Scale bar, 500?nm; inset scale bar, 200?nm. (H) Representative images of Gag/Env-labeled HIV-1 particles adhered to a coverslip. Figures 6J and 6K mmc7.jpg (527K) GUID:?A6361FDB-5757-414A-9BF9-29BACC7193D6 Document S2. Article plus Supplemental Information mmc8.pdf (12M) GUID:?E814BB93-1E95-47E9-A69F-2662CA61F76C Summary HIV-1 disseminates to diverse tissues and establishes long-lived viral reservoirs. These reservoirs include the CNS, in which macrophage-lineage cells, and as suggested by many studies, astrocytes, may be infected. Here, we have investigated astrocyte contamination by HIV-1. We confirm that astrocytes trap and internalize HIV-1 particles for subsequent release but find no evidence that these particles infect the cell. Astrocyte contamination was not observed by cell-free or cell-to-cell routes using diverse approaches, including luciferase and GFP reporter viruses, fixed and live-cell fusion assays, multispectral flow cytometry, and super-resolution imaging. By contrast, we observed romantic interactions between HIV-1-infected macrophages and astrocytes leading to signals that might be mistaken for astrocyte contamination using less stringent approaches. These results have implications for HIV-1 contamination of the CNS, viral reservoir formation, and antiretroviral therapy. or VSV-G were concentrated and spinoculated onto HFA or MDM target cells to generate a robust input signal and were live-cell gated and assessed for viability before fixation and flow cytometric analysis (Figures S2ACS2C). Physique?2B shows that uninfected MDMs expressed no significant signal, whereas the VSV-G PV yielded 96% positive cells inhibited to 38% by chloroquine, an antagonist of endosomal acidification (Physique?2C). HIV-1BaL PV gave 57% fusion with MDMs that was receptor-mediated, because the CD4 blocking mAb Q4120, SB590885 the CCR5 antagonist TAK779 and the gp41 fusion inhibitor T20 reduced entry signals to background levels, whereas the CXCR4 antagonist “type”:”entrez-protein”,”attrs”:”text”:”AMD31000″,”term_id”:”985631993″AMD31000 failed to inhibit (Physique?2D). When HFAs were exposed to the same PV stocks, VSV-G PV transduced 99% of cells reduced to 49% by chloroquine, whereas HIV-1BaL gave close to background signals that were not further reduced by receptor antagonists or T20 (Figures 2G and 2H). PV carrying the X4 LAI failed to produce a significantly inhibitable signal in MDMs or HFAs, confirming a lack of entry into either cell type (Physique?2H; Physique?S2). Altogether, these results indicate that unlike VSV-G, HIV-1 Env is unable to mediate fusion with astrocytes. However, we did detect low-frequency (2%) -lactamase signals in HFAs that were not significantly reduced by entry inhibitors (Physique?2H). We hypothesized that this apparently non-specific substrate conversion arose from the spinoculation and/or fixation process associated with?the reported HIV-1 binding activity of astrocytes (Chauhan and Khandkar, 2015, Chauhan et?al., 2014, Clarke et?al., 2006, Deiva et?al., 2006, Gray et?al., 2014, Hao and Lyman, 1999, Liu et?al., 2004). To exclude this, we used a SB590885 altered BlaM-Vpr assay that generates real-time data in live cells without spinoculation or fixation (Putcharoen et?al., 2012). BlaM-Vpr HIV-1 without Env (HIVEnv) or pseudotyped with the R5 HIV-1JRFL Env or VSV-G were incubated with SB590885 CCF2-AM substrate-loaded cells at SB590885 4C for 30?min before washing, warming to 37C, and imaging (Physique?3A). The ratio of uncleaved to cleaved substrate was quantified pixel by pixel and plotted against time, using the signal derived from HIV-1Env virions at the final time point as a background control for no fusion. Individual MDMs showed a positive BlaM fusion signal from 20?min DFNA56 onward when exposed to HIVJRFL and HIV-1VSV-G, with 30% and 16% red cells, respectively, by the final time point (Figures 3BC3D). By contrast, although HIV-1VSV-G yielded 35% red cells at 140?min in HFAs, none were detectable at any time point with HIV-1JRFL (Figures 3EC3G). Further evidence for an inability of HIV-1JRFL to fuse with HFAs was obtained in real time using single-particle tracking. Gag-GFP HIV-1 virions double-labeled with the red fluorescent DiD membrane dye, resulting in yellow fluorescent particles, were incubated with cells for 30?min at 4C, washed, and imaged every 8C12?s at 37C for the times shown (Physique?4). Virion fusion leads to DiD diffusion into the limited endosomal membrane, turning.