HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function.

KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting ln-cRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migrationregulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection. KEYWORDS Kaposi's sarcoma-associated herpesvirus, KSHV, human immunodeficiency virus, HIV, HIV-1 Tat, long noncoding RNA, lncRNA K aposi's sarcoma (KS) is a multicentric malignancy of endothelial origin and the most common AIDS-defining neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus also known as human herpesvirus type 8 (HHV-8) (1). As a result of the AIDS pandemic, KS has become one of the most common cancers in developed countries, where it causes significant morbidity and mortality. However, KSHV infection is necessary but not sufficient for KS development, and other cofactors are required for malignant transformation (2,3). Among them, human immunodeficiency virus (HIV) infection is the main cofactor, and supporting evidence includes the following: (i) the incidence of KS is 1 in 100,000 in the general population (4) but increases approximately 20,000-to 50,000-fold in HIV-infected patients (5,6) and (ii) the highly active antiretroviral therapy (HAART) targeting HIV has markedly decreased the incidence of KS (7,8). However, although HAART has markedly decreased the incidence of KS (9,10), up to 50% of AIDS-associated KS (AIDS-KS) patients never achieve total remission under HAART (11). Therefore, AIDS-KS remains the second most frequent tumor in HIV-infected patients worldwide (7,8,12), and development of new intervention strategies is still needed.
Although it has been proposed that immunodeficiency itself is the main determinant of KS, the incidence of KS in patients with HIV infection is 300-fold higher than in persons with other types of immunosuppression (6). Moreover, individuals with KSHV-HIV coinfection have an increase of 60%/year in the probability of developing KS after HIV infection (13). Since HIV and KSHV infect different cell types (HIV infects primarily CD4 ϩ T cells and tissue macrophages, and KSHV infects primarily endothelial and B cells), two crucial paracrine mechanisms have been proposed by which HIV promotes KS pathogenesis: (i) promotion of cytokine production from HIV-infected CD4 ϩ T cells and (ii) secretion of HIV proteins, particularly HIV Tat (reviewed in reference 14). The detection of HIV Tat in spindle cells of AIDS-associated KS lesions (15) further supports the second notion.
Studies have initially focused on the potential role of HIV Tat protein in KS formation due to the discovery of a dermal lesion resembling KS in HIV Tat transgenic mice (16) and the promotion of endothelial cell growth (17), migration (18), angiogenesis (19), and tumorigenesis (20) by extracellular HIV Tat. The synergistic effects of HIV Tat and KSHV in modulating KS formation that have been identified include the following: (i) increasing KSHV infectivity in endothelial cells (21); (ii) increasing the expression (22), signaling activity (23,24), and tumorigenicity (24) of vGPCR; (iii) promoting viral interleukin-6 (vIL-6)-induced angiogenesis and tumorigenesis (25); and (iv) enhancing K1-induced cell proliferation and angiogenesis (26). The acceleration of kaposin A (27)and vIL-6 (25)-mediated tumorigenesis by HIV Tat has also been observed in fibroblasts. Interestingly, except for kaposin A, the viral genes modulated by HIV Tat are all KSHV lytic genes, suggesting that the lytic cycle of KSHV is necessary to support KS formation in HIV-infected individuals.
Mechanistic studies have revealed that HIV Tat potentiates the oncogenic activity of KSHV proteins mainly through the regulation of the PI3K/Akt pathway (23,25,27). In addition to signal transduction, one recent report showed that HIV Tat may synergize with a KSHV viral protein to induce the expression of microRNAs (miRNAs), a type of small noncoding RNAs (ncRNAs), which consequently leads to the induction of angiogenesis (26). Beyond miRNAs, long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides (nt) (28), are emerging as a new large class of functional ncRNAs (29). lncRNAs commonly originate from intergenic regions (known as lincRNAs) but share many features with mRNAs, including RNA polymerase II transcription, 5=-methylguanosine capping, 3=-polyadenylation, and RNA splicing (30). However, they are expressed at a lower level than mRNAs (31) and often display high tissue specificity, implying that they may function in a cell type-specific manner (29). Accumulating data suggest that lncRNAs can exhibit diverse biological functions through binding to protein, DNA, or RNA and serve as scaffolds or decoys. It is not surprising that aberrant lncRNA expression is associated with various diseases, including cancer. To date, thousands of lncRNAs have been annotated by genome-wide studies, yet the precise function has been determined for only a small population.
Recently, lncRNAs have been linked to viral infection as well (32,33). The lncRNA uc002yug.2 enhances HIV-1 reactivation through upregulation of HIV Tat protein expression (34). The lncRNA NRON represses HIV replication through interacting with and promoting the ubiquitylation and degradation of HIV Tat (35). These findings prompted us to hypothesize that HIV Tat may potentiate KS formation through binding to lncRNAs upregulated during KSHV reactivation. To study this, we first performed a transcriptome analysis in latent and lytic KSHV-infected cells treated with or without HIV Tat and identified 60 lncRNAs that were upregulated upon KSHV reactivation in both the presence and absence of HIV Tat. RNA immunoprecipitation (RIP) and RNA pulldown assays showed that one lncRNA identified in this screen, LINC00313, interacted with HIV Tat and polycomb repressive complex 2 (PRC2). Importantly, overexpression of LINC00313 significantly inhibits the migration, invasion, and tube formation of HMEC1 cells and human umbilical vein endothelial cells (HUVECs), and this inhibition may be rescued by HIV Tat. Consistent with our cell culture data, transcriptional profiling followed by Ingenuity Pathway Analysis (IPA) showed that LINC00313 tends to inhibit cell movement and migration, and inhibition of these pathways was no longer observed after HIV Tat treatment. Together, our study revealed a novel HIV Tatinteracting lncRNA, LINC00313, that links the KSHV and HIV virus-virus interaction in mediating KS progression.

RESULTS
Genome-wide transcriptome profiling reveals the functional impact of HIV Tat in KSHV latent and lytic reactivated SLK cells. To explore the role of HIV Tat in KSHV-infected endothelial cells, the ability of our purified recombinant HIV Tat protein (see Fig. S1A in the supplemental material) to enter SLK cells was first examined by immunostaining. The results showed that HIV Tat was mainly localized to the nucleus (Fig. S1B), which is consistent with the detection of nuclear-localized HIV Tat after ectopic expression (Fig. S1C) and in KS clinical samples (Fig. S1D). Following this confirmation, a high-throughput RNA sequence (RNA-seq) analysis was performed using RNAs isolated from iSLK-BAC16 cells, a doxycycline (Dox)-inducible recombinant KSHV-harboring SLK cell line, that was treated with 1 g/ml Dox in the presence or absence of 0.2 g/ml purified HIV Tat. As shown in Fig. 1A, following alignment of the raw reads to the Hg38 human genome using CLC Genomics Workbench 11 and annotation of the transcripts with RefSeq82 using Partek Genomic Suite v.7.0 (Partek, St. Louis, MO), we identified 13,160 mRNAs (reads per kilobase of transcript per million mapped reads [RPKM] Ͼ 0.05) that were expressed in iSLK-BAC16 cells under at least one of the treatment conditions. When the gene expression profiles between latent infected and lytic reactivated iSLK-BAC16 cells that were treated with or without HIV Tat were compared, around 1,000 to 4,000 genes were regulated (Ն1.5-fold change) by viral reactivation (Dox versus control, 31%) and HIV Tat treatment (HIV Tat versus control, 10%) (Fig. 1B). Unsupervised hierarchical clustering analysis showed that latent infected and lytic reactivated samples were grouped in separate clusters and that HIV Tat treatment was clustered with its corresponding untreated group, demonstrating that HIV Tat affects host gene expression in both the latent and lytic phases of the KSHV life cycle (Fig. 1C).
To explore the potential mechanisms that HIV Tat uses to modulate gene expression in SLK cells during the KSHV lytic cycle, genes that were differentially expressed upon KSHV reactivation with or without HIV Tat treatment (Fig. 1B, upper pie chart) were subjected to pathway analysis using Ingenuity Pathway Analysis (IPA) software. We used an activation Z-score to identify the most important downstream signaling pathways modulated by HIV Tat upon KSHV reactivation and considered a Z-score greater than or equal to 2 as significant activation and a Z-score less than or equal to Ϫ2 as significant inhibition of the functional activity identified. As shown in Fig. 1D, genes altered upon KSHV reactivation regulate 8 main functions connected to 26 cell processes. Among them, 13 pathways lost their Z-score significance after HIV Tat treatment (Fig. 1D). These pathways include disinhibition of cell tubulation, proliferation and metastasis, and deactivation of cell death.

Soluble HIV Tat protein promotes cell motility and viral reactivation in KSHV lytic reactivated SLK cells.
Following the pathway analysis, cell proliferation, migration, and invasion were evaluated in iSLK-BAC16 cells treated with Dox (1 g/ml) and/or HIV Tat (0.2 g/ml). Cells without any treatment were used as a control. Our data showed that KSHV reactivation significantly reduced cell number on day 5 compared to the control and that HIV Tat treatment might slightly recover the cell number during reactivation ( Fig. 2A and Fig. S2A). This observation agrees with our IPA analysis showing that enriched cell death pathways during KSHV reactivation were diminished by HIV Tat treatment (Fig. 1D). Consistent with previous reports (18), the addition of HIV Tat had a stimulatory effect on cell migration ( Fig. 2B and C). More importantly, HIV Tat slightly increased KSHV reactivation-induced cell migration ( Fig. 2B and C) and significantly enhanced the properties of invasion of iSLK-BAC16 cells ( Fig. 2D to E).
We further analyzed viral titers at 72 and 96 h after Dox-induced KSHV reactivation. Consistent with previous reports indicating that HIV Tat was able to enhance KSHV lytic reactivation in B lymphoma cells (36,37), our data showed that soluble HIV Tat can also enhance the virus production in SLK cells at 96 h after KSHV reactivation but not in latent control cells ( Fig. 2F; see Fig. S2B in the supplemental material). These data suggested that internalized soluble HIV Tat protein may promote angiogenesis-related functions in KSHV-infected cells through enhancement of KSHV reactivation. However, we cannot exclude the possibility that HIV Tat may enhance angiogenesis through other regulatory mechanisms, including effects on latently infected cells.
Transcriptome profiling of lncRNAs in iSLK-BAC16 cells upon KSHV reactivation. In recent years, thousands of lncRNAs have been identified by high-throughput sequence technology. Increasing numbers of reports suggest that lncRNAs participate in multiple cellular processes. In an attempt to identify lncRNAs whose expression is differentially regulated in cells upon KSHV reactivation and HIV Tat treatment, we reannotated the RNA-seq reads with NONCODE v5.0 using Partek Genomic Suite v.7.0 (Partek, St. Louis, MO), followed by changing the NONCODE designations (IDs) to RefSeq IDs. We identified 1,469 lncRNAs (RPKM Ͼ 0.01) that were expressed in iSLK-BAC16 cells under at least one treatment condition (Fig. 3A). Among them, 325, 219, and 227 lncRNAs were differentially regulated (Ն2-fold change) by Dox (23%), HIV Tat (16%), and Dox-plus-HIV Tat treatment, respectively (Fig. 3B). To identify lncRNAs that were regulated by HIV Tat under KSHV reactivation conditions, the lncRNAs that were differentially regulated under Dox treatment in the absence (325) and presence (227) of HIV Tat were compared, and 115 lncRNAs were identified (Fig. 3C). Unsupervised hierarchical clustering analysis grouped these lncRNAs into 4 distinct clusters (Fig. 3D). The Dox-upregulated lncRNAs that were upregulated (7) or downregulated (38) by HIV Tat under KSHV reactivation conditions were grouped into clusters 1 and 2, respectively. Clusters 3 and 4 contained Dox-downregulated lncRNAs that were upregulated (39) or downregulated (8) by HIV Tat treatment upon KSHV reactivation, respectively. As shown in Fig. 3E, the 115 lncRNAs were plotted by fold change (log 2 ) in expression upon KSHV reactivation, and the top 5 up-and downregulated lncRNAs were listed. However, using reverse transcriptase quantitative PCR (RT-qPCR) analysis, we could  S3); we therefore focused our efforts on this lncRNA. LINC00313 is upregulated during KSHV reactivation and interacts with HIV Tat. Since lncRNAs can bind to various proteins to form functional complexes and HIV Tat is a well-known RNA-binding protein, we further asked whether HIV Tat may modulate cellular function by interacting with KSHV reactivation-upregulated lncRNAs. To this end, an RNA immunoprecipitation (RIP) experiment was performed. Following confirmation of LINC00313 upregulation upon KSHV reactivation (Fig. 4B), RIP was conducted in SLK cells overexpressing Flag-tagged HIV Tat. lncRNA LINC00640 that was expressed at a relative high level in SLK cells was included as a control (Fig. 4A). Following RIP, immunoblotting was first performed to confirm the successful immunoprecipitation of HIV Tat (Fig. 4C, left panel). RIP followed by RT-qPCR showed the enrichment of LINC00313 by Flag-HIV Tat but not the Flag-tagged control (Fig. 4C, right panel). These data indicate that HIV Tat may potently bind to endogenous LINC00313 in SLK cells. This interaction was further confirmed by an in vitro RNA pulldown assay. Total cell lysates (TCLs) prepared from SLK cells overexpressing the Flag tag or Flag-tagged HIV Tat were incubated with biotinylated LINC00313 RNA and subjected to the pulldown procedure using streptavidin beads. Immunoblotting results confirmed the interaction between HIV Tat and LINC00313 (Fig. 4D).
Exogenous LINC00313 inhibits endothelial cell motility and tube formation. To probe the angiogenic role of LINC00313 by using an overexpression strategy, the expression level of LINC00313 was first analyzed in human endothelial cell lines HMEC1 and HUVEC, together with SLK cells. RT-qPCR data showed that HMEC1 cells and HUVECs express relatively low levels of LINC00313 (Fig. 5A), and they were used for ectopic expression experiments. Following transduction of HMEC1 cells and HUVECs with control or lentivirus-expressing LINC00313, the successful overexpression of LINC00313 was first confirmed by RT-qPCR (Fig. 5B). The LINC00313-overexpressed HMEC1 cells and HUVECs treated with or without HIV Tat were subjected to migration,

HIV Tat antagonized LINC00313 function through inhibition of the LINC00313-PRC2 interaction.
To elucidate the mechanisms responsible for LINC00313 that were opposed by HIV Tat, the genes regulated by LINC00313 overexpression in the presence or absence of HIV Tat were revealed by an RNA-seq survey of HMEC1 cells (Fig. 6A). The analysis indicated that more genes were downregulated (1,152; 8%) by LINC00313 overexpression than were upregulated (761; 5%) in comparison with the control (Fig. 6B, left pie chart). Interestingly, following HIV Tat treatment, more genes were upregulated (855; 6%) than downregulated (770; 5%) ( Fig. 6B, right pie chart). With additional dissection of LINC00313-downregulated genes, we found that 526 (45.6%) of the genes became upregulated upon HIV Tat treatment and only 130 (11.2%) of the genes remained downregulated (Fig. 6C). Bioinformatics analysis of LINC00313-downregulated genes (1,152) using IPA showed a total of 54 pathways (Table S1) that were significantly enriched with Z-score hits of Ն2.0. Among them, only 4 pathways (Table S1) were identified after HIV Tat treatment. This result suggests that HIV Tat may relieve LINC00313-mediated transcription repression.
Among chromatin remodeling complexes shown to be recruited by lncRNAs, PRC2, a histone H3 lysine 27 (H3K27) methyltransferase complex, is one of the most well studied (40). Since H3K27me3 is a well-known repressive histone mark (41), we hypothesized that LINC00313 may be a novel PRC2-interacting lncRNA. HIV Tat may interact with LINC00313 and disrupt this association. To study this, we performed an RNA pulldown experiment. TCLs prepared from SLK cells overexpressing Flag-tagged EED, EZH2, and SUZ12 alone or with hemagglutinin (HA)-tagged HIV Tat were incubated with biotinylated in vitro-transcribed LINC00313 RNA and subjected to the pulldown procedure using streptavidin beads. Consistent with our hypothesis, immunoblotting results showed that LINC00313 interacts with components of PRC2 (Fig. 7A). Interestingly, the interaction between LINC00313 and EZH2 was inhibited by HIV Tat overexpression (Fig. 7A). These data suggest that LINC00313 may mediate transcription repression through recruitment of the PRC2 complex. HIV Tat may interact with LINC00313 and disrupt this association. This notion was further supported by RT-qPCR analysis of two LINC00313-repressed genes, those encoding corticotropin releasing hormone binding protein (CRHBP) and tumor suppressor protein p63 (TP63) (Fig. 7B), whose expression patterns confirmed Tat-dependent derepression of LINC00313mediated transcriptional effects.

DISCUSSION
Preexisting HIV infection followed by secondary KSHV infection induces KS development in up to 50% of HIV-positive patients, demonstrating that KSHV is one of the most potent oncogenic human viruses in this special setting (42). However, KSHV   (24)(25)(26)(27)(43)(44)(45). Previous reports indicated that viral proteins secreted by HIV-infected cells may be taken up by KSHV-infected cells and consequently dysregulate the KSHV latent to lytic infection switch (39,46) and promote disease progression (24-27, 43, 45). However, it should be note that KS tumor cells are almost exclusively latently infected (1)(2)(3)(4)(5). Thus, the effects of HIV secretory proteins may be limited to early stages of KS pathogenesis, when lytic replication can promote KS, or to a subpopulation of infected cells undergoing lytic replication against a larger background of latently infected tumor cells. It has long been known that HIV Tat promotes a KS-related phenotype, including migration, angiogenesis, and tumorigenesis (18)(19)(20). Studies focused on the interaction between KSHV and HIV Tat showed that the synergistic effect of HIV Tat and KSHV in KS formation can be explained, in part, by an increase in KSHV infectivity of endothelial cells (21). In addition, both intracellular, overexpressed HIV Tat protein and extracellular, treated HIV Tat protein have been shown to enhance KSHV lytic reactivation in B lymphoma cells (36,37). While HIV load may correlate with the production of HIV Tat, HIV infection has also been identified as responsible for inducing KSHV reactivation (47)(48)(49). Since persistent lytic reactivation of KSHV is a prerequisite for KS pathogenesis, these results implicate the induction of KSHV reactivation by HIV Tat as a participant in promoting KS pathogenesis (21,50). In line with this, accumulating reports have shown that both soluble HIV Tat and ectopically expressed HIV Tat are able to synergize KSHV latent and lytic viral protein-mediated angiogenesis and tumorigenesis (22,24,25). However, most of the KSHV reactivation studies have been done in primary effusion lymphoma (PEL) cells, not in endothelial cells, the cell type of KS tumor origin. Moreover, the knowledge gained has been largely dependent on overexpression of individual KSHV proteins and not on the whole viral context. Here, by using viruscontaining SLK cells, we confirmed that KSHV reactivation increased cell migration and invasion ability and that these phenotypes can be further enhanced by HIV Tat (Fig. 2B to E). Our data showed that HIV Tat was also able to enhance KSHV reactivation in SLK cells after prolonged treatment (Fig. 2F). Surprisingly, bioinformatics analysis of transcriptome data from KSHV-containing SLK cells undergoing lytic reactivation showed inhibition of tubulation and metastasis (Z-score less than or equal to Ϫ2) (Fig. 1D). This result suggests that though many KSHV lytic proteins have angiogenesis potential, in the whole virus context, KSHV reactivation tends to modulate genes negatively associated with cell migration and tube formation. This may also explain why KSHV infection alone induces KS inefficiently. The loss of tubulation and metastasis pathways after HIV Tat treatment (Fig. 1D) suggests that HIV Tat may enhance an angiogenesis phenotype through interference with those pathways. Although our initial KSHVrelated studies were carried out in SLK cells of epithelial origin, we subsequently confirmed these findings using endothelial cell lines HMEC1 and HUVEC (Fig. 5 and 6).
It should be noted that HIV Tat protein has been detected in the serum of HIV-infected patients at levels in the nanomolar range (38,51). The levels of HIV Tat protein required for increased KSHV infectivity (10 nM) were 10-to 15-fold higher than the HIV Tat levels detected in serum collected from HIV-infected patients (21). Moreover, soluble HIV Tat protein used to study its synergistic effect with KSHV Orf-K1 protein was 0.2 g/ml (13 nM) (26). It is likely that the concentration of HIV Tat in KS is underestimated, since the amount of Tat protein may be much higher in the vicinity of HIV-infected cells (52). Moreover, the proportion of Tat protein might be concentrated on the surface of the cell membrane via adsorption by heparan sulfates (53,54). Following our immunofluorescence assay (IFA) result showing that HIV Tat can be efficiently internalized into SLK cells after incubation of cells with 0.2 to 1 g/ml (13 to 65 nM) of HIV Tat, 0.2 g/ml of HIV Tat was used in this study.
Interestingly, previous studies have shown that miRNAs play important roles in mediating the synergistic effect of HIV auxiliary proteins in KSHV-mediated angiogenesis in tumor progression (26,43,45). Emerging evidence shows that in addition to small noncoding RNAs such as miRNAs, mammalian cells also have lncRNAs with multiple biological functions. lncRNAs have been shown to regulate gene expression through direct interaction with other biomolecules, such as proteins and miRNAs, and function either as scaffolds to recruit proteins to or as sponges to remove miRNAs and proteins from their targets. Interestingly, HIV Tat is a well-known RNA binding protein and has recently been found to interact with lncRNA NRON (35). Here, we identified KSHV reactivation-upregulated LINC00313 (Fig. 3D) as a novel HIV Tat-interacting lncRNA (Fig. 4). lncRNAs have been long proposed to function as epigenetic regulators that recruit histone-modifying complexes, such as PRC2 (40), and consequently repress gene expression. Transcriptome analysis revealing more genes to be downregulated than upregulated by LINC00313 overexpression (Fig. 6B) supported the notion that LINC00313 may also function as a repressive epigenetic regulator. More importantly, ectopic expression of LINC00313 inhibited genes involved in cell movement and migration pathways (Fig. 6), as well as the migration and tube formation ability of endothelial cells (Fig. 5), and this inhibition can be rescued by treatment with HIV Tat (Fig. 5 and 6). Taken together, these data suggest that HIV Tat may interact with LINC00313 and consequently inhibit its function. However, since our RT-qPCR data showed that the LINC00313 level was significantly reduced upon HIV Tat treatment (Fig.  4A), we cannot exclude the possibility that HIV Tat may also modulate the transcription and/or stability of LINC00313. Our data here provide the first evidence that HIV Tat may also be capable of inducing angiogenesis and tumorigenesis in KSHV-infected cells by targeting KSHV reactivation-upregulated lncRNAs.
In summary, our work revealed LINC00313 as an lncRNA that is upregulated during KSHV reactivation, which inhibits endothelial cell migration and tube formation. Our biological studies together with bioinformatics analysis suggest that HIV Tat can interact with LINC00313 and rescue the endothelial cell migration and tube formation ability inhibited by LINC00313. These results not only provide a potential mechanism by which HIV Tat enhances endothelial cell angiogenesis function under KSHV reactivation conditions but also suggest that the lncRNA-HIV Tat axis may be responsible for the HIV-KSHV interaction that contributes to KS progression. Further studies will be needed to understand how LINC00313 is upregulated upon KSHV reactivation and whether other KSHV-regulated lncRNAs are also involved in HIV Tat-mediated KS pathogenesis.
For transfection, SLK cells were seeded at 2 ϫ 10 5 cells/well in a 6-well plate. Two micrograms of DNA was transfected in each well using X-tremeGENE HD DNA transfection reagent (Roche; catalog no. 06366236001) according to the manufacturer's instructions. For transduction, the control or pLenti4-CMV/TO-LINC00313 plasmid (0.4 g) was cotransfected with packaging plasmids pCMV-ΔR8.91 (0.3 g) and pCMV-VSV-G (0.1 g) into 293T cells by TransFectin lipid reagent (Bio-Rad; 170-3351). At 72 h after transfection, supernatants were collected, filtered, mixed with Polybrene at a final concentration of 8 g/ml, and applied to HMEC1 cells or HUVECs. Infection was accomplished by spin inoculation at 1,000 ϫ g for 2 h at room temperature (RT), followed by incubation at 37°C in a 5% CO 2 incubator for another 2 h.
RIP assay. RNA immunoprecipitation (RIP) was performed using a Magna RIP RNA-binding protein immunoprecipitation kit (Millipore; catalog no. CR203146) by following the manufacturer's protocol. Briefly, SLK cells were transfected with pcDNA3-Flag or pcDNA3-Flag-Tat for 48 h and used for RIP experiments with the anti-Flag M2 magnetic beads. The resultant immunoprecipitants and 10% total cell lysates (TCLs) were subjected to immunoblotting using anti-Flag antibody. The coprecipitated RNAs were extracted by acid phenol-chloroform, pH 4.5 (Ambion; AM9722). The levels of LINC00313 and LINC00640 were examined by real-time RT-qPCR.
In vitro transcription and RNA pulldown assay. To synthesize biotinylated LINC00313 RNA by in vitro transcription using a MEGAscript T7 transcription kit, the DNA template was prepared by digestion of 20 g pcDNA-Flag-LINC00313 DNA with XhoI at 37°C for 2 h, followed by ethanol precipitation with sodium acetate (pH 5.2). The in vitro transcription was performed by incubating 3 g of linearized DNA template with T7 RNA polymerase at 37°C for 2 h according to the manufacturer's instructions. The in vitro-transcribed RNA was purified using a MEGAclear kit (Ambion; AM1908). TCLs prepared from SLK cells transfected with pcDNA3-Flag or pcDNA3-Flag-Tat for 48 h were incubated with 1 g of biotinlabeled LINC00313 RNA with rotation at 4°C for 3 h, followed by the addition of streptavidin beads for another 1 h. The RNA-bound protein was detected by immunoblotting using anti-Flag antibody. Three percent of TCLs was used as the input.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.3 MB.