The combined antiretroviral therapy (cART) or HAART has improved the life span of people living with HIV (PLWH), however, the major hurdle in achieving complete cure of HIV is the persistence of latent reservoirs i.e. the cells harboring quiescent HIV provirus (1) (2) (3) . Resting CD4+ T cells are identified as one of major latent reservoirs; however, HIV infections are anatomically found in all vital organs including lungs (4, 5) , but the presence of latent nonimmune cell HIV reservoirs in these tissues remains unclear. These putative reservoirs may contribute significantly to the comorbidities of the organ. Frequency and the early onset of OLDs such as chronic bronchitis (CB) and chronic obstructive pulmonary disease (COPD) are significantly higher among PLWH (6) (7) (8) (9) . Airway mucus hypersecretion is associated with chronic bronchitis and COPD (10) and we have shown that, even after cART, lungs from HIV-infected humans and SIV-infected macaques contain large amounts of mucus (11) . In the lung, the mucus is produced by specialized secretory lung epithelial cells (goblet cells), which are also the key effectors of chronic bronchitis and COPD (12) . We have demonstrated that HIV-gp120 stimulates mucus formation in normal human bronchial epithelial (NHBE) cells (11) and epidemiological evidences suggest that HIV is an independent risk factor for the development of COPD (13, 14) .
The role of epithelial cells as one of the cellular targets of HIV infection is not clearly resolved. Foreskin epithelial cells express HIV coreceptors (15) and when the cells are infected with herpes simplex virus 2, the cells internalize HIV virions (16) . Although the virions do not replicate, they can be transferred to lymphocytes by coculturing (17) . On the other hand, NHBE cells express CXCR4 (C-X-C motif chemokine receptor-4) and the X4-tropic gp120 induces mucus in these cells (11) . Similarly, X4-tropic, but not R5-tropic, HIV impairs epithelial barrier integrity in NHBE cells (18) . Also, cynomolgus monkeys (CMs) infected with simian-adapted HIV (SHIV) contain large number of mucus containing goblet cells and a significant percentage of SHIV-infected airway epithelial cells are HIV-gp120-positive (19) ; however, whether HIV productively infects airway epithelial cells is still debatable (18, 20) . This question can be answered definitively, if the HIV-infected bronchial epithelial cells are shown to contain activatable integrated HIV provirus. In this study, we demonstrate that, following HIV infection, NHBE cells produce HIV p24 and become carriers of latent HIV provirus that can be activated by treating the virus-containing cells with lipopolysaccharide (LPS) and/or vorinostat. Moreover, DNA from HIV-1 infected NHBE cells contains HIV-specific genes and HIV-and SHIV-infected lung epithelial cells express HIV-specific RNAs.
Besides CD4, the CXCR4 and CCR5 (C-C motif chemokine receptor-5) are known HIV coreceptors (21) . The expression of CXCR4 on NHBE cells has been amply documented (11, 22, 23) , and we have also previously demonstrated that following infection, NHBE cells produce mucus in response to X4-but not to R5-tropic HIV-gp120 (11) ; however, the expression CCR5 on these cells is not unequivocal (18, 20) . During carcinogenesis, epithelial cells have been shown to express CCR5 that enhances their resistance to cytotoxicity (24) . We first analyzed the expression pattern of CD4 and other HIV co-receptors in normal disease-free NHBE cells.
Based on the qPCR and immunoblot analysis, NHBE cells expressed CD4 that was more than fifty-fold higher at mRNA levels ( Fig. 1A) and around five-fold higher at protein levels ( Fig. 1B) , respectively, in comparison with A549 cells (a human lung adenocarcinoma cell line of lung epithelial origin, used for experimental reference). These NHBE cells also showed high immunopositivity for CD4 (Fig. 1C) and CXCR4 ( Fig. 1D ) with 3-fold more abundance of CD4 compared to CXCR4 levels (Fig. 1E) . On the other hand, CCR5 was expressed at very low levels in naïve NHBE cells cultured in submerged conditions; however, these cells showed an induced expression of CCR5 following 48 h stimulation with a Th2 cytokine, IL-13 at 1 ng/ml (Suppl Fig. S1A) . The IL-13-induced CCR5 expression (Suppl Figs. S1B and S1C) correlated with susceptibility of NHBEs to R5-tropic HIV-1 (HIVBaL), leading to higher HIV-gp120 positivity (Suppl Fig. S1D ) in IL-13 treated cells compared to the non-treated (NT) controls.
Next, we analyzed the CD4 and CXCR4 receptor expression in the lung epithelium of nonhuman primates. Archived lung tissues from the cynomologus macaques (CMs) that were exposed to CS for 27 weeks and/or infected with SHIV at 11 th week to obtain 4 experimental groups: fresh air control (FA), CS-exposed, SHIV-infected, and CS+SHIV, as described recently (19) . All SHIV-infected macaques received daily injections of cART (Tenofovir and Emtrictabine) starting at 2 weeks post infection until euthanasia. We analyzed the expression of HIV secretory protein, transactivator protein (Tat) which regulates HIV transcription inside the host. There was very high expression of immunoreactive Tat was observed in lung epithelial cells of SHIVinfected CMs ( Fig. 2A) that have been successfully treated with cART, and the expression was significantly higher (more than four-fold) in the lungs of CS-exposed and SHIV-infected (CS+SHIV) macaques (Fig. 2B) . Further immunofluorescence analyses showed that, compared to control, the airway epithelial cells (pan-cytokeratin or pCK-positive cells) from the CSexposed group had significantly upregulated expression of CD4 (Fig. 2C) and CXCR4 (Fig. 2D) in CS+SHIV macaques, supporting our in-vitro findings in human NHBEs (Fig. 1) . The expression of HIV co-receptor, CCR5 (Suppl. Fig. S2 ) was also confirmed in-vivo in the CM lung epithelium.
To confirm the of HIV-1 uses classical HIV receptors and co-receptors to infect, 3D-cultured NHBE on air-liquid interface (ALI) were pre-incubated with anti-CD4 and/or anti-CXCR4 antibodies for 1 h at 37°C and then infected with CXCR4-tropic HIV-1LAV. At 24 h post-infection, cellular RNA was isolated and assayed for HIV-specific LTR RNA levels. As seen in Figure 3A , compared to control immunoglobulin-treated cells, anti-CD4, anti-CXCR4, or anti-CD4+anti-CXCR4 significantly reduced the HIV-RNA expression. However, the level of the mucin MUC5AC was more sensitive to anti-CXCR4 than anti-CD4, but the combination (anti-CD4+anti-CXCR4) potently reduced the viral RNA expression (Fig. 3B) . We have previously shown that HIV-gp120-induced mucus formation and MUC5AC expression in NHBE cells is suppressed by blocking CXCR4 (11) . Along with CD4, HIV coreceptors have been strongly implicated in optimal HIV-gp120 binding and viral infection (25) . These results suggest that HIV-1 enters NHBE cells via CD4 and CXCR4, but the mucus formation primarily depends on the binding of the virus to the cell surface and may not necessarily require a productive viral infection.
To determine whether HIV infects and establishes latency in differentiated NHBE cells, NHBE cells were grown on ALI and infected with HIV-1LAV either apically or basolaterally in trans-well cell culture. After 2 h post-infection, media was removed, and the trans-well filters were washed on both sides 4 times with warm ALI culture media and each wash was tested for HIV-p24 to confirm the removal of input virus. As shown in Figure 4A there was no detectable p24 in the 4 th wash and was considered as 0 h time point. Cells were incubated in fresh medium and, at the indicated times, media aliquots were collected from both apical and basolateral sides and assayed for HIV p24. Cells were infected with basolateral side, The level of p24 were increased in the cultures infected basolaterally reaching maximum (30 pg/ml) at 1 day (24 h) post-infection; thereafter, the p24 levels started declining, reaching very low levels by day 7 (Fig. 4A) . On the other hand, the p24 levels of the apically infected cultures remained undetectable throughout this period (data not shown). These results suggest that the airway epithelial cells are infected by HIV via the basolateral but not the apical side and may partly contribute to the lack of HIV transmission via the oral route.
In tissue reservoirs, HIV is known to establish latency and, in cell cultures, the virus is activated by various reactivation stimuli, including histone deacetylase (HDAC) inhibitors (26, 27) and several toll-like receptor (TLR) agonists like LPS (28) (29) (30) . To determine if HIV infection of NHBE cells establishes viral latency in the surviving cells from 7-day post-infection Transwell cultures, the cells were treated with the latency reversing agents, LPS (1 µg/ml) and/or the HDAC inhibitor, vorinostat (300 nM) that is shown to activate the HIV provirus in vivo and in vitro (31, 32) . At 24 h after the treatment, the HIV-1 Gag p24 levels were significantly upregulated (Fig. 4B) . This accords well with the observation that vorinostat treatment of HIV provirus containing T cells from patients breaks their latency, leading to increased HIV-specific RNA in the cell (27) . Activation of HIV provirus is invariably associated with cell apoptosis (33) and, indeed, following each of these treatments, essentially all the cells on the Transwell filters were dead (data not shown). These observations clearly show that HIV infects and establishes latency in differentiated NHBE cells. Besides, the respiratory tract is constantly exposed to viruses and bacteria and many of these activate TLRs on the cells. Therefore, it is feasible that respiratory infections will promote activation of HIV proviruses that are integrated within the airway epithelial cells, making the lung epithelial cells as an important reservoir of HIV.
For a productive HIV infection, the reverse transcribed HIV DNA enters the nucleus to integrate into the cellular DNA and this integration of the provirus into the host genome is a central event in HIV pathogenesis (34) . HIV proviruses integrate at many sites in the host genome (35) and, in resting cells, the integrated viral genome may remain essentially silent, leading to latency and viral replication primarily through clonal expansion (36) . Therefore, to identify a HIV target, it is imperative to show that the viral DNA is integrated into the cellular DNA. To demonstrate this, we focused on genes that follow the 5'-LTR and precede the 3'-LTR of HIV (i.e., gag and Nef genes, respectively). We amplified these HIV genes from the DNA isolated from control (non-infected) and HIV-1-infected differentiated NHBEs and sequenced the amplified products. Briefly, DNA was isolated from control and HIV-infected NHBE cells at 24h post-infection. Two approaches were used to identify viral genes in the host cell DNA.
In the first approach, we amplified HIV gag by nested PCR. A single band at 1.52 Kb was present on the 1.0% agarose gel that was absent in the control DNA (Fig. 5A) . The product was extracted from the gel and sequenced. The sequence was >99% identical to human HIV-1 gag by blast analysis using both forward primers (Fig. 5B) and reverse primers (Suppl. Fig. S2A) .
Similarly, Nef gene was amplified by nested PCR and isolated by gel electrophoresis. A single band on the gene at 730 bp (Suppl. Fig. S2B ) was sequenced and showed >99%identity with published HIV-1 Nef sequence. The primers for the nested PCR and the sequence are provided in the online supplemental data (Suppl. Fig. S2C ).
In the second approach, we employed a two-step, Alu-gag PCR assay to confirm the presence of integrated HIV-1 proviral DNA. This was done by isolating DNA from uninfected controls and HIV-1 infected NHBE cells and subjected to nested PCR approach as described schematically (Fig. 5C) , Briefly, the 1 st round PCR was performed with forward primer that anneals to Alu repeat elements in the human genome and the reverse primer anneals to HIV-1 gag gene. In the second round of PCR, the 1 st PCR amplicon was used as a template to amplify a 130 bp region of the 5'-LTR of HIV-1 genome as identified by agarose gel analysis (Fig. 5D) .
Finally, DNA sequencing was performed to confirm that 130 bp amplicon generated from the 5' LTR region of HIV-1contains intact sequences of LTR-gag (Fig. 5E) The Forward Primer sequence detected in the reverse complement sequence generated using reverse primer and the Reverse Primer sequence detected in the sequence generated using forward primer (Fig. 5F) , confirming the genomic integration of HIV provirus in NHBEs.
We have previously demonstrated that HIV-and SIV-infected lungs from humans and monkeys, respectively, contain HIV-gp120 immunoreactive cells (11) . More recently, we have shown that a significant number of airway and alveolar epithelial cells in SHIV-infected CMs are HIV-gp120-positive and the number of these cells are essentially doubled in SHIV+CS-exposed lungs (19) . To determine that HIV replicates in airway epithelial cells in-vivo, we performed RNA FISH by RNAScope® technology (Advanced Cell Diagnostics, Biotechne Inc.) using commercially available HIVgag-pol probes (ACD, Bioteche Inc.). As seen in Fig. 6A , HIVspecific RNA is present in SHIV-infected pan-cytokeratin (pCK) positive lung epithelial cells and this expression was three-fold higher in the animals, which were also exposed to CS i.e.
CS+SHIV group (Fig. 6B) . Similarly, unlike the uninfected controls, the archived airway sections of HIV-infected human subjects contain significant amount of HIV-specific RNA (20-fold higher than uninfected) and the RNA persisted (14-fold higher than uninfected) in HIV subjects on HAART (HIV+HARRT group) (Figs. 6E and 6F) . These results suggest that lung epithelial cells in humans and macaques are targets of HIV infection.
In this study, we report that differentiated NHBE cells normally express receptors or coreceptors CD4 and CXCR4, and when exposed to X4-tropic HIV, the cells produce HIV p24. 
Normal Human Bronchial Epithelial Cells (NHBE) cells were obtained from MatTek Incorp (EpiAirway™, Ashland, MA). For all air-liquid interface (ALI) cultures of primary NHBEs (Lonza Inc., Basel, Switzerland), cells were plated onto collagen IV-coated 24-mm Transwell-clear culture inserts (Corning Costar Corporation, Cambridge, MA) at a density of 5x10 5 cells/cm 2 in BEGM media (Lonza Inc) and subsequently in ALI media as described previously (43) . The apical surface of the cells was exposed to air and cells were cultured for another 21 days till they were fully differentiated.
The X4-tropic viral strain HIV-1LAV and R5-tropic viral strain HIV-1BaL were employed in these studies. NHBE ALI cultures grown on transwells were infected apically and basolaterally with of either X4-tropic HIV-1LAV or R5-tropic HIV-1BaL (5 ng/ml p24 equivalent) as described earlier (44) . 16 hours post infection, cells were washed apically and basolaterally with PBS four-times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as day 0 to confirm that all input virus had been removed. Culture supernatants were collected on day 3 and p24 antigen levels were determined using p24 ELISA (ZeptoMetrix Corp. Cat # 0801200) as per manufacturer's instructions. For chronic HIV exposure, NHBE ALI cultures were infected with X4-tropic HIV strain (IIIB) or R5-tropic HIV strain (BaL) (2.5 ng/ml p24 equivalent) were allowed to proceed for 7 days.
For immunohistochemical staining, deparaffinized and hydrated lung tissue sections were washed in 0.05% v Brij-35 in PBS (pH 7.4) and immunostained for antigen expression as described previously (19) . Briefly, the antigens were unmasked by steaming the sections in 10 mM Citrate buffer (pH 6.0) followed by incubation in a blocking solution containing 3% BSA, 1% Gelatin and 1% normal donkey serum with 0.1% Triton X-100 and 0.1% Saponin and were stained with antibodies to CD4 (Abcam, #ab133616), CXCR4 (Abcam, #ab181020), CCR5 (Abcam, #ab11466), HIV-Tat (Abcam, #ab63957) and pan-CK (#4545, Cell Signaling Technologies, Danvers, MA). The NHBE cells grown on coated coverslips or the coated Labtek-II slides (Thermo Fisher Inc.) were fixed in 4% paraformaldehyde and washed in 0.05% v Brij-35 in PBS (pH 7.4) and immunostaining was performed as described previously (45) . Briefly, the cells were blocked using a solution containing 3% BSA, 1% Gelatin and 1% normal donkey serum with 0.1% Triton X-100 and 0.1% Saponin and were stained CD4, CXCR4, CCR5 and pan-CK, as described above. The immunolabelled cells/tissue sections were detected using respective secondary antibodies conjugated fluorescent dyes (Jackson ImmunoResearch Lab Inc., West Grove, PA) and mounted with 4',6-diamidino-2-phenylindole (DAPI) containing Fluormount-GTM (SouthernBiotech, Birmingham, AL) for nuclear staining. Immunofluorescent images were captured using BZX700 Microscopy system (Keyence Corp., Japan) and analyzed using NIH Image J software.
Total RNA was isolated from the experimental cells using RNAeasy kit (Qiagen, Germantown, MD) as per manufacturer's instruction. RNA concentration was determined using the Synergy Relative quantities were calculated by normalizing averaged CT values to CDKN1B or GAPDH to obtain ΔCT, and the fold-change (ΔΔCT) over the controls were determined as described previously (45) .
Cell extracts were prepared using RIPA buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1 mM EDTA and 1% protease inhibitor cocktail). Protein concentration was determined by BCA kit (Pierce; Rockford, IL) and 50 µg protein was analyzed by western blotting as described previously (43) . Antibodies used were for CCR5 (Abcam, #ab11466) and for β-actin (Sigma Co. St. Louis, MO). Proteins were detected using ECL and visualized by chemiluminescence (Perkin Elmer, Waltham, MA) using the BioRad Chemidoc Imaging system (Hercules, CA).
We utilized the methods as described previously (46) in which two-step Alu-gag PCR assay was Round PCR: 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15s, annealing at 50°C for 15s and extension at 72°C for 3 min 30s; 2 nd round PCR: 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15s, annealing at 60°C for 15s and extension at 72°C for 30s.
Finally, PCR amplicons were resolved on a 2% agarose gel (Promega Corporation, Madison, USA) pre-stained with Ethidium bromide (0.5 μg/ml) and gel images were documented using a Bio Rad gel documentation system (BIORAD, USA). Next, the PCR amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Germany) as per the manufacturer's instructions and subjected to Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit 
Grouped results were expressed as means ± SEM. Data were analyzed using GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA). Grouped results were analyzed using twoway analysis of variance. When significant main effects were detected (P < 0.05), Fishers least significant difference test was used to determine differences between groups. 