAAATATAA AP1 GABP NFκB SP1x3 TAAAAAE

TACCA AP1 AP1 NFκB NFκB SP1x3 TATAATACCA AP1 AP1 NFκB NFκB SP1x3 TATAAF

TACAA AP1 NFκB NFκB SP1x3 TATAATACAA AP1 NFκB NFκB SP1x3 TATAAG

elements

A C DB

104

105 2.98 1.71

0

102

103

104

105 1.69 0.821

0 052397 40

102

103

104

105 1.71 0.732

0 052397 50

102

103

104

105 1.65 0.444

0 020997 9

B

0 102 103 104 105

0

102

103

0.084795.2

0 102 103 104 105

0.052397.40 102 103 104 105

0.052397.50 102 103 104 105

0.020997.9

103

104

105 2.21 1.35

103

104

105 2.49 1.11

103

104

105 1.27 0.704

Ch

erry

AE G F

0 102 103 104 105

0

102

10

0.07996.40 102 103 104 105

0

102

10

0.047796.30 102 103 104 105

0

102

10

0.031598

LTR - eGFP

CM

V -

m

C D

Red

2

3

4

Yel

low

Rat

io

DMSOPMA/Iono

2

2.5

3

3.5

4

4.5

chan

ge

R-Y

Rat

ioP

MA

/DM

SO

)

***

ns ***nsns

Yel

low

0

1

Red

-

LTR subtype

1

1.5

2

B-L

AI A C D

AE F G

Fo

ld c (P

LTR subtype

***

on Novem


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AFigure 6

Donor 1 Donor 2 Donor 3

CD4+ T cells – Activated (IL-2 + PHA)

RGHRGHinfected

ry

Mockinfected

LTR - eGFP

CM

V -

mC

her

LTR eGFP


CD4+ T cells – Non-activated (IL-2)B

RGH

rry

infected

LTR - eGFP

CM

V -

mC

her

Mockinfected

0 5

1

1.5

2

osi

tive

cel

ls

eGFP- mCherry+ eGFP+ mCherry+C

0

0.5


% P

o

1.72 1.45 1.22Red-Yellow Ratio:

CD4+ T cells - Activated (IL-2 + PHA)

on Novem


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dahabieh et al. 2013 1 a double-fluorescent hiv-1 reporter shows

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