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Development of an HIV reporter virus that identifies latently infected CD4+ T cells

Summary There is no cure for HIV infection, as the virus establishes a latent reservoir, which escapes highly active antiretroviral treatments. One major obstacle is the difficulty identifying cells that harbor latent proviruses. We devised a single-round viral vector that carries a series of versatile reporter molecules that are expressed in an LTR-dependent or LTR-independent manner and make it possible to accurately distinguish productive from latent infection. Using primary human CD4+ T cells, we show that transcriptionally silent proviruses are found in more than 50% of infected cells. The latently infected cells harbor proviruses but lack evidence for multiple spliced transcripts. LTR-silent integrations occurred to variable degrees in all CD4+ T subsets examined, with CD4+ TEM and CD4+ TREG displaying the highest frequency of latent infections. This viral vector permits the interrogation of HIV latency at single-cell resolution, revealing mechanisms of latency establishment and allowing the characterization of effective latency-reversing agents.


INTRODUCTION
An HIV/AIDS cure remains elusive because HIV establishes a hard-to-eradicate latent reservoir. Despite the intense efforts over the past forty years, one of the main obstacles to HIV eradication is the difficulty identifying the cells that harbor transcriptionally silent proviruses forming a long-lasting, antiretroviral-resistant, latent reservoir.
CD4 + T cells are the primary targets of HIV infection and contribute to establishing and maintaining the latent reservoir (Churchill et al., 2016). Subsets of these cells differ in their susceptibility to infection and their ability to traffic, exert effector functions, proliferate, and survive (Zerbato et al., 2016;Kulpa et al., 2019;Grau-Exposito et al., 2019). A more detailed and cell type-specific understanding of latency establishment is, therefore, of great relevance.
HIV latency can result from progressive epigenetic silencing of productive infections (Colin and Van Lint, 2009;Siliciano and Greene, 2011;Pearson et al., 2008;Weinberger et al., 2008;Hashemi et al., 2016;Jefferys et al., 2021;Mbonye and Karn, 2014). However, evidence suggest that a substantial portion of HIV integrations occur in an LTR-silent manner (Dahabieh et al., 2013;Chavez et al., 2015;Calvanese et al., 2013). This shift in our understanding of HIV latency establishment was made MOTIVATION HIV latency is a major obstacle to the eradication of HIV/AIDS. Identification of the infected cells harboring LTR-silent proviruses is technically complicated and labor intensive. Cell culture systems that identify latently infected primary human cells at the single-cell level are necessary to study the mechanisms of latency establishment in a cell-type-specific manner. Moreover, such an experimental system would also allow the discovery of small molecules that either lock or reactivate proviruses. The pMorpheus HIV vector system represents an advancement in the field, as it provides a flexible toolbox for dissecting HIV latency in the context of a single-cycle virus encoding all the accessory genes.
possible by the use of HIV vector constructs, which encode fluorescent reporters expressed in either an HIV LTR-dependent or LTR-independent manner (Dahabieh et al., 2013;Calvanese et al., 2013).
A number of different dual-reporter vectors have been used in the past to investigate latency establishment (Battivelli et al., 2018;Calvanese et al., 2013;Cavrois et al., 2017;Chavez et al., 2015;Dahabieh et al., 2013Dahabieh et al., , 2014Hashemi et al., 2016;Kim et al., 2019;Matsuda et al., 2015;Cai et al., 2021). They all rely on LTR-dependent or LTR-independent differential reporter expression. We reasoned that positioning the reporters in distinct reading frames undergoing different splicing events would reduce the potential for promoter interference. Moreover, having a full-length functional gag and all accessory proteins, including Nef, is necessary to obtain a physiologically relevant assessment of HIV latency establishment. Last, a constitutively active PGK promoter ensures high expression of the latency protein tags while avoiding rapid silencing by the HUSH complex (Jones et al., 2009;Kim et al., 2019;Cai et al., 2021;Tchasovnikarova et al., 2015). Of note, the resulting HIV molecular clone, pMorpheus-V5, lacks a functional Env, which eases biosafety precautions for downstream applications with live cells and allows pseudotyping with different HIV envelopes.
Detection of infected cells at the single-cell level in a manner that is independent of HIV LTR activity allows probing for potential differences in latency establishment in specific CD4 + T cell subsets. Here we show that primary human CD4 + T cells harboring transcriptionally silent proviruses are more frequent than productively infected cells. This was true for all seven CD4 + T cell subsets investigated in this study, but CD4 + regulatory T cells (CD4 + T REG ) and CD4 + effector memory T cells (CD4 + T EM ) had the highest frequency of LTR-silent infection. We propose that the pMorpheus-V5 system encoding all the viral accessory proteins as well as different reporter proteins will facilitate obtaining new mechanistic insights into latency establishment and reactivation in different primary human cells, including but not limited to primary human CD4 + T cells.

pMorpheus-V5 construction and identification of productively and latently infected cells
To investigate the cellular processes that govern establishment of HIV latency at the single-cell level in human primary CD4 + T cells, we developed a viral reporter vector (pMorpheus-V5) suitable to detect the LTR-dependent or LTR-independent expression of four reporters, two of which, HSA and V5-NGFR, are membrane bound ( Figure 1A). A PGK promoter-driven reporter cassette encoding V5-NGFR was inserted into the Env open reading frame deleting a portion of the HIV envelope. Upon proviral integration, but independently of LTR promoter activation, V5-NGFR localizes to the surface of the infected cell. The LTR-dependent expression cassette includes sequences encoding HSA and mCherry that were cloned upstream of an internal ribosomal site followed by Nef. Thus, LTR-dependent expression of HSA, mCherry and Nef is linked (Young et al., 2018). Cells productively infected with pMorpheus-V5 express four reporters: V5-NGFR driven by PGK as well as HSA and mCherry driven by the HIV-LTR. In contrast, latently infected cells express only V5-NGFR. The combination of fluorescence reporter and membrane bound tags allow easy and specific identification of the different cell population using flow cytometry ( Figures 1B and 1C). Cells expressing only HSA and mCherry (lower right quadrant in the cartoon) should not be found, as all productively infected cells harbor proviruses and thus would be positive for V5-NGFR ( Figure 1B). To assess the ability to reliably distinguish cells harboring LTR-silent or LTR-active proviruses, we infected primary human CD4 + T cells, stimulated either with IL-2 alone or activated by a combination of IL-2 and a-CD3/ CD28 coated beads, with pMorpheus-V5. Dual-tropic envelope pSV III-92 HT593.1 (Gao et al., 1996) was used to pseudotype pMorpheus-V5 in order to broaden the range of CD4 + T cell types susceptible to infection, including naive CD4 + T cells as well as CD4 + memory stem cells. It is important to understand latency establishment in these specific CD4 + T cell types known to express low levels of CCR5 yet critical for the formation of the latent reservoir (Buzon et al., 2014;Mavigner et al., 2018;Zerbato et al., 2019). Of note, pMorpheus-V5 is a single-cycle viral vector, which does not support spreading infection. Primary human CD4 + T cells were infected with pMorpheus-V5 for five days. Day 5 post-infection was chosen because optimization experiments revealed it to be the time point when productive infection peaked. We used flow cytometry or cytometry by time-of-flight (CyTOF) for the analysis (Table S1). The cell viability was comparable between infected and uninfected cells under each experimental condition (Figures S1A, S1B, S2A, and S2B). NGFR and V5 as well as HSA and mCherry provided comparable percentages of latently or productively infected cells as measured using either flow cytometry or CyTOF ( Figure 1C, flow cytometry; Figure S2C,CyTOF). The absence of HSA-mCherry alone expressing cells indicates that promoter interference is not a problem with the current vector system.
As HIV-1 Nef is known to down-regulate the CD4 receptor (Garcia and Miller, 1991;Guy et al., 1987), we next compared the expression of CD4 in latently and productively infected CD4 + T cells with that of mock-infected cells. As expected, HIV Gag p24-positive CD4 + T cells expressed substantially less CD4 compared with p24-negative cells or mock-infected cells ( Figures 1D, 1E, S1C, S1D, S2D, and S2E). Down-regulation of CD4 was also observed in HSA POS or mCherry POS cells but not in NGFR POS or V5 POS -only cells. Thus, CD4 expression in CD4 + T cells harboring only LTR-silent proviruses (as identified by NGFR POS /V5 POS but HSA NEG /mCherry NEG expression) remained comparable with that of mock-infected cells ( Figures 1D and 1E; Figures S1C and S1D [flow cytometry] and S2D and S2E [CyTOF]).
Taken together, infection with pMorpheus-V5 permits to distinguish between latently infected and productively infected primary CD4 + T cells using either flow cytometry or CyTOF.

Detection of HIV proviruses and transcripts in latently
infected primary human CD4 + T cells We next measured the level of integration and multiply spliced HIV transcripts in LTR-silent and LTR-active infected CD4 + T cells ( Figure 3A). Briefly, we infected IL-2 + a-CD3/CD28-stimulated primary human CD4 + T cells from three different healthy donors with pMorpheus-V5 in the presence and absence of the FDA-approved integrase inhibitor raltegravir. Five days Cell Reports Methods 2, 100238, June 20, 2022 3 Article ll OPEN ACCESS later, culture supernatants were collected for p24 analysis, and the live CD4 + T cells were sorted using fluorescence-activated cell sorting (FACS) (latently infected: V5-NGFR POS /HSA-mCherry NEG ; productively infected: V5-NGFR POS /HSA-mCherry POS ; negative: V5-NGFR NEG /HSA-mCherry NEG ). As anticipated, raltegravir prevented both latent and productive infections ( Figure 3B).
Genomic DNA was extracted from sorted cells to measure integration using Alu-1 PCR (Butler et al., 2001), while RNA was extracted from the same samples to detect multiply spliced HIV tat/rev transcripts using qRT-PCR assay (Pasternak et al., 2008;Zerbato et al., 2021) ( Figure 3A). As a positive control, we used the T cell line J-Lat, which harbors a single provirus, stimulated with PMA (16 nM) and ionomycin (0.5 mM) for three days. We found that the levels of integrated provirus were comparable between latently and productively infected cells ( Figure 3C). Raltegravir treatment effectively blocked HIV integration. A small portion of the cells lacking expression of all four reporters (''negative cell population'') did contain integrated proviruses. Such positive qPCR signal could derive from integrated defective viruses, wherein the reporters have been deleted, or cellular pathways silenced expression of both reporter cassettes.
Multiply spliced HIV transcripts were detected only in the productively infected CD4 + T cells (V5-NGFR Pos /HSA and mCherry Pos ) and not in the LTR-silent (V5-NGFR Pos ) or Figure 2. Identification of latently and productively infected CD4 + T cells (A) Experimental design to analyze CD4 + T cells infected with pMorpheus-V5. CD4 + T cells from four healthy donors were stimulated with IL-2 or a combination of IL-2 with a-CD3/CD28 antibody-coated beads for 3 days prior to infection with pMorpheus-V5. The frequency of latently and productively infected cells was determined 5 dpi using flow cytometry. (B) Frequency of latently (V5 POS , NGFR POS ; yellow) and productively (mCherry POS , V5 POS , NGFR POS , p24 POS ; green) infected CD4 + T cells stimulated with IL-2 alone or IL-2/a-CD3/CD28. Cells were analyzed using flow cytometry 5 dpi. Each of the four healthy donors is identified by symbol. Error bars represent the average ± SD, and statistical significance was established using unpaired t test. (C) Relative proportion of pMorpheus-V5 latently (yellow) or productively (green) infected CD4 + T cells stimulated with IL-2 is shown. The data represent the average of the four healthy donors. Average LTR-silent 58% ± 4% versus average LTR-active 42% ± 4% (p = 0.0014, unpaired t test). (D) Relative proportion of pMorpheus-V5 latently (yellow) or productively (green) infected CD4 + T cells activated with a-CD3/CD28. The data represent the average of the four individual healthy donors. Average LTR-silent 52% ± 8% versus average LTR-active 48% ± 8% (p = 0.61, t test).
reporter-negative CD4 + T cells ( Figure 3D). Similarly, the raltegravir-treated cells lacked expression of multiply spliced HIV transcripts, while such expression in stimulated J-Lat cells was comparable with that of productively infected primary human CD4 + T cells.
Collectively, these data reinforce the notion that the difference between LTR-active and LTR-silent cells is not due to differences in the level of integrated proviruses but rather to low HIV gene expression rate, as revealed by the assessment of HIV early gene Tat/Rev RNA expression.
Identification of CD4 + T cell subpopulations that preferentially support HIV latency We next used CyTOF (Kotecha et al., 2010) to determine whether CD4 + T cell subsets differ in their propensity to support latency establishment. Primary human CD4 + T cells stimulated with IL-2 alone or IL-2-a-CD3/CD28 were infected with pMorpheus-V5 and stained for CyTOF. We first barcoded the individual infections with CD45 antibodies and then stained with a custom antibody cocktail recognizing the HIV-encoded reporters as well as 29 other cellular markers ( Figure S3A).
Multi-dimensional analysis identified the protein tags of latent infection (V5-NGFR POS only) in more than 50% of the cells analyzed ( Figures 4A, 4C, 4D, 5A, and 5B). The overall frequency of infection (both latent and productive) was 6.05% (donor 1) and 2.31% (donor 2) in IL-2-treated cells. Stimulation of CD4 + T cells with IL-2-a-CD3/CD28 increased the percentages of productively infected cells to 5.02% and 5.92%. The percentages of latently infected cells (V5-NGFR POS ) were 2.3% and 6% (IL-2 alone) and 5% and 5.9% when stimulated with IL-2-a-CD3/ CD28. The productively (V5-NGFG POS , mCherry POS , HSA POS , p24 POS ) infected cells instead amounted to 1.6% and 1.3% Figure 3. Quantification of proviruses and multiply spliced HIV transcripts in FACS-sorted CD4 + T cells (A) Representation of the experimental design to analyze provirus integration or multiply spliced transcripts in productive or latently infected cells from three different healthy donors. CD4 + T cells were activated with IL-2 and a-CD3/CD28 antibody-coated beads for 3 days prior to infection with pMorpheus-V5. CD4 + T cells were infected with pMorpheus-V5 in the presence and absence of raltegravir. Latently (orange), productively (green), and the reporter negative cell populations were sorted using FACS 5 dpi with pMorpheus-V5. RNA and DNA were extracted from the same samples. (B) Gag p24 concentration of culture supernatants was measured using ELISA at 5 dpi. Bar graph shows the production of p24 in pMorpheus-V5 or mock-infected cells in the presence and absence of raltegravir (RAL). Each donor is identified by a specific symbol. The average of three donors is shown (±SD) (****p < 0.0001, unpaired t test). (C) HIV integration was measured using a two-step quantitative Alu-PCR assay using total DNA from each sorted cell population (latent infection, yellow; productive infection, green; reporter expression-negative cells, gray). The bar graph depicts relative amounts of integrated proviruses. J-Lat 6.3 cells were used as a positive control. The mean ± SD of three independent experiments/donors are shown. Sorted cells from the same donor (N = 3) are identified by the same symbol. with IL-2 alone and 2.1% and 5.5% with IL-2-a-CD3/CD28 stimulation ( Figure 4A). In line with our previous data generated using flow cytometry, the relative percentage of productively infected cells increased overall with activation (compare IL-2-a-CD3/ CD28 with IL-2 alone in Figure 4B), while the proportion of latently infected cells was higher in the cells treated only with IL-2 ( Figures 4C and 4D).
We visualized the latently and productively infected cells at the single-cell level using viSNE, which uses t-stochastic neighbor embedding (t-SNE) to generate a two-dimensional map of the latency protein tag expression in different CD4 + T cell populations ( Figure 4E).
The CyTOF antibody panel used allowed the identification and visualization of the distribution of seven different CD4 + T cell subsets using viSNE ( Figure S4E). Of note, activation with IL-2 + a-CD3/CD28 modified the distribution of the CD4 + T cell subsets in the absence of pMorpheus-V5 infection ( Figures S4A-S4C).
Across the CD4 + T cell subsets, the proportion of cells harboring LTR-silent proviruses (V5-NGFR pos only ) was higher than that of productively infected cells (Figures 5A and 5B). We noted that LTR-silent infections were more common in cells stimulated with IL-2, while cell activation with IL-2 + a-CD3/ CD28 reduced the relative proportion of latently infected cells ( Figures 5B and 5C). viSNE maps further visualize LTR-silent and LTR-active proportions in each distinct CD4 + T cell populations ( Figures S5A and S5B). Of note, the vast majority of infected naive T cells harbored latent proviruses. We observed that CD4 + T EM and CD4 + T REG have the highest total number of latently infected cells, and IL-2/a-CD3/CD28 stimulation increases the percentage of productive infection in all subsets with the exception of T EM . Taken together, our data are in agreement with previous reports (Buzon et al., 2014) suggesting that latent infections occur across all seven CD4 + T cell subsets analyzed with CD4 + T EM and CD4 + T REG contributing heavily to the latent reservoir formation. Naive T cells are regarded as being refractory to infection, but several studies have shown that in vivo naive T cells bear integrated provirus, suggesting that they can be infected and are part of the latent reservoir (Venanzi Rullo et al., 2020;Zerbato et al., 2019). Indeed, we observe that a portion of naive cells support infection up to the level of integration (latent infection in naive T cells 1.53%-2.745% [IL-2] and 1.44%-2.395% [IL-2 + a-CD3/CD28]). Further studies will determine the extent to which naive T cells play a role in the establishment of HIV-1 latent reservoir.

Expression of activation and exhaustion markers confirms the latent and productive nature of infections with pMorpheus-V5
Expression of the activation markers CD69 and HLA-DR associated with productive infection (Marini et al., 2008;Sahu et al., 2006) but not with latent infection (Moso et al., 2019). We therefore analyzed the expression of CD69 and HLA-DR in latently and productively infected CD4 + T cells upon pMorpheus-V5 infection. We observed that both markers were expressed at higher levels in productively infected CD4 + T cells compared with latently infected, the negative cell populations, or mock-infected cells ( Figures 6A and 6C). In the absence of overt T cell activation, all the memory T cell populations showed higher expression of these two activation markers when productively infected (Figure 6B). The same was true following stimulation with IL-2 + a-CD3/CD28, with the exception of CD4 + T transitional memory (T TM ) cells. This T cell subset displayed only a marginally higher expression of CD69/HLA-DR in productively infected cells compared with latently infected, the negative populations, or mock-infected cells ( Figure 6D). Importantly, latently infected CD4 + T cells showed CD69/HLA-DR expression profiles that were comparable with the negative cells or mock-infected cells, providing validation for the fact that latently infected cells identified by pMorpheus-V5 infection are fundamentally distinct from the productively infected cells.
Recent studies have indicated that, among other markers, Tim-3 is enriched in CD4 + T cells harboring latent proviruses (Evans et al., 2018). We therefore analyzed the expression of this exhaustion marker on CD4 + T cells latently and productively infected with pMorpheus-V5. We found that Tim-3 was enriched in latently infected CD4 + T cells compared with productively infected cells as well as negative and mock-infected cells. The Tim-3 enrichment of latently infected cells was more pronounced in cells treated with IL-2 alone than with IL-2 + a-CD3/CD28 (Figures 7A and 7C). When analyzing the different CD4 + T cell populations, we found that the most pronounced differences were observed in the IL-2-only treated CD4 + -naive T cells and in the CD4 + T TM, with an average 6.3-fold difference (range 5.9-6.7) and 8.2-fold (range 3.3-13), respectively ( Figure 7B). Overall, Tim-3 expression increased in all populations with IL-2 + a-CD3/CD28 treatment except for the mock-infected cells ( Figure 7D).
Taken together, our CyTOF experiments detail the CD69/HLA-DR and Tim-3 expression profiles of the CD4 + T cell subsets undergoing productive and latent infection. Furthermore, the stark differences in expression of these T cell activation and exhaustion markers represent an important independent confirmation of the ability of pMorpheus-V5 to distinguish productive from latent infections.

DISCUSSION
A more comprehensive understanding of the mechanisms supporting HIV latency is needed to guide HIV cure strategies. The ability to detect and enrich for latently infected primary human cells is essential to dissect mechanisms controlling HIV latency establishment, maintenance, and provirus reactivation. Dual-color HIV reporters are powerful tools to distinguish latently and productively infected cells from uninfected cells (Calvanese et al., 2013;Dahabieh et al., 2014;Battivelli et al., 2018). However, many of these dual-reporter HIV vectors are suboptimal with respect to detecting latently infected cells because of promoter interference between the LTR and the LTR-independent promoter controlling expression of the phenotypic markers for productive and latent infection (Battivelli et al., 2018;Calvanese et al., 2013;Chavez et al., 2015;Dahabieh et al., 2013Dahabieh et al., , 2014Hashemi et al., 2016;Kim et al., 2019;Matsuda et al., 2015).
In this study, we generated a quadruple-reporter HIV vector (pMorpheus-V5) that efficiently identifies productive or latently infected primary CD4 + T cells (Figures 1A and 1B) with negligible promoter interference (Figures 1C and S2C). In addition, pMorpheus encodes all the accessory proteins, including Nef. Indeed, the down-regulation of the CD4 + receptor exclusively on the surface of cells harboring LTR-active proviruses indicates that pMorpheus-V5 expresses a functional Nef alby Vpu also does contribute somewhat to CD4 down-regulation (Wildum et al., 2006).
Upon infection with pMorpheus-V5, cells harboring latent proviruses are distinguished from productively infected cells using flow cytometry or CyTOF ( Figures 2B-2D, 4A, 4C, and 4D). On average, 64% of the non-activated infected cells and 51% of the activated infected cells harbored an LTR-silent provirus (total: six different healthy donors; Figures 2C, 2D, 4C, and 4D; Table S1). These data indicate that the level of latency establishment in primary CD4 + T cells might have been previously underestimated (Battivelli et al., 2018). This could be because the two reporter expression cassettes in pMorpheus-V5 (LTR-dependent and LTR-independent) are distantly positioned in a manner that minimizes promoter interference. Indeed, some reporter viral vectors (Battivelli et al., 2018) carry both reporters in the Nef ORF, possibly mediating promoter interference between the HIV-1 5 0 LTR and the LTR-independent EF1-a promoter (Greger et al., 1998). Furthermore, the strength of the PGK promoter present in pMorpheus-V5, which might overcome the action of epigenetic factors that cause HIV latency through LTR silencing, could contribute to these observations. For instance, data reported about the impact the HUSH complex, that mediates trimethylation of H3K9 on both endogenous and exogenous retro-elements (Tchasovnikarova et al., 2015;Robbez-Masson et al., 2018), and thought to play a major role in latency (Chougui et al., 2018;Yurkovetskiy et al., 2018), show that the PGK promoter, unlike retroviral LTRs and other cellular promoters, is only minimally affected such activity (Tchasovnikarova et al., 2015).
Our data indicate that HIV latency was established upon integration given that ''early transcription'' of multiply spliced RNA of regulatory proteins, Tat and Rev, is absent in latently infected cells ( Figure 3D). Additionally, in both latent and productive infections, we show similar levels of integration ( Figure 3C). It is worth noting that a small portion of the cells lacking expression of all four reporters (''negative cell population'') harbor integrated proviruses but lack evidence of HIV multiple splice transcripts. This might be due to the presence of integrated defective proviruses or epigenetic silencing of both promoters. Figure 6. Validation of latent and productive infection by measuring early activation markers CD69 and HLA-DR in different CD4 + T cell subsets (A) CD69/HLA-DR expression on latently and productively pMorpheus-V5-infected primary human CD4 + T cells stimulated with IL-2 was determined using CyTOF. Bar graphs represent levels of CD69 and HLA-DR in total CD4 + T cells. Each donor is indicated by a unique symbol. The infection status of each CD4 + T cell subset is color coded in the figure. The average of two individual donors is shown. (B) CD69/HLA-DR expression on latently and productively pMorpheus-V5-infected CD4 + T subsets stimulated with IL-2 was determined. The levels of CD69+/ HLA-DR+ in mock-infected, reporter-negative cells, latently infected, and productively infected CD4 + T cell subsets stimulated were measured using CyTOF. The average of two individual donors is shown. (C) CD69/HLA-DR expression on latently and productively pMorpheus-V5-infected primary human CD4 + T cells stimulated with IL-2 and a-CD3/CD28 was determined using CyTOF. Bar graphs represent levels of CD69 and HLA-DR in total CD4 + T cells. Each donor is indicated by a unique symbol. Results are from two independent donors. (D) CD69/HLA-DR expression on latently and productively pMorpheus-V5-infected CD4 + T subsets stimulated with IL-2 and a-CD3/CD28. The levels of CD69/ HLA-DR in mock-infected, reporter-negative cells, latently infected, and productively infected CD4 + T cell subsets were measured using CyTOF. The average of two individual donors is shown.
Cell Reports Methods 2, 100238, June 20, 2022 9 Article ll OPEN ACCESS Indeed, defective proviruses are common, especially in patients undergoing antiretroviral treatment (Bruner et al., 2016). A broad range of deletions have been observed in the genome of these proviruses, some of which could still provide antigenic activity (Bruner et al., 2016;Pollack et al., 2017). It will be of interest to determine whether these ''reporter-negative CD4 + T cells'' display a distinct integration landscape or are enriched for defective proviruses.
We identified the CD4 + T cells subsets most prone to latency establishment using CyTOF (Figures 5A and 5B). By measuring expression of activation and exhaustion markers, CD69/HLA-DR as well as Tim-3, we also present independent validation that V5-NGFR-only positive cells are bona fide latently infected ( Figures 6A-6D and 7A-7D). In the future, it will be of interest to study the kinetics of latency establishment and confirm CD4 + T cells subset specific latency by assess to what extent Figure 7. Investigation of immune exhaustion maker Tim-3 in latently and productively infected cells in different CD4 + T cell subsets (A) Tim-3 expression on latently and productively pMorpheus-V5-infected primary human CD4 + T cells stimulated with IL-2 was determined using CyTOF. Bar graphs represent levels of Tim-3 in total CD4 + T cells. Each donor is indicated by a unique symbol. The infection status of each CD4 + T cell subset is color coded in the figure. Results are from two independent donors. (B) IL-2 stimulated CD4 + T cells were infected with pMorpheus-V5. The levels of Tim-3 POS in mock-infected, reporter-negative cells, latently infected and productively infected CD4 + T cell subsets stimulated were measured using CyTOF. The average of two individual donors is shown. (C) Tim-3 expression on latently and productively pMorpheus-V5-infected primary human CD4 + T cells stimulated with IL-2 and a-CD3/CD28 beads was determined using CyTOF. Bar graphs represent levels of Tim-3 in total CD4 + T cells. Each donor is indicated by a unique symbol. Results are from two independent donors. (D) Tim-3 expression on latently and productively pMorpheus-V5-infected CD4 + T subsets stimulated with IL-2 and a-CD3/CD28 beads was determined. The levels of Tim-3 POS in mock-infected, reporter-negative cells, latently infected, and productively infected CD4 + T cell subsets were measured using CyTOF. The average of two individual donors is shown.
Article ll OPEN ACCESS other exhaustion markers, such as PD-1, TIGIT, and LAG-3, shown to be of relevance to the latent reservoir (Fromentin et al., 2016), are enriched in latently infected cells detected by pMorpheus-V5. We found that CD4 + T EM and T REG are the subsets where HIV latency establishment occurred with higher frequency ( Figures 5A and 5B). Previous reports show that CD4 + T EM cells contain the largest reservoir in which HIV reactivation has been observed (Kulpa et al., 2019;Buzon et al., 2014). CD4 + T EM cells also harbor more intact proviruses (Hiener et al., 2017), while maintaining a higher proliferation rate and a short half-life (Vukmanovic-Stejic et al., 2006;Farber et al., 2014). T REG s also represent an important HIV reservoir because of their potential to expand in chronically HIV-infected patients (Moreno-Fernandez et al., 2012). Moreover, T REG s contribute more to the HIV cellular reservoir than non-T REG s in HIV-infected patients on antiretroviral therapy (ART) (Tran et al., 2008). Their impact on the rapid establishment of the HIV reservoir could be due to their ability to reverse the immune activation status of CD4 + T cells (Moreno-Fernandez et al., 2012). Although with much lower frequency than for the T central memory (T CM ) and T TM compartments, proviruses are almost always detected in naive cells of people with HIV (Chomont et al., 2009;Bacchus et al., 2013;Douek et al., 2002;Lambotte et al., 2002;Zerbato et al., 2016). Similarly, we observed establishment of latency in the CD4 + -naive T cells subset following either stimulation or activation ( Figures 5A-5C). Indeed, CD4 + -naive T cells were the more prone subset to harbor LTR-silent proviruses. These results suggest that CD4 + -naive T cells may serve as an important reservoir of LTR-silent proviruses.
The molecular mechanisms supporting these differences require further targeted investigations, which now become feasible using pMorpheus-V5. For example, cell-to-cell transmission, through the formation of virologic synapses is a critical mode of HIV infection (Groot et al., 2008;Hubner et al., 2009;Jolly et al., 2004), as it is exponentially more efficient than cellfree transmission and far more antibody neutralization-resistant (Chen et al., 2007). Recently it has also been reported that cellto-cell transmission from an activated CD4 + T cell to a resting one preferentially results in latent infection (Agosto et al., 2018). Although the current version of pMorpheus-V5 generates single round viruses, one can envision studies that will involve a replication competent version of our reporter virus that will enable us to gain new insight in latency establishment in the context of HIV cell-to-cell transmission.
Last not least, pMorpheus-V5 will be an excellent tool for screening for drugs targeting cellular signaling networks and epigenetic complexes (Agosto et al., 2015;Gagne et al., 2019) that selectively prevent or reverse latency establishment in primary human CD4 + T cell subpopulations.
Limitations of the study The pMorpheus system was built with two reporter cassettes inserted in different HIV regions in order to decrease the chance of promoter interference. The PGK promoter was chosen as an LTR-independent promoter because of the constitutive nature of its activity in combination with its low susceptibility to silencing by epigenetic mechanisms such as the HUSH complex (Tchasovnikarova et al., 2015). Nonetheless, there are some charac-teristics of the system that deserve further clarification in the future. For example, the insertion of the NGFR cassette deleted a cryptic exon, contained in the Env ORF and typically present in HIV IIIB. This cryptic exon forms a fusion protein called Tnv of unknown properties. Its deletion has been reported to be without major consequences (Gottlinger et al., 1992), but this deletion could modify the pMorpheus phenotype. Likewise, it might be of interest to investigate whether cryptic splicing is responsible for the absence of PGK driven Nef expression, as Nef is preceded by an IRES that, theoretically, could lead to its translation. The CyTOF experiments allowed us to begin investigating latency establishment in CD4 + T subset-dependent manner. The results obtained provided us with insights about the potential importance of naive CD4 + T cells in the establishment of the latent reservoir and, moreover, provided independent validation of the nature of pMorpheus-V5 infections. Nonetheless, the CyTOF experiments presented here were performed using cells from only two independent healthy donors, resulting in some expected donor variability (Table S1), and future experiments with additional donors are needed to confirm and extend our observations.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Viviana Simon (Viviana.simon@mssm.edu).

Materials availability
Availability of the plasmid encoding the pMorpheus-V5 reporter construct is subject to Material Transfer Agreements.
Data and code availability d All data reported in this paper will be shared by the lead contact upon request. d This paper does not report original code. d Any additional information required to reanalyze the data reported in this manuscript is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Primary CD4 + T cell isolation and cell culture Leukopaks were obtained from anonymous healthy blood donors from the New York Blood Center. Peripheral blood mononuclear cells were purified by Ficoll paque (GE Healthcare) density centrifugation and primary CD4 + T cells were purified by negative selection of CD4 + cells using CD4 + T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. CD4 + T cells were cultured in RPMI 1640 supplemented with 10% FBS (Gibco/Thermo Fisher), 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.1 M HEPES, 2 mM L-glutamine in the presence of recombinant human IL-2 (20 U/mL) (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) at 37 C in a 5% CO 2 humidified incubator.

Construction of pMorpheus-V5 and virus stock production
The backbone of the pLAI2-V5-NGFR-HSA-mCherry-IRES-Nef plasmid (renamed in the text to pMorpheus-V5) is based on the pLAI2 HSA-mCherry-IRES-Nef full length viral construct (Young et al., 2018), which encodes the full-length HIV-1 strain LAI (Peden et al., 1991) as well as the HSA-mCherry-IRES-Nef cassette in the Nef ORF. The cassette encoding the PGK promoter followed by V5-NGFR (Wroblewska et al., 2018) was inserted in the envelope region encompassing the SalI and SmaI restriction sites. The construct was verified by sequencing. The virus stocks were produced by transfecting HEK 293T cells with 20 mg of pLAI2-V5-NGFR-HSA-mCherry-IRES-Nef and 5 mg of expression plasmid encoding the Env of pSV III-92 HT 593.1 subtype B virus R5 aid X4 (dual-tropic). After approximately 20 h, the transfection medium was changed, and the viral supernatants were collected at 48 and 72 hrs after transfection. The viral supernatants were first filtered (0.45 mm) and then concentrated using the Lenti-X concentrator (Takara) following manufacturer's instruction.