Primary HIV-1 isolates  were derived from patients' peripheral blood mononuclear cells (PBMC) by co-culturing patient CD4+ T-lymphocytes with stimulated, CD8+ T-cell depleted PBMC as previously described . Patients were enrolled in the Zurich primary HIV infection (ZPHI) study http://clinicaltrials.gov: NCT00537966, and written informed consent was obtained from all participants. Viral replication was, for all experiments, assessed from culture supernatants by p24 ELISA (adapted from ). TCID50 of primary isolates and CD8+ T-cell depleted PBMC grown HIV-1JR-FL virus stocks was estimated as described .
Cells and infection
PBMC from healthy donors were isolated, CD8+ T-cell depleted, and CD4+ T-lymphocytes were stimulated and cultured as described previously . Cells were infected with HIV-1JR-FL (MOI = 0.01), harvested 7 days post infection and lysed using QIAzol lysis reagent (Qiagen).
For the generation of macrophages, primary human monocytes were isolated from CD8+ T-cell depleted PBMC using positive selection with anti-CD14-coated magnetic beads (Miltenyi Biotech). Monocytes (3 × 106 cells per T25 flask) matured to macrophages in the presence of 0.02 μg/ml human M-CSF (macrophage colony stimulating factor, PeproTech). Macrophages were maintained in RPMI-1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 5% MCM (macrophage conditioned medium, sterile-filtered), 5% human serum, and 0.02 μg/ml M-CSF (the latter three ingredients were added only during the first 6-8 days). After 14 days of maturation, macrophages were infected with HIV-1JR-FL (MOI = 0.01). After 14 days, cells were harvested and lysed using QIAzol lysis reagent (Qiagen).
Isolation of the low molecular weight RNA fraction
Lysed cells were homogenized with QIAshredder (Qiagen), and the extraction of small RNA (< 200 nt) was performed using miRNeasy Mini Kit (Qiagen) according to the manufacturers' instructions. RNA was eluted in 40 μl RNase-free water.
Adaptor addition and cDNA synthesis
An aliquot (15 μl) of the low molecular weight fraction of extracted RNA was C-tailed for 15 min at 37°C using 7.5 units E.coli Poly(A) Polymerase (New England Biolabs) and 0.75 mM CTP (Connectorate). The synthesis of C-tails was blocked by addition of 0.5 mM Cordycepin (Sigma-Aldrich) and 2.5 units E.coli Poly(A) Polymerase, and incubation for 15 minutes at 37°C. At the same time, C-tailed RNA was treated with 15 U DNase (Roche). Afterwards, precipitation was performed by adding 1 volume isopropanol, 0.2 M sodium acetate, and 4 μl precipitation carrier Dr. Gentle (Takara) and centrifuged for 30 min at 16°C and 16,000 g. The pellet was washed with 80% ethanol and eluted in 20 μl H2O. Subsequently, the 5'-end was ligated to an 2' O-methylated RNA adaptor 5'-AUCGGAACAUCCAGACAUAACA-3' using 40 U T4 RNA ligase (New England Biolabs), 4 μM adaptor RNA, and 60 U RNaseOut (Intvitrogen) (15 min at 37°C and over night at 16°C). This was followed by precipitation as described above and elution in 10 μl H2O. cDNA was generated using M-MuLV Reverse Transcriptase (Finnzymes) and the 3' linker primer mf331 5'-ACCAGAGTGCGAGTAGGAAGATTGGGGGGGGG-3' partly complementary to the C-tail of the RNA. Briefly, RNA and 5 μM primer were denaturated for 5 min at 95°C followed by incubation on ice for at least 2 min. The enzyme-buffer-dNTP (400 U M-MuLV Reverse Transcriptase, 2.5 mM dNTP) mixture was added, and the reaction was incubated for 60 minutes at 37°C. Amplification of 2 μl cDNA was executed with JumpStart Taq ReadyMix (Sigma-Aldrich) for 15 cycles using 1 μM 5' adaptor primer mf311 5'-ATCGGAACATCCAGACATAACA-3' and 1.5 mM MgCl2 (95°C-5'; 15 × (95°C-5″; 52°C-5″; 72°C-40″)).
A second round of PCR with 25 cycles was performed using 1 μl of a 1:10 dilution of the first PCR product. Again JumpStart Taq ReadyMix (Sigma-Aldrich) supplemented with 1.5 mM MgCl2 and 1 μM of each 5' and 3' adaptor primers mf311 and mf315 5'-ACCAGAGTGCGAGTAGGAAGATT-3' was used (95°C-2'; 25 × (95°C-5″; 52°C-5″; 72°C-20″)). Amplicons were precipitated with isopropanol and dissolved in TENT5/200 (10 mM Tris-HCl, 5 mM EDTA, 0.2 M NaCl, 0.1% Triton).
Generation of HIV-1 DNA/streptavidin beads for selection of HIV-1 sncRNAs
The HIV-1JR-FL plasmid  was used as template and amplified with HIV-1 specific biotinylated primers, using the HotStartTaq Master Mix Kit (Qiagen) supplemented with 1.5 mM MgCl2 (95°C 15'; 40 × (95°C 10″- 57°C 10″-72°C 4'); 72°C 7'). Five amplicons were generated using the following primers that are biotinylated at the 5'-end: 1) TAR to gag (position according to HIV-1HXB2 (GenBank accession number K03455): 455-1,658) with the primers mf271 5'-GGTCTCTCTGGTTAGACCAGATTTGA-3' and sk39 5'-TTTGGTCCTTGTCTTATGTCCAGAATGC-3', 2) gag to pol (1,273-4,837) with mf219 5'-AAGGCTTTCAGCCCAGAAGTAATACCCATGTT-3' and mf255 5'-ATGTCTACTATTCTTTCCCCTGCA-3', 3) pol to env (4,758-6,741) with mf254 5'-CAAATGGCAGTATTCATCCACAA-3' and mf237 5'-ATTCTTCCTGATCCCCTTCACTCTCAT-3', 4) env (5,956-8,421) with mf1 5'-CTTAGGCATCTCCTATGGCAGGAA-3' and mf2 5'-TTCCTTCGGGCCTGTCGGGTCCC-3', and 5) splice acceptor 7 (sA7) to the 3'LTR (8,317-9,700) with mf214 5'-TTTTTGCTGTACTTTCTATAGTGAATAGAGTTA-3' and cr2 5'-TGACTAAAAGGGTCTGAGGGATCTCTAGTTACCAG-3'. All primers were used in a final concentration of 1 μM. The PCR products were purified (Qiaquick, Qiagen) and eluted in 10 mM Tris-HCl (pH 8.5).
Biotinylated DNA was attached to streptavidin beads (Roche). Either 400 ng of biotinylated DNA from each PCR were used separately, or in combination (5 × 400 ng) for preparation of the beads. Briefly, 25 μg beads were washed with TENT100 buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5, 0.1% Triton), and resuspended in 75 μl 2 × TENT100. Denaturated amplicons (5', 95°C) were added to the beads, and the volume was adjusted to 150 μl with H2O. DNA was immobilized by 30 minutes incubation with the beads at 37°C. Streptavidin-biotinylated, single-stranded DNA complexes were achieved by heating to 90°C for 1 minute. The attachement-dehybridization procedure was repeated once. Streptavidin-biotinylated-ssDNA complexes were washed 3 times with TENT1000 (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 7.5, 0.1% Triton) and 3 times with TENT100. They were stored in TENT100 at 4°C.
Selection of HIV-1 sncRNAs
For the hybridization of amplified HIV-1 sncRNAs to the Streptavidin-biotinylated-ssDNA complexes, 10 μl of these beads (> 1010 molecules of each HIV-1 ssDNA hybridization probe) were added to the amplified HIV-1 sncRNAs and incubated for 3 minutes at 95°C followed by a cool down to 50°C over night on a head to tail wheel. Beads were washed 4 times with pre-warmed (50°C) TENT5/200 buffer. Annealed amplified HIV-1 sncRNAs were eluted from the beads by adding 15 μl Tris-HCl buffer (10 mM Tris-HCl, pH 8.5) and heating for 5 minutes at 95°C. Beads and eluted sncRNA were separated by magnetic separation. HIV-1 sncRNAs were amplified using JumpStart Taq ReadyMix (Sigma) supplemented with 1.5 mM MgCl2 and 1 μM of each adaptor-specific primers mf311 and mf315 (95°C-2'; 30 × (95°C-5″; 52°C-5″; 72°C-30″)). Amplicons were size-selected using a 3% MetaPhor agarose gel. DNA with a length of 50-110 bp was extracted from gel using GenElute Agarose Spin Columns (Sigma). When two selection steps were performed, eluate was precipitated with isopropanol and the hybridization and size selection steps were repeated. Eluates were precipitated with isopropanol and eluted in 15 ul H2O.
Cloning and sequencing of HIV-1 sncRNAs
Amplified and selected HIV-1 sncRNAs were ligated into the vector pDrive using the QIAGEN PCR Cloning kit (Qiagen). Single clones were sequenced in one direction with the primer T7 using BigDye chain terminator chemistry and the automated sequencer ABI 3100 (Applied Biosystems).
Sequences were controlled for the presence of both adaptor sequences, which were subsequently deleted to obtain the sncRNA sequence. This analysis was performed using the software BioEdit . All sncRNA sequences were aligned to the reference strains HIV-1HXB2 and HIV-1JR-FL using the software DNAstar (DNA-star Madison). Sequences with > 90% homology to the reference strain HIV-1JR-FL were considered HIV-1 specific. FASTA  was chosen for further nucleotide similarity searches. Classification of small RNA sequences was based on sequence analysis using the GenBank database http://www.ncbi.nlm.nih.gov/genbank/, the miRNA registry database http://www.mirbase.org/, and the human tRNA database http://gtrnadb.ucsc.edu/. Secondary structures of selected HIV-1 sncRNA were predicted with RNAstructure 5.2 . SncRNA sequences smaller than 16 nucleotides were not included in our analysis.
Statistical analyses were performed using GraphPad Prism5.0 software. The two-tailed Chi square test and the Wilcoxon rank sum test were used for binary and cardinal data, respectively. p < 0.05 was considered statistically significant.
Transfection of primary macrophages with HIV-1 sncRNAs
Maturated macrophages were generated and infected with HIV-1JR-FL as described above. Seven days after infection cells were transfected with HIV-1 sncRNAs using jetPRIME transfection reagent (Polyplus-Transfection). Briefly, medium was replaced by Opti-MEM® I Reduced Serum Media (Invitrogen) and the transfection mix was added to the cells according to the manufacturer's instructions. After four hours, 10% FCS (Invitrogen) was added. The next day the transfection medium was replaced by RPMI-1640 supplemented with 10% FCS and 1% penicillin/streptomycin. The following oligonucleotides were used for sncRNA transfection: sncRNALTR6; sncRNAenv183; sncRNAenv184; sncRNAenv185 (Supplementary dataset 1). Control siRNA labelled with AlexaFluor488 (AllStars negative controls, Qiagen), here named as nonsense siRNA, was used as control for the transfection efficiency and negative control for virus inhibition, whereas siRNA-M184pol was chosen as positive control as previously described . Western blot analysis for detection of the interferon type I inducible MxA protein was carried out as previously described using a mouse monoclonal antibody directed against MxA .
Detection of HIV-1 sncRNAs in cells infected with primary HIV-1 isolates
HIV-1 sncRNAs detected in most or all of the libraries were screened in terms of their presence in primary cells infected with primary HIV-1 isolates. Infection of CD8+ T-cell depleted PBMC, RNA isolation, C-tailing, and reverse transcription were performed as described above. HIV-1 sncRNAs were amplified, using 1 uM of primer mf315, 1 uM of respective HIV-1 sncRNA-specific primer (mf382 5'- ATAAAGCTTGCCTTGAGTG-3', mf178 5'- ATGGTAGAACAGATGCATGAGGATATAAT-3', CA-179 5'- CGTTCACTAATCGTCCGGATCTGTC-3') and JumpStart Taq DNA Polymerase (Sigma). PCR was performed as follows: 95°C-2'; 50 × (94°C-10″; 55°C-10″; 72°C-40″). Amplicons were loaded on 3% MetaPhor agarose gel and separated by electrophoresis mobility.
Quantification of HIV-1 sncRNAs in HIV-1 infected cells
In order to quantify mi/sncRNA in macrophages and CD8+ T-cell depleted PBMC, the small RNA fraction was extracted from cell lysate, C-tailed, reverse transcripted, and amplified as described above. Mi/sncRNA were amplified by using 1 μM of the adaptor primer mf315 and 1 μM of the corresponding mi/sncRNA specific primer (hsa-miR-21 5'-TAGCTTATCAGACTGATGTTGA-3', hsa-miR-223 5'-TGTCAGTTTGTCAAATACCC-3', mf382 5'-ATAAAGCTTGCCTTGAGTG-3', VV153 5'-CGTTCACTAATCGTCCGGAT-3'). For quantification via qPCR, EvaGreen™ Dye (Biotium) was used for cellular miRNAs and 0.3 and 0.2 μM of LNA probes CA20tq 5'-tgTgTgcCcGt-3' and VV154 5'-TGTctctgtctCT-3', respectively, were used for HIV-1 derived sncRNA, LNA bases are indicated as lower case letters.