thymic hiv-2 infection uncovers posttranscriptional control of viral replication in human thymocytes

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Thymic HIV-2 infection uncovers post-transcriptional control of viral replication in 1

human thymocytes 2

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Helena Nunes-Cabaço#, Paula Matoso, Russell B. Foxall, Rita Tendeiro*, Ana R. Pires, Tânia 4

Carvalho, Ana I. Pinheiro, Rui S. Soares and Ana E. Sousa# 5

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Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, 7

Portugal. 8

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Running Head: HIV-2 Infection of the Human Thymus 10

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Word count (Abstract/Importance): 190/131 12

Word count (Text): 3566 13

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#Address correspondence to: 15

Helena Nunes-Cabaço ([email protected]) 16

Ana E. Sousa ([email protected]) 17

Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa 18

Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal 19

Phone +351 217 999 525; Fax +351 217 999 527 20

21

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*Present address: Rita Tendeiro, Division of Infection and Immunity, UCL Institute of Immunity 23

and Transplantation, Rowland Hill Street, London NW3 2PF, UK 24

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JVI Accepts, published online ahead of print on 3 December 2014J. Virol. doi:10.1128/JVI.03047-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 26

A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals, characterized by 27

low-to-undetectable viremia throughout the disease course despite the establishment of 28

disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 29

infection. Although the clinical spectrum is similar in the two infections, HIV-2 is associated 30

with a much slower rate of CD4 T-cell decline and limited impact on the mortality of infected 31

adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-32

cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive 33

thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 34

co-receptors. We found that HIV-2 infected both thymic organ cultures and thymocyte 35

suspensions, as attested by total HIV DNA and cell-associated viral mRNA levels. 36

Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, 37

irrespective of HIV-2 co-receptor tropism and cell differentiation stage, in agreement with the 38

low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human 39

thymus but leads to reduced viral production by thymocytes, providing a new model to identify 40

therapeutic targets for viral replication control. 41

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IMPORTANCE 43

HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to 44

the characteristic CD4 T-cell loss. HIV-2 is associated with a very slow rate of progression to 45

AIDS, and is therefore considered a unique naturally-occurring model of attenuated HIV 46

disease. HIV-2-infected individuals feature low-to-undetectable plasma viral load, in spite of 47

the numbers of circulating infected T-cells being similar to those found in patients infected 48

with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human 49

thymus. We showed that HIV-2 is able to infect the thymus, but that the HIV-2 replication 50

cycle in thymocytes is impaired. We propose that this system will be important to devise 51

immunotherapies that target viral production, aiding the design of future therapeutic strategies 52

for HIV control. 53

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INTRODUCTION 55

The thymus is the primary organ for T-cell production and, despite the age-associated 56

decline, thymic function is maintained until late in life (1, 2). Thymic activity is vital in clinical 57

settings requiring de novo T-cell generation, such as HIV infection (1, 3, 4). Accordingly, 58

impairment of thymic output impacts on the rate of HIV-1 disease progression, whilst the 59

degree of immunological reconstitution achieved after antiretroviral therapy has been shown 60

to rely on thymus recovery (1, 3, 4). Moreover, a “functional” cure for HIV infection is thought 61

to entail a diverse T-cell repertoire, which can only be generated by the thymus. 62

HIV-1 targets the thymus in both children and adults, resulting in severe disruption of the 63

thymic microenvironment, as demonstrated by the morphological changes and thymocyte 64

depletion reported in thymuses of HIV-1-infected individuals (5, 6). Several studies, either 65

based on HIV-1 infection of the human thymus in vitro (7-9), or in vivo using the SCID/hu 66

mouse model (10, 11), have indicated that both direct infection of thymic cells and indirect 67

viral effects upon the microenvironment play a role in HIV-1-associated thymic pathology. 68

Furthermore, viral entry, replication kinetics and cytopathicity of HIV-1 in human thymocytes 69

have been shown to be highly dependent on viral tropism, due to the predominance of 70

CXCR4 (X4) vs. CCR5 (R5) expression in the human thymus (9, 12, 13). Thymic disruption 71

has also been described in non-human primate models of SIV infection (14). 72

Here we addressed, for the first time, the direct impact of HIV-2 infection on the human 73

thymus. This is particularly relevant because HIV-2-infected individuals feature slow rates of 74

CD4 T-cell decline and disease progression (15-17). Moreover, they typically have low-to-75

undetectable viremia, this being observed even in AIDS patients with <200 CD4 T cells/µl 76

(18, 19). The low levels of circulating virus account for the reduced horizontal and vertical 77

transmission observed in HIV-2 infection (20, 21), as well as for its geographical confinement 78

to West Africa and connected countries, such as Portugal. Despite the high prevalence of 79

HIV-2 in several regions of West Africa, such as in Guinea Bissau (8% in adults and up to 80

20% in people over 40 years of age) (22), there is no significant impact on the mortality of 81

infected adults. HIV-2 thus constitutes a unique naturally-occurring model of attenuated HIV 82

disease, valuable for the study of HIV pathogenesis. 83


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In spite of the low-to-undetectable HIV-2 plasma viral load, HIV-2 and HIV-1-infected patients 84

at equivalent stages of CD4 T-cell depletion feature comparable levels of cell-associated viral 85

burden (18, 23, 24), indicating the establishment of disseminated HIV-2 reservoirs. They also 86

feature similar levels of T-cell activation (19), suggesting distinct control of viral replication in 87

the presence of cell activation in HIV-2 and HIV-1 infections. 88

Our previous data support preservation of thymic function in HIV-2 infection, as estimated by 89

signal-joint (sj)/TREC quantification (3) in circulating T-cells of HIV-2 infected patients (25). 90

However, there are no studies on the direct impact of HIV-2 infection on the human thymus, 91

either in vivo, due to the difficulty of obtaining thymic tissue from HIV-2-infected patients, or in 92

vitro. We show here that HIV-2 was able to infect the human thymus, but that this was 93

associated with limited viral replication, irrespectively of viral co-receptor tropism and 94

thymocyte differentiation stage. HIV-2 infection of the human thymus thus offers a novel 95

approach to investigate the mechanisms underlying the establishment of HIV reservoirs and 96

the control of viral replication. 97

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MATERIALS AND METHODS 99

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Ethical Statement 101

Thymic specimens (from newborns to 4-year-old children) were obtained from routine 102

thymectomy performed during pediatric corrective cardiac surgery at Hospital de Santa Cruz, 103

Carnaxide, Portugal, after parent’s written informed consent. Buffy coats from healthy donors 104

were provided by Instituto Português do Sangue e da Transplantação, after written informed 105

consent. The study was approved by the Ethical Boards of Faculty of Medicine of the 106

University of Lisbon and of Hospital de Santa Cruz, Portugal. 107

108

HIV stocks 109

Viruses used are described in Table 1. Viral stocks were propagated in pools of isolated 110

peripheral blood mononuclear cells (PBMC) stimulated for 3 days with phytohemagglutinin 111

(PHA; 5μg/ml, Sigma) and maintained with human recombinant IL-2 (10U/ml; Dr. Maurice 112

Gately, Hoffmann – La Roche Inc, NIH AIDS Reagent Program), as described (28). Virus in 113

cell-free culture supernatants was quantified by measuring reverse transcriptase (RT) activity 114

using Lenti-RT Activity Kit (Cavidi). 115

116

HIV infection of thymocyte suspensions 117

Total thymocytes were recovered through tissue dispersion and separation on Ficoll-Paque 118

PLUS (GE Healthcare) density gradient. The CD4 single-positive (SP) population was sorted 119

from total thymocytes as CD3high

CD8neg

cells (purity>98%), using a FACSAria High-Speed 120

Cell Sorter (BD Biosciences). Viral stocks were ultra-centrifuged for 30min at 50,000g and 121

4ºC (Beckmann L8 Ultracentrifuge), resuspended in complete medium (RPMI1640 with 10% 122

fetal bovine serum (FBS), 2mM L-glutamine, 50U/ml penicillin/streptomycin and 50g/ml 123

gentamycin (all from GIBCO/Invitrogen) plus 3g/ml polybrene (Sigma)) and added to the 124

cells at 0.3ng RT/106 thymocytes. Total and CD4SP thymocytes were cultured for 3-4h at 10

8 125

and 3.5x107 cells/ml, respectively, in absence or presence of virus. After infection, cells were 126

washed and cultured at 107 cells/ml in complete medium supplemented with IL-2 (20U/ml), IL-127

4 (20ng/ml; R&D) and IL-7 (10ng/ml; R&D) at 37ºC. A quarter of the media was replaced 128


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every 3-4 days. On day 10 thymocyte number per well was determined using 10M latex 129

beads (Coulter), and cells were analyzed by flow-cytometry or stored as pellets at -80ºC. Viral 130

production in supernatants was quantified at day 10 post-infection of 131

CD11cneg

CD14neg

CD123neg

TCRneg

thymocytes by measuring RT activity using SYBR 132

Green–product enhanced RT (SG-PERT), as described (29, 30). 133

134

HIV infection of Thymic Organ Cultures (TOC) 135

Thymic tissue blocks (1-2mm diameter) were placed on Millicell organotypic inserts (Millipore) 136

in a 6-well plate containing 1ml of TOC medium (complete medium with 15% FBS, 10mM 137

HEPES, 1mM sodium pyruvate and 1% MEM non-essential amino-acids, all from 138

GIBCO/Invitrogen). TOC were placed at 37ºC, 5%CO2 overnight and half of the medium 139

replaced prior to infection. HIV infection was performed by placing a 5l drop containing 3ng 140

RT of virus on top of each TOC. A third of the media was replaced every 2-3 days. On day 141

10/11 post-infection TOC were placed in 4% formaldehyde or mashed. Thymocytes were 142

analyzed by flow-cytometry or stored as cell pellets at -80ºC. The role of Env proteins was 143

assessed by culturing TOC for 7 days on Isopore membranes (Millipore) placed in TOC 144

medium containing 1g/ml of purified recombinant glycoproteins: gp105ROD (MRC EVA621), 145

gp120BaL (NIH Cat. No.4961) and gp120IIIB (MRC EVA607); or anti-CD4 mAb (BD 146

Biosciences) as control. 147

148

Flow-cytometry 149

Surface staining was performed for 20min at room temperature and always included Fixable 150

Viability Dye (eBioscience) for dead cell exclusion. Thymocytes were fixed, permeabilized and 151

stained using an intracellular staining kit (eBioscience), as described (31). Anti-human 152

monoclonal antibodies (mAb) used were (clones in brackets): CD3 (UCHT1), CD4 (RPA-T4), 153

CD8 (RPA-T8), CD11c (3.9), CD14 (61D3), CD16 (eBioCB16), CD19 (HIB19), CD123 (6H6) 154

and TCR(B1.1) from eBioscience. The following anti-Gag mAb were used: KC57 (Beckman 155

Coulter), anti-p24 (Kal-1; Dako), anti-p24 (AG3.0 or 183-H12-5C, NIH); anti-p27 156

(ARP396/397; Dr.Szawlowski, NIBSC-Centre for AIDS Reagents, Medical Research Council). 157

AlexaFluor®488 goat anti-Mouse IgG(H+L) antibody (Molecular Probes) was used for 158


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secondary detection. Cells were acquired using LSRFortessa (BD Biosciences) and data was 159

analyzed in FlowJo (TreeStar). 160

161

Immunohistochemistry 162

Fixed TOC were embedded in paraffin and cut into 3μm sections (Minot Microtome Leica 163

RM2145). Epitope-retrieval was performed at pH9 (Leica Biosystems buffer) for 15min using 164

a microwave (800W). Samples were stained with the appropriate anti-Gag primary antibodies, 165

incubated with a peroxidase/DAB detection system (EnVision, Dako) and counterstained with 166

Harris’ hematoxylin (BioOptica). Images were acquired using a Leica DM2500 microscope. 167

168

Quantification of total HIV DNA and gag mRNA by real-time PCR 169

Total HIV DNA was quantified in cell lysates prepared by treating cell pellets with 100g/ml 170

proteinase K in 10mM Tris-HCl for 1h at 56ºC, followed by 10min enzyme inactivation at 171

95ºC. For gag mRNA quantification, 200ng of total RNA, purified using mirVana (Ambion), 172

were used to synthesize cDNA using oligo(dT)20 and SuperscriptIII (Invitrogen), according to 173

manufacturer’s instructions. Real-time PCR was performed using Platinum Taq plus ROX or 174

Taqman Gene Expression Master Mix (Applied Biosystems), with primers and probes 175

described in Supplementary Table 1. Standard curves were generated from serial dilutions of 176

cDNA prepared from mRNA of 3 different thymuses (for GAPDH quantification) or of plasmids 177

containing the amplicons of HIV-1 Gag and CD3 (kind gift from Rémi Cheynier) (32) or HIV-2 178

Gag. HIV-2 Gag plasmid was generated by inserting a sequence including LTR/Gag from 179

HIV-2ROD into the pGEM®-TEasy vector (Promega). Quantification was performed using an 180

Applied Biosystems 7500 Fast Real-Time PCR System. 181

182

Statistical Analysis 183

Statistical analysis was performed using GraphPad Prism v5.01 (GraphPad Software Inc.). 184

Two-sample data were compared using Wilcoxon-matched pairs test. Data from more than 185

two samples were compared using Friedman test or Kruskal-Wallis test with Dunn’s multiple 186

comparison post-test. P values <0.05 were considered significant. 187


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RESULTS 188

189

HIV-2 infects the human thymus 190

We investigated HIV-2 infection of the human thymus in vitro using both TOC, which preserve 191

the thymic microenvironment and have been shown to be permissive to HIV-1 infection (7, 8), 192

and thymocyte suspensions. TOC and thymocyte suspensions were infected with HIV-2 or 193

HIV-1 primary isolates with selective R5 or X4 co-receptor specificity and cultured for 10 days. 194

RT activity was used to normalize viral input, as HIV-1 and HIV-2 RT enzymes were shown to 195

possess similar specific activities (33). 196

We found that HIV-2 was able to infect both thymic tissue and total thymocyte suspensions, 197

as indicated by the levels of total HIV DNA, which includes unintegrated and integrated 198

proviral DNA, in HIV-2-infected samples (Figure 1A). Furthermore, we observed that HIV-2 199

infection of both thymic tissue and total thymocyte suspensions was less co-receptor 200

dependent than HIV-1 infection. 201

Next we sort-purified mature CD4SP thymocytes, which constitute a target of HIV-1 (11), and 202

cultured them for 10 days after infection with the above-mentioned viruses. In contrast to TOC 203

and thymocyte suspensions, HIV-2 infection of CD4SP thymocytes resulted in co-receptor-204

dependent levels of total HIV DNA (Figure 1A). The limited ability of HIV-2 R5 to infect the 205

CD4SP subset was in contrast to our observations in TOC and total thymocyte suspensions 206

(Figure 1A), suggesting that HIV-2 R5 targeted other human thymocyte populations. 207

We next assessed the levels of ongoing viral transcription in HIV-2-infected TOC and 208

thymocytes by measuring viral gag mRNA. Both HIV-2 R5 and X4 isolates generated gag 209

mRNA levels comparable to those found upon infection with the HIV-1 X4 isolate (Figure 1B), 210

indicating efficient integration and transcription of HIV-2 in TOC and thymocyte cultures. Of 211

note, HIV-2 gag mRNA levels showed co-receptor dependency in the case of purified CD4SP 212

thymocytes, but not in TOC or thymocyte suspensions (Figure 1B), in line with the total HIV 213

DNA levels that were observed in the same samples. 214

In summary, our results indicated that HIV-2 was able to successfully enter, reverse 215

transcribe and integrate in human thymocytes in TOC and in suspension, and that viral 216

transcription was ongoing in these systems. 217


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218

Limited viral production in HIV-2 infection of human thymocytes 219

Next we assessed viral production in HIV-2-infected thymic tissue using 220

immunohistochemistry. Gag protein was detected in HIV-2 R5- and X4-infected TOC after 221

culture (Figure 2A), using validated antibodies (Supplementary Figure 1) (34). Gag protein 222

expression was both cell-associated and extracellular (Figure 2B), with the latter likely 223

corresponding to virus produced by HIV-infected cells during TOC culture, as no Gag staining 224

was observed on day 1 (Figure 2A). Of note, Gag protein levels were often indistinguishable 225

in X4 and R5 HIV-1 TOC infections (Figure 2A), despite the differences in the levels of cell-226

associated viral burden (Figure 1), indicating lack of correlation between the amount of virus 227

detected by immunohistochemistry and the levels of thymocyte viral production. Therefore, 228

although our immunohistochemistry data confirmed the ability of HIV-2 to productively infect 229

the human thymus, it was not possible to infer the specific contribution of thymocytes vs. 230

thymic stroma to the viral protein detected in the histological analysis. 231

We thus evaluated viral production at the single-cell level by flow-cytometry. Notably, we 232

found very low levels of intracellular Gag protein in cells isolated from HIV-2-infected TOC or 233

in thymocyte suspensions, irrespective of the virus used (Figure 3) and time-point analyzed 234

(Supplementary Figure 2). In contrast, Gag protein expression was clearly detected in cells 235

infected with HIV-1, particularly with the X4 isolate (Figure 3), excluding the possibility that the 236

low Gag detection in HIV-2 infections was due to sample processing. Of note, intracellular 237

Gag protein was also barely detected upon thymocyte infection with the lab-adapted strain 238

HIV-2ROD (Supplementary Figure 2), despite the high levels of total HIV DNA detected 239

(181,499copies/106 cells in the example presented). Low Gag levels upon HIV-2 infection 240

were particularly evident in CD4SP thymocytes, which featured extremely low levels of 241

intracellular Gag after infection with HIV-2 primary isolates or HIV-2ROD (Figure 3 and 242

Supplementary Figure 2). 243

The antibody used (KC57) has been previously shown to be specific for both HIV-2 and HIV-1 244

Gag (35). We further confirmed the ability of the antibody to efficiently detect HIV-2 Gag by 245

flow-cytometry in activated PBMCs infected with the HIV-2 strains used in the current study 246

(Supplementary Figure 3). Moreover, KC57 staining was comparable to that obtained with 247


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other available anti-Gag antibodies in HIV-2-infected PBMCs or thymocytes (Supplementary 248

Figures 2 and 3), confirming low viral production in HIV-2-infected thymocytes. 249

The low levels of intracellular Gag protein translated into reduced viral production in HIV-2 250

infections, as indicated by the lower levels of virus in the supernatant of HIV-2-infected 251

thymocyte cultures than in their HIV-1-infected counterparts (RT activity: HIV-1 R5: 252

5.97±3.23pg/ml; HIV-1 X4: 1318±583.5pg/ml; HIV-2 R5: 0.31±0.15pg/ml; HIV-2 X4: 253

8.54±3.40pg/ml; n=3). 254

Overall, we found that HIV-2 infection of human thymocytes occurred without significant viral 255

production per cell, despite evidence of cell-associated viral burden and active ongoing viral 256

transcription. 257

258

Impact of HIV-2 infection on thymocyte populations 259

Finally, we assessed the impact of HIV-2 on the distribution of thymocyte populations in 260

infected TOC and thymocyte suspensions. TOC infected with X4-using viruses, whether HIV-261

2 or HIV-1, featured a dramatic decrease in CD4+ thymocytes as compared to R5 viruses 262

(Figures 4A and 4B). This was also observed in thymocyte suspensions infected with HIV-1 263

X4, but not with HIV-2, which did not result in significant alterations of the thymocyte 264

phenotype in culture (Figures 4C and 4D). One possible explanation for these results is that 265

down-regulation of the CD4 co-receptor is distinctly induced by HIV-2 and HIV-1. Accordingly, 266

our analysis of productively infected cells indicated that HIV-2 was less efficient than HIV-1 at 267

down-regulating CD4 (Figure 4E). 268

The effects on subset distribution were mirrored by the cytopathic effect of the respective 269

virus: both HIV-2 and HIV-1 X4 but not R5 viruses were cytopathic in TOC (Figure 4F), whilst 270

HIV-1 X4 had a small effect on cell viability in thymocyte suspensions (Figure 4G). The low 271

cell recovery in HIV-2 X4-infected TOC, together with the imbalance in cell subsets and the 272

apparent low impact of HIV-2 infection on CD4 down-regulation, suggested that HIV-2 X4 273

infection of TOC led, directly or indirectly, to the death of CD4+ thymocytes. This occurred 274

despite the lack of impact of HIV-2 X4 infection on total thymic cells suspensions, supporting 275

the contribution of other cell populations present in the whole tissue. 276


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Given our previous data supporting immunosuppressive properties of the HIV-2 envelope 277

proteins in peripheral blood lymphocytes mediated, at least in part, by monocytes (36-38), we 278

also evaluated whether the alterations observed in HIV-2 X4-infected TOC were induced by 279

direct action of viral envelope proteins. We cultured TOC in the presence of recombinant HIV-280

2 and HIV-1 Env proteins, either binding to X4 (HIV-2 gp105ROD and HIV-1 gp120IIIB) or R5 281

(HIV-1 gp120Ba-L). In our system, Env proteins did not induce CD4+ thymocyte depletion or 282

subset imbalances (Figure 5), indicating that the effects observed may require viral entry 283

and/or post-entry steps. 284

Altogether, our data support a cytopathic effect of HIV-2 X4 on the human thymus that was 285

not observed in thymocyte suspensions, suggesting a dependency on the stromal 286

compartment. Importantly, HIV-2 R5 had no significant cytopathic effect on either TOC or 287

thymocyte suspensions. 288

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DISCUSSION 290

We addressed here, for the first time, the ability of HIV-2 to infect the human thymus. We 291

showed that HIV-2 is able to infect thymic tissue and thymocyte suspensions in the absence 292

of exogenous stimulation, although with a very low level of viral production per thymocyte, 293

supporting the existence of post-transcriptional control in viral replication. 294

HIV-2 infection of the human thymus was confirmed by the levels of cell-associated HIV DNA 295

and gag mRNA measured following HIV-2 infection of either TOC or thymocyte suspensions. 296

The high levels of total HIV DNA observed were consistent with previous reports where 297

unintegrated plus integrated proviral DNA were quantified (39, 40). HIV-1 infection of human 298

thymocytes was shown to be co-receptor-dependent (9, 12), and our data recapitulated these 299

results. Conversely, we showed that HIV-2 infection of TOC and thymocyte suspensions was 300

much less dependent than HIV-1 on co-receptor tropism, both in terms of cell-associated HIV 301

DNA and viral RNA, which could be related to the broader co-receptor usage reported for 302

HIV-2 as compared to HIV-1 (41). 303

On the other hand, HIV-2 cytopathicity in human thymic tissue was co-receptor-dependent, 304

with X4 virus inducing significant T-cell death in TOC, which was not observed for HIV-2 R5 305

virus. This was in line with a previous study of HIV-2 infection of lymphoid tissue, where 306

lymphocyte depletion also occurred with X4 but not R5-tropic isolates (42). Importantly, we 307

showed that HIV-2 X4 cytopathicity in TOC was not due to the direct action of HIV-2 Env, 308

indicating a requirement for viral entry and/or post-entry steps for CD4+ thymocyte depletion 309

in the human thymus. 310

The observed cytopathicity caused by HIV-2 X4 in tissue but not in thymocyte suspensions 311

could be related to the infection of components of the thymic stroma that are present in TOC. 312

Thymic stromal cells, including DCs, macrophages, and even thymic epithelial cells, have 313

been reported to be permissive to HIV-1 infection (43-45). It is not known whether HIV-2 314

infects the thymic stroma and, due to alterations in tissue morphology that occurred during the 315

culture process, we were not able to directly infer on the type of infected cells from our 316

immunohistochemistry data. However, in contrast to HIV-1, HIV-2 encodes the lentiviral 317

accessory protein Vpx, which targets the restriction factor sterile alpha motif and 318

histidine/aspartic acid domain–containing protein 1 (SAMHD1) for degradation, a factor that 319


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has been shown to limit the productive infection of HIV-1 in myeloid cells (46). Moreover, 320

additional cell targets may be considered, given the broader co-receptor usage reported for 321

HIV-2 (41). We are currently addressing the possibility that infection of thymic stroma 322

components might have distinct consequences in HIV-1 and HIV-2 infections. 323

We had previously reported that thymic function, estimated via sj/βTREC measurement, was 324

better preserved in HIV-2 as compared to HIV-1-infected patients (25). It is likely that most of 325

these individuals were infected with R5-tropic HIV-2 (25). Importantly, we showed here that 326

HIV-2 R5 infection did not significantly impact on TOC or thymocyte suspensions, in terms of 327

cytopathicity or subset distribution, or induce CD4 down-regulation in the latter. Nevertheless, 328

the elevated levels of total HIV DNA and viral mRNA support the potential establishment of 329

HIV-2 viral reservoirs in human thymocytes. It would thus be important to investigate whether, 330

as reported for HIV-1 (32), CD4+ recent thymic emigrant T-cells from HIV-2-infected patients 331

may constitute viral reservoirs. 332

Our data suggest that, in our system, distinct regulation of the replicative cycle of HIV-2 and 333

HIV-1 occurs at the post-transcriptional/translational level. The discrepant levels of Gag 334

protein in the two infections markedly contrasted with the similarly high levels of total HIV 335

DNA and viral mRNA documented. HIV-2 and HIV-1 have been reported to differ significantly 336

in their mechanisms of translation initiation (47). For instance, HIV-2 was described to have 337

lower translational efficiency than HIV-1 due to differences in the 5’-untranslated region (5’-338

UTR) of viral genomic RNA (48, 49). In agreement with our observations, lower Gag protein 339

production in the presence of equivalent T-cell-associated viral gag mRNA levels were 340

reported in HIV-2 as compared to HIV-1 infection of T-cell lines and macrophages (49). 341

Importantly, and in relation to our data, this was not due to lower stability of the Gag protein, 342

nor to higher rate of viral particle release (49). Interestingly, the authors raised the possibility 343

that host factors may be differentially regulating the initiation of Gag translation in HIV-2 and 344

HIV-1 (49). HIV-2 infection of the human thymus provides, to our knowledge, the first model of 345

HIV-2 post-transcriptional regulation in ex vivo T-cells using primary isolates, providing a 346

unique opportunity to study the molecular factors and mechanisms involved in the regulation 347

of HIV translation. 348


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Human thymocytes represent an ideal system for the study of HIV latency due to their ability 349

to become infected in the absence of external activation (50). Models using peripheral CD4+ 350

T-cells require cellular activation followed by quiescence induction (51), thus inducing 351

molecular modifications that may impact the processes under study. The system described 352

here, utilizing HIV-2 infection of the human thymus in the absence of exogenous stimulation, 353

thus provides an important cellular model for the study of latency and reservoir generation in 354

HIV pathogenesis. 355

Our results regarding HIV-2 infection also highlight the potential importance of post-356

transcriptional control of viral production, possibly through regulation of translation, in viral 357

pathogenesis. Further studies using this model will enable the discovery of potential 358

molecular targets to be used as the basis for new immunotherapies aimed at achieving a 359

“functional” cure for HIV. 360

361

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ACKNOWLEDGEMENTS 363

This work was supported by “Fundação para a Ciência e a Tecnologia” (FCT) and by 364

“Programa Operacional Ciência e Inovação 2010” (POCI2010), grant PTDC/SAU-365

MII/66248/2006 to AES. HNC, RBF, RT and RSS received scholarships from FCT co-366

financed by POCI2010. 367

The authors thank Dr. Miguel Abecasis and Dr. Rui Anjos for human thymus sample 368

collection; Dr. Rémi Cheynier, Dr. Andreia Amaral and Dr. Íris Caramalho for discussion; Dr. 369

Francisca Matos and nurses and staff at Hospital de Santa Cruz for technical assistance; Dr. 370

Nuno Taveira, Dr. Rémi Cheynier and Dr. Nicolas Manel for reagents; patients and families. 371

372

373

CONFLICT OF INTERESTS 374

The authors declare no conflicting financial interests. 375

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FIGURE LEGENDS 546

547

Figure 1: HIV-2 infects human thymic tissue and isolated thymocytes. Thymic organ 548

cultures (TOC) were infected with R5- or X4-using HIV-2 or HIV-1 primary isolates and 549

cultured for 10/11 days on Millipore inserts. Single cell suspensions of total thymocytes or 550

CD4SP thymocytes, sorted as CD3high

CD8neg

cells, were also infected with the above-551

mentioned viruses and cultured for 10 days. Graphs show total HIV DNA (A), as assessed by 552

quantitative real-time PCR, or viral gag mRNA (B), as assessed by real time RT-PCR 553

(normalized to GAPDH expression). Each dot represents a single thymus. Lines indicate 554

median values. *: P<0.05; **: P<0.01. 555

556

Figure 2: Viral production in HIV-2-infected TOC. TOC infected with R5- or X4-using HIV-2 557

or HIV-1 primary isolates were analyzed for viral production by immunohistochemistry, using 558

anti-Gag antibodies (brown) and hematoxylin counterstain (blue). (A) Viral production at days 559

1 and 11 after infection with HIV-2 (top panels) or HIV-1 (bottom panels), as assessed using 560

anti-p27 (ARP396/397 from MRC) or anti-p24 (Kal-1 from DAKO) mAb, respectively. (B) 561

Extracellular (arrow) and cytoplasmic (arrowhead) Gag expression in R5 or X4 HIV-2-infected 562

TOC at day 11 (ARP396/397 anti-p27 antibody). 563

564

Figure 3: Limited replication of HIV-2 in human thymocytes. Viral production at the single-565

cell level was determined by flow-cytometry using the anti-Gag antibody KC57 10 days post-566

infection of TOC, total thymocytes or CD4SP thymocytes with R5- or X4-tropic HIV-1 and 567

HIV-2 primary isolates. Representative dot plots of intracellular Gag expression (A) and 568

frequency of productively infected KC57+ thymocytes (B) in each condition are shown. Flow 569

cytometric analysis was performed after exclusion of dead cells and aggregates. Each dot 570

represents a single thymus. Lines indicate median values. *: P<0.05; **: P<0.01. 571

572

Figure 4: Distinct cytopathic impact of HIV-2 infection on TOC and thymocytes. HIV-573

infected TOC (A and B) or thymocyte suspensions (C and D) were cultured for 10 days. 574

Representative dot plots of CD4 and CD8 expression in cells in TOC (A) and in total 575


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thymocyte suspensions (C), with graphs showing the mean frequency of thymocyte subsets in 576

all thymuses analyzed (B, n=4, and D, n=8; respectively). TOC analysis was performed in 577

CD14neg

CD16neg

CD19neg

CD123neg

cells. Dead cells and aggregates were excluded from the 578

flow-cytometric analysis. (E) Frequency of CD4neg

cells within Gag+ (KC57

+) thymocytes in 579

HIV-infected thymocyte suspensions. (F) Fold-change in the frequency of live cells in TOC 580

relative to uninfected control, as assessed by flow-cytometry. (G) Fold-change in the number 581

of live thymocytes in culture relative to uninfected control. Lines indicate median values. *: 582

P<0.05. 583

584

Figure 5: HIV-2 X4 envelope per se does not impact on thymocyte distribution in TOC. 585

TOC were cultured for 7 days with medium only (control) or in presence of the recombinant 586

envelope proteins HIV-2 gp105ROD, HIV-1 gp120Ba-L or gp120IIIB (all at 1g/ml) or anti-CD4 587

mAb. Dot plots show a representative example (one of 3 independent experiments with 588

different thymuses) of the frequency of subsets in TOC, as determined by CD4 and CD8 589

expression. 590

591


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Table 1: HIV viruses used in the study. 592 593

Viruses Major co-receptors used Source

Primary isolates HIV-192US660 R5 NIHa

HIV-192HT599 X4 NIHa,b

HIV-260415K R5 NIHa,c

HIV-220.04d X4 Dr. Nuno Taveira

Lab-adapted strains HIV-1NL4-3 X4 MRCe

HIV-2ROD10 R5, X4 MRCe

594 aNIH AIDS Reagent Program, Division of AIDS, NIAID, NIH; from The Multi-center AIDS Cohort Study;

bprovided by 595

Dr. Neal Halsey; cprovided by Dr. Feng Gao and Dr. Beatrice Hahn;

dpreviously represented as PTHCC20/2004 (26) 596

and 19/2004 (27); eNIBSC - Centre for AIDS Reagents, UK, Medical Research Council. 597

598

599


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thymic hiv-2 infection uncovers posttranscriptional control of viral replication in human thymocytes

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