Study subjects

This study was approved by the Johns Hopkins Institutional Review Board and informed consent was obtained from all participants before study enrollment. Peripheral blood was obtained from healthy volunteers and ART suppressed patients with viral loads < 20 copies HIV-1 RNA/mL. Monocytes and resting CD4+ T cells were obtained from donor PBMCs as described below. Characteristics of study participants are given in Table 1. Collection of BAL samples were discontinued in March 2020 due to the extreme risks pertaining to the Covid-19 pandemic. Therefore, sample numbers are unavoidably reduced from the original planned enrollment for the study.

Isolation of primary cells from donor PBMCs

Monocytes (MC) were purified from peripheral blood mononuclear cells (PBMC) using a Ficoll-Hypaque density gradient followed by negative selection using a Pan monocyte isolation kit (Miltenyi Biotech). Then MCs were differentiated into monocyte-derived macrophages (MDM) by culturing MC under adherent conditions for seven days using MDM-AS medium containing RPMI 1640 (Gibco) supplemented with 10% donor autologous serum, 1% Pen/Strep (HyClone), 0.3 mg/mL glutamine and 10 mM HEPES, plus 10 ng/mL M-CSF (R&D Systems). Fully differentiated MDMs were maintained in media containing RPMI 1640 plus 10% dialyzed FBS, 1% Pen/Strep.

CD4+ T cells were isolated from PBMC population using the CD4+ isolation kit (CD4+ T cell Isolation kit II, Miltenyi Biotec, San Diego, CA). CD4+ T cells were first cultured for three days using stimulating conditions [RPMI supplemented with 10% donor autologous serum, 1% Pen/Strep (HyClone), 0.3 mg/mL glutamine, 1 mg/mL of Phytohemagglutinin (Gibco) and, ciprofloxacin (5 mg/mL)].

The dividing cell line HAP1 (Horizon Discovery) was seeded at 2 million cells per T-75 flask. The culture medium containing RPMI-1640 medium plus 10% heat inactivated FBS, 1% Pen/Strep. Cells were grown until 70% confluence with medium changes every 48 h. The cells were released from the flask using 5 mL of 0.25% trypsin–EDTA (Gibco), followed by washing three times with PBS (Gibco).

Processing of alveolar macrophages (AM) from bronchoalveolar lavage procedure (BAL)

Bronchoscopy and broncho-alveolar lavage (BAL) were performed on six participants including three healthy donors, a single HIV-1 infected patient pre- and post-ART treatment and two HIV-1 patients on ART (Table 1). To obtain alveolar macrophages (AM), bronchoscopy and lavage were done, as described elsewhere [54]. Briefly, BAL samples were filtered using a sterilized gauze pad and transferred into 50-mL centrifuge tubes. A pellet was obtained using a short spin (250×g for 5 min) and was resuspended in serum-free RPMI 1640 medium (Gibco) containing antibiotics (Pen/Strep, gentamicin, and amphotericin B) and incubated at 37 °C for 24 h without shaking. Following incubation, the cells were washed and resuspended in culture medium containing RPMI-1640 medium plus 10% heat inactivated FBS, 1% Pen/Strep. Cells were then counted and diluted to 106 cells/mL for further use. Cells were then pelleted and used for dNTP extraction, or in vitro infection, or quantitative viral outgrowth assay (QVOA) as described below.

Generation of RNA standards for TCR β RNA assay

A fragment of TCR β containing both V-region and C-region was subcloned into a mammalian expression vector. The plasmid was transformed to DH5α competent cells then spread onto an agar plate containing corresponding antibiotics. A mini prep was performed followed by plasmid purification and quantification. Two sets of real time PCR primers targeting both V-region (68 bp) (rt-FWD 5′-ACACGTGAAATGCTCTTTGCG-3′, rt-REV 5′-TTACTCCTGCGCCTCTGTGTC-3′) and C-region (65 bp) (rt-FWD 5′-TGGCTTCTGGCACTCCTTG-3′, rt-REV 5′-GCCATGTGAAGACAGAGGCA-3′) were used to generate standard curve.

Quantitation of TCR β RNA

AM and MDM purity was checked for T-cell contamination by RT-qPCR using TCR-β primers. TCR β RNA was quantitated by RT-qPCR using the above-described primers. Cycling conditions were as follows: 95 °C for 5 min, and 40 cycles of 94 °C for 10 s, 55 °C for 15 s, and 60 °C for 30 s.18S rRNA was simultaneously detected with the TCR β RNA to control for cell counts. To determine the average number of TCR β copies, AMs and MDMs from three donors, were isolated and purified using the above-described methods. The purity of the cells was confirmed by marker staining, and the relative number of copies of TCR β RNA was compared to CD4+ T cells.

Characterization of isolated cell populations

The function and cell identity of AM, MDM, and CD4+ T cells was determined by morphology, marker staining, functional assays, and RT-qPCR (see above). The purity of AM cells was analyzed by marker staining using anti-CD68 antibody ([EPR20545] (ab213363), abcam) and pHrodo™ Green E. coli BioParticles™ Conjugate for Phagocytosis (P35366, Thermofisher). Using this method, we obtained AMs with a purity of > 95.8% and are functional of > 91.6% with neglectable T cell contamination (0.9% for AMs and 1.6% for MDMs).

Single nucleotide polymerase extension assay

The extraction of total dNTPs from ~ 1 to 2 million cells and quantification of dUTP and dTTP were performed as previously described [31, 32, 55], except that HIV-1 reverse transcriptase (RTase) was used in the extension assay (Millipore-Sigma). The extension reactions were performed at 37 °C for 40 min and included ~ 6–1200 fmol of total dNTP, 120 or 1200 fmol of DNA template-primer for extracts obtained from non-dividing or dividing cells, respectively, and 2 units of RTase (one unit converts 1 nmol of labeled dTTP into acid-insoluble material in 20 min at 37 °C, pH 8.3). The substrate (22 mer) and the product (23mer) of the extension reaction were separated by electrophoresis on a denaturing PAGE (8 M urea, 1× TBE, 15% acrylamide) and then imaged (Typhoon 9500 imager; GE Healthcare) and the images were quantified using Image Lab software (Bio-Rad). The extension buffer consisted of 20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 2 mM DTT, and 0.1% BSA. Extensive validation experiments were performed using [dUTP + dTTP] mixtures with known dUTP/dTTP 1:1 ratio to establish a detection limit and a linear correlation between the known dNTP concentrations and the ratio determined from the SNE assay. The amount of dUTP per million cells was determined by first calculating the pmol dUTP in the SNE reaction = [pmol DNA template-primer] [fractional extension (no dUTPase) − fractional extension (add dUTPase)]. The total pmol dUTP in the methanol extract was calculated after correction for dilutions and the fraction of the total extract analyzed in the SNE reaction. The total pmol of dUTP was then divided by the cell number used to prepare the extract. The cell number was normalized to the genomic RPP30 copies as the standard (diploid for AM and MDM, and haploid for HAP1).

Expression levels of UBER and dNTP metabolic enzymes using real-time qPCR

RNA was extracted from AM, MDM or HAP1 cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. About 500 ng of RNA was used to synthesize cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368813) following the manufacturers protocol. The cDNA was used as input material and the Rotor Gene qPCR probe kit (Qiagen, #204374), mRNA copies of infected AM and MDM or uninfected controls were measured and normalized to an internal control gene (18S rRNA). Thermal cycling conditions for qPCR consisted of 95 °C for 5 min, and 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s. The relative expression levels were calculated using the 2−ΔΔCT method, where the ΔΔCt is the difference in ΔCt between the target and reference samples:

$$\Delta \Delta }_}} = \, \Delta }_},}/}}} - \Delta }_},}}} = \left( }_},}}} }_},}}} } \right)_}/}}} - \left( }_},}}} }_},}}} } \right)_} }} .$$

(1)

Viruses and infections

The macrophage tropic replicative HIVBal was obtained from the NIH AIDS Reagent Program (NIH-ARP #11414) and propagated in MOLT-4/CCR5 cells. Culture supernatants of infected MOLT-4/CCR5 cells were collected at 14 dpi, centrifuged to clear cellular debris, passed through 0.45 μm filters, aliquoted, and stored at − 80 °C. Viral titer was determined by the ELISA p24 antigen assay (Lenti-X p24 Rapid Titer Kit, TaKaRa). Infections of fully differentiated MDM were performed after adherent growth in the presence of M-CSF for seven days. Prior to addition of virus to MDM, the media was replaced with RPMI + 10% dFCS and omitting M-CSF. Infections of purified AM were performed after adherent purification. AMs were seeded at 1 × 105 cells per well in a 24-well plate and kept at 37 °C, 5% CO2. The culturing medium was removed, and cells were washed twice with PBS, then infected with HIVBal strain at the multiplicity of infection (MOI) of 1. Then the cells were washed with PBS and fresh medium was replaced.

Determination of viral copy number using real-time qPCR

Viral copies were determined using qPCR. A standard curve was generated using tenfold serial dilutions of DNA extracted from J-lat cells (NIH-ARP). Real-time qPCR was performed using Rotor-Gene Probe PCR kit (Qiagen, #204374) on Rotor-Gene qPCR instrument. PCR targeted the early or late HIV reverse transcripts (ERT or LRT). Amplification was performed using a two-step program: initial heating at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s. For determination of viral copies per cell, simultaneous quantification of RPP30 gene was performed using published primers and a specific probe as previously described. All primers and probes used in this study are listed in Additional file 1: Table S3.

Determination of HIV proviral DNA using Alu-gag nested PCR

To determine copy numbers of HIV proviral DNA, a nested PCR method was used. The first PCR was performed using a forward primer (Alu-F, 0.2 μM) that targeted genomic alu sequences randomly located near integrated proviruses and an HIV-specific gag reverse primer (Gag-R, 1.2 μM). Other PCR conditions were 200 μM of each dNTP, 1X LongAmp taq buffer (NEB), 5 units of LongAmp taq DNA polymerase (NEB) and 50 ng of DNA sample extracted from infected cells in a 50 μL final reaction volume. Amplification was performed using the following thermocycler program: initial activation heating 94 °C for 2 min, followed by 20 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 65 °C for 3 min and a final extension reaction at 65 °C for 10 min. The PCR product is diluted 20-fold and 5 μL of the diluted PCR product is used as an input material for the second PCR reaction, which is performed using LRT forward and reverse primers and probe using the Rotor-Gene Probe PCR kit (Qiagen) as described above. Proviral copy numbers were determined using the J-lat cell integration standard as previously described [21, 31, 32]. Genomic DNA copy numbers were determined using the RPP30 reference gene using the same amount of input DNA used to measure proviral copy numbers.

Uracil content of viral DNA

Uracil content of viral DNA was determined using uracil excision qPCR method (Ex-qPCR) as previously described with some modifications as described below [21, 31, 32]. Ex-qPCR was used to determine uracil-containing fraction of viral DNA. The sample was split into two equal portions and one portion was treated with UDG. Briefly, 0.2 units of UDG (NEB) was added into the qPCR master mix to excise uracils from viral DNA. The qPCR reaction was modified to include the UDG reaction time and heat-cleavage of the resulting abasic sites. Thermocycler program for this reaction was: 37 °C for 30 min (UDG reaction), 95 °C for 5 min (abasic site cleavage) and 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s. Primers and probe sets used to amplify viral DNA are indicated under the specific experiments listed in Additional file 1: Table S3. Primers and probe sets targeting RPP30 were used to calculate Frac UDNA using the ∆∆Ct method.

CD4+ T cells viral outgrowth assays (CD4-QVOA)

Peripheral blood mononuclear cells (PBMCs) from healthy HIV-1-infected patients (under suppressive cART with undetectable plasma viral loads) were separated from whole blood by a standard Ficoll gradient. CD4+ T cells were obtained using a negative CD4 magnetic selection kit (Stemcell Technologies), according to the manufacturer’s instructions. To obtain activated T-cell blasts, the cells were resuspended in Roswell Park Memorial Institute (RPMI) medium (RPMI 1640 medium, 10% fetal bovine serum, 2 mM L-glutamine, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (Invitrogen)) and treated with 5 μg mL−1 PHA (Sigma–Aldrich) and 10 U mL−1 IL-2 (Roche) for 48 h at 37 °C. The cells were then re-suspended in RPMI medium, seeded in successive 1:5 or 1:10 serial dilutions in triplicate wells of a 6-well plate or 24-well plate (starting from 1 × 106 cells), and reactivated for 48 h at 37 °C with 5 μg mL−1 PHA. Next, the cells were washed, resuspended in fresh medium containing 10 U mL−1 IL-2, and co-cultured with MOLT-4/CCR5 cells (0.5 × 106 cells) for 2 weeks at 37 °C, with fresh medium containing IL-2 replenished after 1 week. HIV-1 in the co-culture supernatants was measured using RT-qPCR. Infection frequencies were determined by maximum likelihood statistics as described previously, using the application at http://silicianolab.johnshopkins.edu, and expressed as infectious units per million (IUPM).

AM viral outgrowth assays (AM-QVOA)

BAL samples were collected from HIV-1 infected patients and AMs were isolated and purified as described above. The cells were then re-suspended in RPMI medium, seeded in successive 1:5 or 1:10 serial dilutions in triplicate wells of a 6-well plate or 24-well plate (starting from 1 × 106 cells), and kept for 72 h at 37 °C with T20 entry inhibitor (10 μM) to prevent reinfection. Next, the cells were washed and co-cultured with MOLT-4/CCR5 cells (0.5 × 106 cells) for 2 weeks at 37 °C, with fresh medium containing 1 μg/mL lipopolysaccharide (Sigma-Aldrich, L5293) replenished after 1 week. These culture conditions have been previously reported to be favorable for the detection of virus outgrowth from macrophages [56]. HIV-1 in the co-culture supernatants was measured using RT-qPCR. Infection frequencies were determined by maximum likelihood statistics as described previously, using the application available online at http://silicianolab.johnshopkins.edu, and expressed as infectious units per million (IUPM).

MC viral outgrowth assays (MC-QVOA)

Blood samples were collected from HIV-1 infected patients and MCs were purified and differentiated with T20 entry inhibitor (10 μM) to prevent reinfection as described above. The cells were then re-suspended in RPMI medium, seeded in successive 1:5 or 1:10 serial dilutions in triplicate wells of a 6-well plate or 24-well plate (starting from 1 × 106 cells) and co-cultured with MOLT-4/CCR5 cells (0.5 × 106 cells) for two weeks at 37 °C, with fresh medium containing 1 μg/mL lipopolysaccharide (Sigma-Aldrich, L5293) replenished after 1 week. HIV-1 in the co-culture supernatants was measured using RT-qPCR and IUPM was calculated as above.

HIV-1 reinfection assay

MOLT-4/CCR5 cells (20,000/well) were subjected to spin-inoculation for 2 h with diluted supernatant containing varying copies of HIV-1 RNA from positive QVOA wells. Supernatants were collected at days 0, 4, 10, and 15 after spin-inoculation. RNA was isolated from supernatant, and HIV-1 RNA was quantitated by RT-qPCR and number of replicating HIV-1 particles was calculated by maximum likelihood statistics as described above using the application available online at http://silicianolab.johnshopkins.edu.

Next-generation Amplicon-EZ sequencing of HIV RNA

HIV-1 RNA from the QVOA supernatants was isolated using PureLink™ Viral RNA/DNA Mini Kit (Invitrogen, #12280050) and RT-PCR was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368813) following the manufacturers protocol. After cDNA synthesis, a 359 bp segment of the env region was amplified using nested PCR (outer primers E90 and Nesty8 and inner primers DLoop and E115, as described previously) [57]. Thermocycler settings were the same for both outer and inner env PCR reactions: 94 °C for 3 min, followed by 35 cycles and 40 cycles of: 94 °C for 30 s, 55 °C for 30 s and then 68 °C for 1 min, followed by a final extension at 68 °C for 5 min for the first and second PCR, respectively. The PCR products were gel extracted using the QIAquick Gel Extraction Kit (Qiagen, #28706) and quantified using a Nanodrop 2000. The amplification product (20 ng/uL, 50 uL total) were sent to GENEWIZ for targeted amplicon sequencing. Sequencing adapters are listed in Additional file 1: Table S4.

DNA library preparations were performed using NEBNext Ultra DNA Library Prep kit following the manufacturer’s recommendations (Illumina, San Diego, CA, USA). Briefly, end repaired adapters were ligated after adenylation of the 3′ends followed by enrichment by limited cycle PCR. DNA libraries were validated and quantified before loading. The pooled DNA libraries were loaded on the Illumina instrument according to manufacturer’s instructions. The samples were sequenced using a 2 × 250 paired-end (PE) configuration. Image analysis and base calling were conducted by the Illumina Control Software on the Illumina instrument.

Unique sequence and abundance raw sequence data were demultiplexed using bcl2fastq version 2.17.1.14. Read pairs were trimmed for adapter sequences and low quality basecalls using Trimmomatic version 0.36. Reads were discarded if they were less than 30 bases long. Each read pair was then merged using the bbmerge tool from the bbtools software toolkit. Reads that could not be merged were discarded from further analysis. The target sequence between conserved flanking primers was extracted from each merged pair. The forward and reverse reads were joined and assigned to samples based on barcode and truncated by cutting off the barcode and primer sequence. Quality filtering on joined sequences was performed (Additional file 1: Table S4). The sequences are grouped according to their abundance and identities (single nucleotide differences were considered different).

Determination of viral tropism

Viral tropism was predicted according to the V3 region of the most abundant sequence of each QVOA by applying the 11/25 rule with a false-positive rate of 10% [58], and the Position-Specific Scoring Matrix (PSSM) approach (https://indra.mullins.microbiol.washington.edu/webpssm) (Additional file 1: Table S1) [59].

Fluorescence microscopy

AMs or MDMs were treated at 37 °C for 4 h with 10 μM pHrodo Green E. coli BioParticles (Invitrogen, P35366), which can be phagocytosed only by functional macrophages and are nonfluorescent at neutral pH, but which are fluorescent in the acidic pH of phagosomes. The cells were fixed using cold methanol for 15 min then stained at room temperature for 60 min with anti-CD68 antibodies and DAPI for 5 min. Images were taken on an Olympus IX81 fluorescence microscope and analyzed using Fiji ImageJ.

Statistics

The data uncertainties are expressed as the mean ± 1 SD. Unless otherwise indicated in the figure legends, data are derived from three independent biological replicates using cells isolated from three donors. Experiments using cells from individual donors were performed in technical triplicates. Data were analyzed using the Prism 8.0 statistical program from GraphPad Software. Pairwise statistical analysis of test groups was performed by a two-tailed unpaired Student’s t-test, assuming equal variances between groups. Due to the Covid pandemic, all BAL clinical procedures were prohibited in March 2020 which prevented collection of the entire planned set of samples. Due to this unanticipated event, the uncertainty in some of the measurements with patient samples is unavoidably greater.