1. Microbiology and Infectious Disease
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CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release

Research Article
Cite this article as: eLife 2019;8:e35546 doi: 10.7554/eLife.35546
13 figures and 2 tables

Figures

Figure 1 with 3 supplements
Differential effects of CCL2 levels in the medium on HIV-1B and HIV-1C replication in MDMs.

(A, B) MDMs were infected with 10ng/1×106 cells each of either HIV-1BADA (panel A) or HIV-1CIndieC1 (panel B) and the cultures propagated in the presence of CCL2 (0.25 µg/ml), α-CCL2 (2.5 µg/ml) or isotype antibody (2.5 µg/ml). Mock indicates no virus added and UT refers to no CCL2 or antibodies. Data are represented as mean ± SEM (n = 3). The experiment was performed three times with MDMs from three separate donors with two replicates per experiment. Means ± SEM are plotted. (C) Media and cells were collected at day 15, virus in the supernatant was pelleted via 20% sucrose cushion. Both the cell lysate and purified virus were analyzed in a Gag-p24 Western immunoblot.

https://doi.org/10.7554/eLife.35546.002
Figure 1—figure supplement 1
CCL2 measurement in HIV-1 infected MDM, CEM-CCR5 culture media and HeLa cells.

(A and B). CCL2 measurement in HIV-1 infected MDM culture media. Aliquots of culture media were collected during the multi-day propagation of HIV-infected MDMs depicted in Figure 3 and were assayed for CCL2 using Luminex assay. Data are represented as mean ± SEM (n = 2) – the experiment has been performed at least 3 times. (B) The CCL2 values in HIV-1B ADA and HIV-1 IndieC1 with CCL2 addition are separately plotted as these values are much higher due to added CCL2. (C) Supernatant from uninfected and HIV-1B ADA infected CEM-CCR5 cells (untreated, CCL2, α-CCL2 antibody, and isotype antibody treated cells) were assayed for CCL2 production. CCL2 was measurable only in CCL2 treated cells. Data are represented as mean ± SEM (n = 3). (D) The supernatants from HeLa cells (untreated, CCL2, α-CCL2 and isotype treatment) were assayed for CCL2 production. The untreated, CCL2 and isotype conditions showed comparable CCL2 levels and α-CCL2 antibody treatment reduced the CCL2 level to 35.8 pg/mL. Data are represented as mean ± SEM (n = 3). p value untreated vs CCL2:0.15 (ns), untreated vs isotype:0.35 (ns), untreated vs α-CCL2:<0.0001 (****).

https://doi.org/10.7554/eLife.35546.003
Figure 1—figure supplement 2
Assessing expression levels of CCR2b mRNA and protein in cells employed in this study.

(A) RNA was extracted from MDMs, CEM-CCR5 and HeLa cells. The amplification and Ct values were analyzed using QuantStudio Real-Time PCR software v1.3. (B) U87-CCR2 cells, primary astrocytes, CEM-CCR5, HeLa cells and macrophages were probed for total CCR2 expression through immnoblotting. Immunoblot was developed using Licor Odyssey fc and Imagestudio software.

https://doi.org/10.7554/eLife.35546.004
Figure 1—figure supplement 2—source data 4

Fold change in CCR2b mRNA plotted in Figure 1—figure supplement 2A.

https://doi.org/10.7554/eLife.35546.010
Figure 1—figure supplement 3
Inhibition of virus replication by α-CCL2 can be reversed by exogenous CCL2.

HIV-1B ADA infected macrophages were treated with CCL2, α-CCL2 or Isotype antibody for 24 days. Samples labeled HIV-1B α-CCL2_CCL2 were first treated with α-CCL2 for 9 days, cells washed α- CCL2 replaced with CCL2 until day 24. Media were collected every 3 days and replenished with fresh media. The p24 levels are shown as mean ± SEM of 2 experiments. The results show that CCL2 addition can reverse the effects of CCL2 immuno-depletion.

https://doi.org/10.7554/eLife.35546.005
Figure 1—figure supplement 3—source data 5

All the p24 values plotted in Figure 1—figure supplement 3.

https://doi.org/10.7554/eLife.35546.011
Differential CCL2-responsiveness of HIV-1B and HIV-1C is also observed in a T cell line albeit to a lower degree.

(A, B) CEM-CCR5 cells were infected with 10ng p24/ 1×106 cells input of either HIV-1BADA (panel A) or HIV-1CIndiec1 (panel B) and the culture was propagated in the presence of no additional treatment (UT) , CCL2 (0.25 µg/ml), α-CCL2 (2.5 µg/ml) or isotype antibody (2.5 µg/ml). Mock indicates no virus added and UT refers to no CCL2 or antibodies. Data are represented as mean ± SEM (n = 3). The experiment was performed three times.

https://doi.org/10.7554/eLife.35546.012
Sequence alignment of HIV-1B and HIV-1C Gag-p6 proteins, and Gag p6-ALIX interaction.

(A) Representative HIV-1 Gag p6 sequences from either HIV-1B or HIV-1C isolates that are prevalent in different geographic regions were selected from the Los Alamos database and aligned using Clustal-X. As shown, HIV-1B Gag-p6 retains both the late domains (PTAP and LYPXnL), whereas HIV-1C Gag p6 sequences have a LY deletion. (B) The pie charts represent the frequencies of clade B (left) or clade C (right) sequences with or without the residue Y (of the LYPX motif) in Gag-p6. Clade-B sequences containing the Y residue are shown in red, the remaining sequences have been combined and shown in green. The frequencies of residues replacing Y are 1.6% (H), 0.9% (none), 0.4% (C), 0.3% (F), 0.3% (S), 0.1% (N) and 0.1% (P) respectively. Clade-C sequences without a Y (LY deletion) are shown in green and the 0.2% of the sequences that retain the Y residue or the 0.2% sequences that contain an F residue are combined and shown in red. The total number of sequences examined in Clade-B or Clade-C are 695 and 475, respectively, after removing redundancy at 95%. (C) Employing lysates of Hela cells transfected with either empty vector (pcDNA 3.1) or ALIX-expressing pcDNA construct, pull-downs were performed using GST by itself or GST fused to either HIV-1B-p6 or HIV-1C-p6. GST-B-p6 binds to ALIX, whereas GST-C-p6 does not, due to absence of LY in the HIV-1C Gag p6.

https://doi.org/10.7554/eLife.35546.014
Figure 3—source data 1

HIV-1 clade C sequences used in the analysis in Figure 3B.

https://doi.org/10.7554/eLife.35546.015
Figure 3—source data 2

HIV-1 clade B sequences used in the analysis in Figure 3B.

https://doi.org/10.7554/eLife.35546.016
Figure 3—source data 3

Sequences that were aligned in Figure 3A.

https://doi.org/10.7554/eLife.35546.017
Figure 4 with 2 supplements
CCL2-mediated changes in virus production requires the presence of Gag-p6 LYPX motif in the virus and ALIX in target cells.

(A) Alignment of Gag-p6 amino acid sequences from the two viruses employed in this study - HIV-1BADA and HIV-1CIndieC1. HIV-1BADA has both late motifs (PTAP and LYPX) while HIV-1CIndieC1 lacks the LY dipeptide in LYPX motif. (B, C) Modulation of virus production by CCL2 levels in the media of HeLa cells transfected with HIV-1BADA (panel B) or HIV-1CIndieC1 (panel C) molecular clones or their late domain mutant versions. (B) Shows results of transfecting HIV-1BADA or its -PTAP, ΔLY or –PTAP/ΔLY variants and (C) Shows HIV-1CIndieC1 or its –PTAP, +LY or –PTAP/+LY variants. For each virus, p24 production 24 hr post transfection is plotted including with no treatment, in the presence of CCL2 (0.25 µg/ml), α-CCL2 antibody (2.5 µg/ml) or Isotype antibody (2.5 µg/mL). Data are represented as mean ± SEM (n = 4). The experiment was performed four times with two replicates in each experiment. (D) CEM-CCR5 cells were transduced with lentiviral vectors carrying either a control scrambled shRNA or an ALIX shRNA 29394 expression cassette. Western blot analysis of lysates of untransduced CEM-CCR5 cells or transduced with either the scrambled shRNA or the ALIX shRNA is shown. The bottom panel shows GAPDH protein as a loading control. (E) CEM-CCR5 cells with ALIX knocked-down were infected with HIV-1BADA, either in the presence of CCL2, α-CCL2 or Isotype antibody followed by measurement of virus replication at 12 days post-infection. The results show that CCL2-responsiveness of HIV-1BADA is dependent on ALIX expression. The experiment was performed three times and the mean ± SEM are plotted.

https://doi.org/10.7554/eLife.35546.018
Figure 4—source data 1

All p24 values plotted in Figure 4B and 4C.

https://doi.org/10.7554/eLife.35546.021
Figure 4—source data 2

All p24 values plotted in Figure 4E.

https://doi.org/10.7554/eLife.35546.022
Figure 4—figure supplement 1
Western blot to measure intracellular capsid protein levels.

HeLa cells transfected with HIV-1B and HIV-1C wildtype and L domain mutants molecular clones were treated with one of the following: 1X PBS, CCL2, -CCL2 and Isotype antibody. Cell lysates were subjected to immunoblotting analysis with anti-p24 and -GAPDH antibodies. The results indicate that a decrease in extracellular p24 was associated with a corresponding increase in the intracellular Gag levels that in viruses with LYPX motif.

https://doi.org/10.7554/eLife.35546.019
Figure 4—figure supplement 2
Effect of CCL2 levels on the infectivity of HIV-1B, HIV-1C and L-domain mutant viruses.

TZMbL cells were infected with equal p24 input (10ng per 1x10 6 cells) for 24 h. Infected cells were collected and lysed as before. Luciferase activity was measured to determine the infectivity of the virus to ascertain the effect of CCL2 levels on virus infectivity. Data are represented as mean ± SEM (n = 3) and show a lack of effect of CCL2 on infectivity.

https://doi.org/10.7554/eLife.35546.020
Figure 4—figure supplement 2—source data 1

Relative ight units (from Luciferase assays) plotted in Figure 4—figure supplement 2.

https://cdn.elifesciences.org/articles/35546/elife-35546-fig4-figsupp2-data1-v2.xlsx
CCL2 does not impact ALIX mRNA levels but increases soluble ALIX and facilitates Gag-p6 binding.

(A) Effect of CCL2 levels on ALIX mRNA levels. Total RNA from HeLa cells treated with CCL2 (0.25 µg/ml) or α-CCL2 (2.5 µg/ml) was extracted, reverse transcribed and the cDNAs employed in real-time polymerase chain reactions. Data are represented as mean ± SEM (n = 3). The experiment was performed three times. (B) Immunoblots showing ALIX levels in the total, soluble and insoluble fractions of HeLa cells. Total ALIX protein levels in untreated (UT), CCL2 treated or α-CCL2-treated HeLa cells are similar (Top panel). However, upon CCL2 treatment, most of the ALIX is in the soluble phase while α-CCL2 treatment shows the reverse (middle panel) ALIX levels in pellet II (which constitutes uncross-linked actin - see Materials and methods) represent the converse of what is seen in the soluble fraction (bottom Panels). Two identically treated batches of cells were used, but with or without Latrunculin treatment. ALIX band intensities are quantified, normalized for GAPDH (Total and Soluble) or Actin (Pellet 2) and plotted to the right of the immunoblot. The experiment has been performed three times. (C) CCL2 treatment facilitates Gag-p6-ALIX interaction. GST protein, either by itself or fused to HIV-1B-p6 or HIV-1C-p6 proteins, were expressed in E. coli and purified using Glutathione agarose beads, and incubated with HeLa cell lysates that were either untreated, treated with CCL2 or with α-CCL2 for 24 hr followed by pull-down experiments (two top panels). The input control panels show that the quantities of all proteins (GST and its p6 fusions, ALIX, TSG101 and GAPDH) in the cell lysates from all treatments were comparable within each set – except ALIX, which appears to be absent upon α-CCL2 treatment (three middle panels). The bottom panel shows that the levels of GST bait proteins used were also at equivalent levels. The experiment was performed three times.

https://doi.org/10.7554/eLife.35546.024
Figure 5—source data 1

The mRNA fold change values for Figure 5A.

https://doi.org/10.7554/eLife.35546.025
Figure 5—source data 2

Densitometric intensity values for ALIX bands in Figure 5B.

https://doi.org/10.7554/eLife.35546.026
CCL2 triggers intracellular redistribution of ALIX in HeLa cells.

(A) Confocal microscopy to visualize ALIX in HeLa cells. Cells treated with nothing (Untreated), CCL2, α-CCL2 or Isotype antibody for 4 hr were stained for ALIX, F-actin and DAPI to determine the cellular localization of ALIX upon modulating CCL2 levels. Fluorescence micrographs show that upon CCL2 depletion (α-CCL2 panels), ALIX is dramatically redistributed from the cytoplasm to F-actin stress fibers, while in the presence of CCL2 (both Untreated and CCL2-treated), ALIX is more diffuse throughout the cytoplasm and near the plasma membrane. A small portion of the merged panels was amplified 5.21X to show that the signals colocalize only in α-CCL2-treated cells (B) To quantify the degree of colocalization of ALIX and F-actin signals under conditions of CCL2 addition and depletion, we calculated the Pearsoncorrelation coefficients between the two fluorophores. For HeLa cells, the data plotted indicates the close proximity of F-actin and ALIX upon CCL2 depletion, while in the presence of CCL2 (untreated and CCL2) ALIX and F-actin signals are distinct from each other. Data are represented as mean ± SEM (n = 50 cells each for HeLa cells).

https://doi.org/10.7554/eLife.35546.027
Figure 6—source data 1

Pearson coefficient values plotted in Figure 6B.

https://doi.org/10.7554/eLife.35546.028
CCL2 triggers intracellular redistribution of ALIX in macrophages.

(A) MDM cells treated similarly as in Figure 6, were stained as described above. Fluorescence micrographs show that upon CCL2 depletion, ALIX is redistributed from the cytoplasm to the F-actin structures (podosomes), while in the presence of CCL2 (Untreated and CCL2+) ALIX is diffuse (present as smaller puncta than podosomes) throughout the cytoplasm and near the plasma membrane. (B) To quantify the degree of colocalization of ALIX and F-actin under conditions of CCL2 addition and depletion, we calculated the Pearson correlation coefficient between the two fluorophores. For MDMs, the data plotted indicates the close proximity of F-actin and ALIX upon CCL2 depletion, while in the presence of CCL2 (untreated and CCL2) ALIX signal is distinct from that of F-actin. Data are represented as mean ± SEM (n = 100 cells each for MDMs).

https://doi.org/10.7554/eLife.35546.029
Figure 7—source data 1

Pearson coefficient values plotted in Figure 7B.

https://doi.org/10.7554/eLife.35546.030
ALIX sequestration to actin cytoskeleton alters Gag trafficking.

(A) Confocal microscopy to visualize HIV-1 p24 in HeLa cells transfected with HIV-1BADA molecular clone. Cells were treated with 1X PBS, α-CCL2 or Isotype antibody for 24 hr were stained for p24, and DAPI to determine the cellular distribution of p24 upon modulating CCL2 levels. Fluorescence micrographs show that upon CCL2 depletion (α-CCL2 panels), p24 signal is diffused evenly throughout the cytoplasm, while in the untreated and Isotype antibody treated cells, p24 appears to be concentrated near the plasma membrane. (B) To quantify the distribution of p24, we quantified p24 pixel intensities starting from plasma membrane to nucleus. Data are represented as mean ± SEM (n = 13) cells.

https://doi.org/10.7554/eLife.35546.031
Figure 8—source data 1

Pixel intensity values of p24 staining plotted in Figure 8B.

https://doi.org/10.7554/eLife.35546.032
Intracellular compartments of HIV-1 assembly are observed when ALIX is sequestered to actin.

(A) Electron micrographs to detect HIV-1 budding particles from HeLa cells producing HIV-1B virus particles. HeLa cells tranfected with HIV-1BADA molecular clone DNA were treated with: nothing (Untreated), α-CCL2 (2.5 μg/mL) or isotype antibody (2.5 μg/mL) for 24 hr post-transfection. The cells were fixed and processed for scanning electron microscopy and imaged at 15,000x, 20,000x and 30,000x magnification. (B) The types of virion budding observed was analyzed from 57 (Untreated), 64 (α-CCL2-treated) or 69 (Isotype antibody-treated) cells and the results tabulated. Anti-CCL2 treated cell images collected show low level budding at the cell surface, but display more robust intracellular HIV-1 particle and sub-viral particles in cytoplasmic vacuoles.

https://doi.org/10.7554/eLife.35546.033
Effect of restoring the LYPX motif on HIV-1C replication.

Activated PBMCs (Panel A) or MDMs (Panel B) were infected with equal inputs of HIV-1BADA, HIV-1CIndieC1 and HIV-1CIndieC1+LY viruses. Aliquots of media were collected every 3 days until day 30 and the p24 levels were determined (PBMCs, n = 2 and macrophage, n = 3). Data are represented as mean ± SEM. Results indicate increased replication capacity of LYPX-containing viruses.

https://doi.org/10.7554/eLife.35546.034
PYxE insertions restore ALIX binding to Gag-p6 and enhance replication capacity due to improved virion release.

(A) PYxE insertions restore ALIX binding to HIV-1C Gag-p6. GST protein, either by itself or fused to HIV-1B-p6, HIV-1C-p6, HIV-1C+PYxE-p6 were expressed and purified from E. coli using Glutathione agarose beads, and incubated with HeLa cell lysates. Pull-downs of the protein complexes were resolved on SDS-PAGE gels and the western immunoblots probed for ALIX and TSG101 (top panel). Input levels of ALIX, TSG101 as well as the GAPDH control are in the middle panels and purified GST-only and GST-p6 fusion inputs are in lower panels. The experiment was performed three times. (B) The effect of PYxE insertion on virus replication fitness. MDMs were infected with equal p24 and media were collected every 3 days till day 30. Media collected were clarified and p24 levels determined via p24 ELISA. Data are represented as mean ± SEM. The experiment was performed three times. (C) The effect of PYxE insertion on virus release. HeLa cells were transfected with molecular clones. Media were collected 24 hours post-transfection and virus pelleted through a 20% sucrose cushion. Cells were pelleted and lysed. Both virus and cell lysates were subjected to a western immunoblot followed by detecting virus-associated p24 and cell-associated p24 and other Gag-derived proteins (pr55, (p41) with α-p24 antibodies. GAPDH was used as a loading control.

https://doi.org/10.7554/eLife.35546.037
Figure 12 with 1 supplement
Replication of viruses bearing LYPX or PYxE insertions is inhibited by silencing ALIX or by ALIX dominant negative protein but enhanced by ALIX over expression.

(A) CEM-CCR5 cells were transduced with scrambled shRNA or anti-ALIX shRNA (29394 or 29395) expression cassettes. After selecting for transduction, the presence of ALIX protein was determined by western immunoblotting. (B) The shRNA-transduced cells were infected with HIV-1B, HIV-1B(∆LY), HIV-1C, HIV-1C+LY, HIV-1C+PYKE or HIV-1C+PYRE viruses. Media were collected every 3 days and p24 levels for day 18 at the peak virus replication determined. Data are represented as mean ± SEM (n = 3). (C) HeLa cells were co-transfected with indicated HIV-1 molecular clones and ALIX wild type or ALIX V domain (ALIX-DN) expression plasmids. At 24 hr post-transfection, p24 levels were measured. Data are represented as mean ± SEM (n = 3).

https://doi.org/10.7554/eLife.35546.039
Figure 12—figure supplement 1
Effect of over-expression of wild type and dominant negative ALIX on intracellular capsid protein.

HeLa cells were either transfected HIV-1B wildtype, HIV-1C wildtype, HIV-1C+LY, HIV-1C+PYKE and HIV-1C+PYRE molecular clones. Cells were also co-transfected with either ALIX dominant negative or ALIX wildtype plasmid DNA. Cell lysates were prepared 24 h post transfection and subjected to immunoblotting with anti-p24 and α-GAPDH antibodies. The intracellular Gag levels were comparable between cells expressing different ALIX forms or that were transfected with different HIV-1 variants. Also see source files.

https://doi.org/10.7554/eLife.35546.040
PYxE insertions restore CCL2-mediated increase in virus production in HIV.

HeLa cells were transfected with equal DNA concentrations of HIV-1BADA, HIV-1CIndieC1 and HIV-1CIndieC1+LY or HIV-1CIndieC1+PYxE molecular clones. Transfected cells were treated with nothing (untreated), CCL2, α-CCL2 or with isotype antibodies. After 24 hr post-transfection, media were collected, clarified and p24 levels determined. Data are represented as mean ± SEM. The experiment was performed three times.

https://doi.org/10.7554/eLife.35546.043

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (H. sapiens)HeLaATCCRRID:CVCL_0030
Cell line (H. sapiens)293TATCCCRL-3216
Cell line (H. sapiens)TZMblNIH AIDS Reagent repository8129
Cell line (H. sapiens)U87-CCR2 (U87 CD4+ CCR2+ cells)NIH AIDS Reagent repository4033
Cell line (H. sapiens)CEM.NKR CCR5 (CEM-CCR5) cellsNIH AIDS Reagent repository4376
Recombinant DNA reagentMolecular clone HIV-1ADA (HIV-1B)Theodore et al., 1996Dr. Keith Peden
 Recombinant DNA reagentMolecular clone HIV-1IndieC1 (HIV-1C)Mochizuki et al., 1999Dr. Udaykumar Ranga, Jawaharalal Nehru Centre for Advanced Research, Bangalore, India
 Recombinant DNA reagentGFP expression plasmid peGFP n1Clontech6085–1For eukaryotic expression under CMV promoter. Includes Kanamycin resistance/Neomycin resistance marker
 Recombinant DNA reagentMISSION Lentiviral Packaging mix pLKOSigma Aldrich PMID: 16929317SHP001Includes two plasmids: pMission Gag-Pol and pCMV VSV-G both - carrying ampicillin resistance marker
 Recombinant DNA reagentpLKO.1 construct expressing shRNA targeting ALIXSigma Aldrich Eekels et al., 2011; PMID: 16929317Clone ID TRCN0000029394Target sequence: 5’ GCTGCTAAACATTACCAGTTT 3’ Carries the puromycin-resistance marker
 Recombinant DNA reagentpLKO.1 construct expressing shRNA targeting ALIXSigma Aldrich Eekels et al., 2011; PMID: 16929317Clone ID TRCN0000029395Target sequence: 5’ CCTGAATTACTGCAACGAAAT 3’ Carries the puromycin-resistance marker
Recombinant DNA reagentpLKO.1 encoding sequences for scrambled shRNA controlSigma Aldrich Eekels et al., 2011; PMID: 16929317SHC001Carries puromycin-resistance marker
Recombinant DNA reagentpBluescript-KSStratageneX52331.1Bacterial cloning plasmid with a polylinker
Recombinant DNA reagentpSL1190AmershamBacterial cloning plasmid with a polylinker
Recombinant DNA reagentHIV-1ADA mutant Gag p6ΔLYThis paperMolecular clone of a HIV-1ADA clade B variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1ADA mutant Gag p6ΔPTAP(+LIRL)This paperMolecular clone of a HIV-1ADA clade B variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1ADA mutant Gag p6ΔPTAP(+LIRL)This paperMolecular clone of a HIV-1ADA clade B variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1ADA mutant ΔLYThis paperMolecular clone of a HIV-1ADA clade B variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1IndieC1 mutant Gag p6+LYThis paperMolecular clone of a HIV-1IndieC1 clade C variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1IndieC1 mutant
Gag p6ΔPTAP(+LIRL)
This paperMolecular clone of a HIV-1IndieC1 clade C variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1IndieC1 mutant Gag p6ΔPTAP(+LIRL)This paperMolecular clone of a HIV-1IndieC1 clade C variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1CIndieC1 + PYKEThis paperMolecular clone of a HIV-1IndieC1 clade C variant created by site directed mutagenesis
Recombinant DNA reagentHIV-1CIndieC1 + PYREThis paperMolecular clone of a HIV-1IndieC1 clade C variant created by site directed mutagenesis
Recombinant DNA reagentpGEX-6PiGE Healthcare28954648For expression of Glutatione S Transferase (GST) protein in E. coli
Recombinant DNA reagentpGEX-6Pi-Bp6This paperFor expression of GST-Bp6 fusion protein in E. coli
Recombinant DNA reagentpGEX-6Pi-Cp6This paperFor expression of purified GST-Cp6 fusion protein E. coli.
Recombinant DNA reagentpGEX-6Pi-C + PYKE p6This paperFor expression of purified GST-C + PYKEp6 fusion protein in E. coli.
Recombinant DNA reagentpGEX-6Pi-C + PYRE p6This paperFor expression of purified GST-C + PYREp6 fusion protein in E. coli.
Antibodyanti-CCL2 (Mouse Monoclonal)PeproTech500-M71-500UG2.5 μg/mL
AntibodyIsotype antibody IgG2a, kappa (Mouse polyclonal)PeproTech500-M002.5 μg/mL
Antibodyanti-ALIX (mouse Monoclonal)Thermo-ScientificMA183977(1:500)
Antibodyanti-TSG101(mouse Monoclonal)GeneTexGTX70255(1:500)
Antibodyanti-beta-actin (mouse Monoclonal)Thermo-ScientificMA515739(1:15000)
Antibodyanti-GAPDH (mouse Monoclonal)Thermo-ScientificMA5157381:10,000
Antibodyanti-HIV p24 (goat)Gift from Dr. David Otts (NCI)Gift from Dr. David Otts (NCI)(1:5000) Dr. David Ott, NCI, Frederick
Antibodyanti-Goat antibodies Alexa 800 nm (Donkey polyclonal)Life TechnologiesA329301:10,000
Antibodyanti-mouse antibodies Alexa 680 nm (goat polyclonal)Life TechnologiesA-210581:10,000
Antibodyanti mouse-Alexa 488 antibody (goat polyclonal)Life TechnologiesA-110011:250
Antibodyanti-goat Alexa 488 (Donkey polyclonal)Life TechnologiesA-110551:500
Peptide, recombinant proteinGSTThis paperRecombinant protein purified using Glutathione agarose beads from lysates of E. coli expressing pGEX-6Pi described in this Table
Peptide, recombinant proteinGST-HIV-1Bp6This paperRecombinant protein purified using Glutathione agarose beads from lysates of E. coli expressing pGEX-6Pi-Bp6 described in this Table
Peptide, recombinant proteinGST-HIV-1Cp6This paperRecombinant protein purified using Glutathione agarose beads from lysates of E. coli expressing pGEX-6Pi-Cp6 described in this Table
Peptide, recombinant proteinGST-HIV-1C-PYKE p6This paperRecombinant protein purified using Glutathione agarose beads from lysates of E. coli expressing pGEX-6Pi-C + PYKE p6 described in this Table
Peptide, recombinant proteinGST-HIV-1C-PYRE p6This paperRecombinant protein purified using Glutathione agarose beads from lysates of E. coli expressing pGEX-6Pi-C + PYRE p6 described in this Table
Peptide, recombinant proteinCCL2PeproTech300-04-50UG250 ng/mL
Peptide, recombinant proteinrhM-CSFPeproTechAF-300–2550 ng/mL
Peptide, recombinant proteinPHASigma112497380011 µg/ml
Peptide, recombinant proteinIL-2PeproTech200–0250 U/ml
Commercial assay or kitHCYTO-MAG kitMilliporeHCYTOMAG-MCP-1For CCL2 measurement via Luminex
Commercial assay or kitQuick Change kitAgilent Technologies210518–5For site directed mutagenesis
Commercial assay or kitLuciferase assay kitPromegaPRE1531For measuring the luciferase activity in TZMbl cell lysates
Commercial assay or kitHIV-1 p24 antigen capture assay ELISAABL, Inc5447For p24 measurement
Software, algorithmQuantStudio Real-Time PCR software v1.3Thermofisher ScientificFor CT value analysis
Software, algorithmVolocity 6.3Perkin ElmerRRID:SCR_002668confocal image analysis
Software, algorithmGraphpad PrismGraphpad Software, IncRRID:SCR_002798Plotting data on graphs
Software, algorithmFIJINIHRRID:SCR_002285Confocal image analysis
Software, algorithmLI-COR image studio softwareLI-COR BiosciencesRRID:SCR_015795Western blot band intensity measurements
Chemical compound, drugGentamicinInvitrogen15750–0600.2% in PBMCs/MDM culture media
Chemical compound, drugCiprofloxacinSigma178504 µg/ml in MDM culture media
Chemical compound, drugPenicillin-StreptomycinFischer Scientific15140–122Provided at 100x, used at 1x concentration in cell culture media (equivalent to 50 to 100 IU/mL Penicillin and 50 to 100 μg/mL Streptomycin)
OtherLatrunculin ALife TechnologiesL12370100 ng/mL
OtherTexas Red PhalloidinLife TechnologiesT74711:500
OtherProlong gold - anti-fade DAPIFisher scientificp36931For nuclear staining and mounting
Strain, strain background (E. Coli)BL21 (DE3)Novagen69450For bacterial expression of pGEX plasmids
Biological sample (H. sapiens)LeukopakNew York Blood CenterNew York Blood center
Table 1
Sequences of primers employed in site-directed mutagenesis.
https://doi.org/10.7554/eLife.35546.045
PrimerSequence
ADA (PTAP to LIRL mutation) primer
Forward
5’CTACAGAGCAGACCAGAGCTGATCAGACTGCCAGAAGAGAGCTTCAGG3’
ADA (PTAP to LIRL mutation) primer Reverse5’CCTGAAGCTCTCTTCTGGCAGTCTGATCAGCTCTGGTCTGCTCTGTAG3’
ADA (LY deletion) primer Forward5’GGAGCCGATAGACAAGGAACCTTTGACTTCCCTC3’
ADA (LY deletion) primer Reverse5’GAGGGAAGTCAAAGGTTCCTTGTCTATCGGCTCC3’
IndieC1 (PTAP to LIRL mutation) primer
Forward
5’CTCCAGAGCAGACCGGAGCTGATCAGACTGCCAGCAGAGAGCTTCAGG3’
IndieC1 (PTAP to LIRL mutation) primer
Reverse
5’CCTGAAGCTCTCTGCTGGCAGTCTGATCAGCTCCGGTCTGCTCTGGAG3’
IndieC1 (LY insertion) primer Forward5’GCCGAAAGACAGGGAACTGTATCCCTTAACTTCCCTCAAA3’
IndieC1 (LY insertion) primer Reverse5’TTTGAGGGAAGTTAAGGGATACAGTTCCCTGTCTTTCGGC3’

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3A, 3B, 4B, 4C, 4E, 5A, 5B, 6B, 7B, 8B, 10A, 10B, 11B, 12B, 12C and 13, Figure 1—figure supplement 1A, 1B, 1C and 1D, Figure 1—figure supplement 2A, Figure 1—figure supplement 3 and Figure 4—figure supplement 2.

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