Immunocapture of cell surface proteins embedded in HIV envelopes uncovers considerable virion genetic diversity associated with different source cell types

HIV particles in the blood largely originate from activated lymphocytes and can overshadow variants which may be expressed from other cell types. Investigations of virus persistence must be able to distinguish cells refractory to viral clearance that serve as reservoirs. To investigate additional cell types that may be associated with in vivo HIV expression we developed a virus particle immunomagnetic capture method targeting several markers of cellular origin that become embedded within virion envelopes during budding. We evaluated the ability of markers to better distinguish cell lineage source subpopulations by assessing combinations of different antibodies with cell-sorted in vitro culture and clinical specimens. Various deductive algorithms were designed to discriminate source cell lineages and subsets. From the particle capture algorithms, we identified distinct variants expressed within individuals that were associated with disparate cellular markers. Among the variants uncovered were minority-level viruses with drug resistance mutations undetected by sequencing and often were associated with markers indicative of myeloid lineage (CD3-/CD10-/CD16+ or /CD14+, and CD3-/CD16-/CD14-/CD11c+ or /HLA-DR+) cell sources. The diverse HIV genetic sequences expressed from different cell types within individuals, further supported by the appearance of distinct drug-resistant variants, highlights the complexity of HIV reservoirs in vivo which must be considered for HIV cure strategies. This approach could also be helpful in examining in vivo host cell origins and genetic diversity in infections involving other families of budding viruses.


Note
If viral load is unknown, determine copies by qPCR or test on a commercial viral load platform.
Insert the Amicon device into the microcentrifuge tube.
Add up to 500 µL Ab ( 0.1 undetermined -0.2 undetermined ) to the filter device and cap it.
Insert the capped Amicon Ultra device into a centrifuge tube and place in the centrifuge rotor.
Remove the device and place it upside down in a clean tube, place in centrifuge, aligning the open cap strap, toward the center of the rotor.

Note
The concentration of Biotinylation Reagent is 5 undetermined .
Immediately add 2 µL of Biotinylation Reagent to the antibody solution with gentle stirring.
Place the column G-50 in a 1.5 ml Eppendorf tube, pre-spin the column for 00:01:00 at 700 x g ( 3000 rpm ).Repeat two times.
Label two of 1.5 ml Eppendorf tube.
Place column in tube 1 and apple the biotinylation reaction mix to the column.
Place column in tube 2 and add 200 up to the column, spin the column for 00:02:00 at 700 x g .collect flow-through (fraction 2).
Prepare standard curve dilutions: Cover the plate with film and incubate 37 °C for 00:30:00 .
Calculate mAb concentration against the standard curve.
Using Immulon II 96 well plate.
Coat three wells with a dilution series of mAb beginning with 1 µL mAb in 99 µL PBS (1:100) continuing with two more 10-fold dilutions.Incubate Overnight at 4 °C .
Add 100 µL equilibration buffer for nucleic acid applications to the column.
Apply HIV-bead-Ab complex onto the top of column, collecting the flowthrough in a clean microfuge tube or eluting directly into the next tube of biotinylated mAb-bead complex.Let reaction pass through the column completely, captured virus will be retained on the column and flow-through will contain non-target virus (see figure below).Gently mix and make sure that all the components are at the bottom of the amplification tube.
Place the reaction in the preheated thermal cycler programmed as described above.Collect the data and analyze the results.
Program the thermal cycler to amplify with the following conditions:

Note
You may check for primary PCR product by gel electrophoresis or real-time detection.Due to the potential for low copy numbers perform nested reactions.
Thaw, vortex briefly to mix and centrifuge each component before use.
For each sample, prepare 48 µL reaction master mix in a PCR workstation as follows:
stop solution to each well.Read the absorbance of each well at 450 undetermined and 550 undetermined .OD values of the 1:100 dilution (first well) of ≥0.6 indicates adequate biotin labelling of antibody.) biotinylated Ab for 00:10:00 at Room temperature on a roller platform.
complex at 8000 rpm, 00:08:00 and then remove supernatant.If working with tissue culture supernatants first DNase treat and inactivate.Add 200 µL HIV-positive material (plasma, CSF, Semen, Culture or flow-through) to the designated bead-Ab complex and incubate for 00:30:00 at Room temperature .Mixing gently on a roller-mixer.
Mini column in a clean 1.5 ml microcentrifuge tube.Discard the old collection tube containing the filtrate.Carefully open the QIAamp Mini column and add 60 briefly to mix and centrifuge each component before use.Prepare 45 µL reaction mast mix in a PCR workstation.template RNA.Final reaction volume is 50 µL .
2m RT PCR: SuperScript™ III One-Step RT-PCR System with Platin… new reaction microfuge tubes and RT-PCR samples to Nested PCR room.Add 2 µL of each RT-PCR sample per tube.Nested PCR (nPCR): Platinum™ SuperFi II PCR Master Mix