In situ vRNA detection

HIV-1 vRNA in cells was probed using RNAscope reagents (Advanced Cell Diagnostics). The manufacturer’s protocol was used with some modifications⁸. Following fixation, cells were dehydrated by removal of DPBS and sequential replacement with 50%, 70% then 100% ethanol, incubating the samples for 5 min at room temperature in each solution. Ethanol solutions were prepared as volume-to-volume ratios in ultrapure water (Synergy; Millipore). The 100% ethanol was replaced with fresh 100% ethanol and incubated at room temperature for a final 10 min. At this point coverslips can be stored in 100% ethanol at −20 °C. To rehydrate cells, the sequence was reversed and the cells were incubated for 2 min at room temperature in each solution; the cells were not allowed to dry in air at any time during the process. Finally, 50% ethanol was replaced with PBS and the cells were hydrated at room temperature for 10 min. Cells were then washed with 0.1% Tween in PBS for 10 min, and twice more in PBS for 1 min. Prior to hybridization, coverslips were immobilized on glass slides; a small drop of nail polish was placed on a glass slide and the coverslip edge was placed on the nail polish drop. Using an ImmEdge hydrophobic barrier pen (Vector Laboratories), a circle was drawn around the coverslip and PBS was added to prevent sample dehydration. The manufacturer’s protease solution (Pretreat 3) was diluted in PBS as appropriate prior to the experiment, (a 1:2 dilution was used when staining incoming virions and a 1:15 dilution when staining for transcription and translation), and incubated in a humidified HybEZ oven (Advanced Cell Diagnostics) at 40 °C for 15 min. Protease solution was discarded and the slides were washed twice by immersion in PBS at room temperature for 1 min. Specific pre-designed anti-sense probes (Supplementary Table ¹) that recognize the HIV-1 vRNA were added to the coverslip, as specified by the manufacturer (Advanced Cell Diagnostics): C1 probes were added directly, C3 probes (PS-3 and PS-5), were diluted 1:50 in probe dilution buffer or in C1 probe. Probes were allowed to hybridize with the samples in a humidified HybEZ oven at 40 °C for 2 h. The probes were then discarded and the coverslips washed twice using the proprietary wash buffer. All wash steps were performed on a rocking platform at room temperature for 2 min, using the proprietary wash buffer. The probes were visualized by hybridizing with preamplifiers, amplifiers, and finally, fluorescent label. Pre-amplifier 1 (Amp 1-FL) was hybridized to its cognate probe in a humidified HybEZ oven at 40 °C for 30 min. Samples were washed twice, then hybridized with Amp 2-FL in a humidified HybEZ oven at 40 °C for 15 min, to suppress background staining. After a further two washes, amplifier (Amp 3-FL) was hybridized to Amp 1-FL in a humidified HybEZ oven at 40 °C for 30 min. Samples were washed twice, then fluorescent label Amp 4-FL was hybridized to Amp 3-FL in a humidified HybEZ oven at 40 °C for 15 min, then washed twice more. For HIV-1 experiments, the labeled probe set Amp 4A-FL was used, labeling the C1 probes with Alexa 488, and the C3 probe (PS-4) with Atto 647. For HIV-1/HIV-2 co-staining experiments, the Amp 4C-FL was used, labeling the C1 probe with Atto 550 and the C3 probe (PS-5) with Alexa 488.

For subsequent protein staining, the RNA-labeled samples were washed with PBS and immunostaining was performed as described below. Otherwise, the final step was to counter-stain nuclei with the manufacturer-supplied 4′,6′-diamino-2-phenylindole (DAPI, Advanced Cell Diagnostics) for 30 s at room temperature, then remove the DAPI, wash twice with PBS and immediately mount the coverslips on slides using Prolong Gold Antifade (Invitrogen).