The MOI per cell in PBMC coculture infection was predicted by timing to be 5.0 infectious viruses. coculture contamination at the earliest time points. Data as in Fig 1C, except CTFR was not used to exclude donor-target fusion events.(TIF) ppat.1005964.s002.tif (1.1M) GUID:?C72D9A67-2564-46C9-B7D0-2DA2FB995765 S3 Fig: Inhibition of Rabbit Polyclonal to ACAD10 additional infection cycles using ATV in cell lines and primary cells. Data is usually from coculture infections, and transmission index (Tx) is usually calculated as the number of target cells infected in the presence of Compound E ATV divided by the number of target cells infected in the absence of ATV. (A) RevCEM clones. (B) MT-4 cells. (C) PBMCs. Shown are means and standard errors of duplicates. One of three independent experiments for each cell type.(TIF) ppat.1005964.s003.tif (1.3M) GUID:?50D0BB69-6F00-42FF-9A1E-D1D8517853B1 S4 Fig: Natural percent of infected target cells in coculture and cell-free infection. Data as in Fig 1C, except no normalization was applied.(TIF) ppat.1005964.s004.tif (1.2M) GUID:?64B366F0-B329-4B24-AA4C-57428C264B21 S5 Fig: NL-AD8 infected donor PBMCs infect PBMCs but are unable to infect G2 targets. Left two bars show contamination of PBMCs by PBMC donors infected with NL-AD8 (reddish) or NL4-3 (blue). Right two bars show the percent of G2 infected after coculture with the same quantity of PBMC donors infected with either NL-AD8 or NL4-3. Shown are means and standard errors of duplicates. One of three independent experiments.(TIF) ppat.1005964.s005.tif (1.2M) GUID:?0EF39B55-5C03-4C95-AB76-25BF168363E8 S6 Fig: Gating strategy to detect CFP, YFP, and CFP/YFP co-infected primary CD4+ T cells. Percent infected cells shown for CFP (top left quadrant), YFP (bottom right quadrant), and CFP/YFP co-infected (top right).(TIF) ppat.1005964.s006.tif (1.2M) GUID:?FCA09B6F-3D23-4F0A-AC23-B51D3122A559 S7 Fig: Gating strategy to detect infected target cell frequency in primary CD4+ T cell infection. Donors were labelled with CFSE and contamination was assayed by circulation cytometry following p24 staining for HIV Gag. Top row is usually coculture contamination, bottom row is usually cell-free contamination. Percent of infected targets in the population (bottom right quadrant) shown in reddish, and values for other subpopulations in black.(TIF) ppat.1005964.s007.tif (1.8M) GUID:?47E97229-C6E9-48AE-AC5C-FA7E031089BE S1 Table: Markers for infection. (TIF) ppat.1005964.s008.tif (1.4M) GUID:?467BBD6C-C4F2-4060-99A7-EF90771B7D0A S1 Movie: Time-lapse microscopy of RevCEM clone infection. Cells were imaged for GFP, mCherry, and CTFR fluorescence using time-lapse microscopy. Time is hours:moments post-infection, bar is usually 20M. Infected GFP+, mCherry+ target cells appear as yellow, CTFR+ donor cells as blue. ATV was added after wash and before the start of the movie to bracket contamination to a 2-hour time window. Hence few new transmissions of viable computer virus occurred during the movie.(MP4) ppat.1005964.s009.mp4 (340K) GUID:?1C582BC0-9D42-4AC5-96C4-47DF5024C59F S2 Movie: Time-lapse microscopy of MT4 cell infection by cell-free HIV. Cells were imaged for YFP and mCherry, fluorescence using time-lapse microscopy. Time is hours:moments post-infection, bar is usually 20M. Infected YFP+, mCherry+ cells appear as yellow. ATV was added after wash and before the start of the movie to bracket contamination to a 2-hour time windows.(MP4) ppat.1005964.s010.mp4 (310K) GUID:?6538DC21-8175-42B9-BF76-98FCA6DF1003 S1 Script: Global fitting of time-lapse data using Gamma distribution. Python.(PY) ppat.1005964.s011.py (9.6K) GUID:?411A48B6-D4BC-4E47-9319-106887F9B4DD S2 Script: Drug sensitivity model. Matlab.(M) ppat.1005964.s012.m (3.0K) GUID:?057A281E-5F8E-4E5F-8103-C4728CAB4C82 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more quick than cell-free contamination. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell collection, as well as single cell staining for contamination over time in main cells. We compared cell-to-cell spread of HIV to cell-free contamination, and limited both types of transmission to a two-hour windows to minimize differences due to computer virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell collection and by Compound E 35% in peripheral blood mononuclear cells relative to cell-free HIV contamination. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing computer virus out of the infecting computer virus pool sets the time for contamination independently of the other co-infecting viruses. Author Summary How quickly contamination occurs should be an important determinant of viral fitness, but mechanisms which could accelerate the starting point of.(C) In shape of coculture versus cell-free infection. fusion occasions.(TIF) ppat.1005964.s002.tif (1.1M) GUID:?C72D9A67-2564-46C9-B7D0-2DA2FB995765 S3 Fig: Inhibition of additional infection cycles using ATV in cell lines and primary cells. Data can be from coculture attacks, and transmitting index (Tx) can be calculated as the amount of focus on cells contaminated in the current presence of ATV divided by the amount of focus on cells contaminated in the lack of ATV. (A) RevCEM clones. (B) MT-4 cells. (C) PBMCs. Demonstrated are means and regular mistakes of duplicates. Among three independent tests for every cell type.(TIF) ppat.1005964.s003.tif (1.3M) GUID:?50D0BB69-6F00-42FF-9A1E-D1D8517853B1 S4 Fig: Organic percent of contaminated target cells in coculture and cell-free infection. Data as Compound E with Fig 1C, except no normalization was used.(TIF) ppat.1005964.s004.tif (1.2M) GUID:?64B366F0-B329-4B24-AA4C-57428C264B21 S5 Fig: NL-AD8 contaminated donor PBMCs infect PBMCs but cannot infect G2 targets. Remaining two bars display disease of PBMCs by PBMC donors contaminated with NL-AD8 (reddish colored) or NL4-3 (blue). Best two bars display the percent of G2 contaminated after coculture using the same amount of PBMC donors contaminated with either NL-AD8 or NL4-3. Demonstrated are means and regular mistakes of duplicates. Among three independent tests.(TIF) ppat.1005964.s005.tif (1.2M) GUID:?0EF39B55-5C03-4C95-AB76-25BF168363E8 S6 Fig: Gating technique to detect CFP, YFP, and CFP/YFP co-infected primary CD4+ T cells. Percent contaminated cells demonstrated for CFP (best remaining quadrant), YFP (bottom level correct quadrant), and CFP/YFP co-infected (best correct).(TIF) ppat.1005964.s006.tif (1.2M) GUID:?FCA09B6F-3D23-4F0A-AC23-B51D3122A559 S7 Fig: Gating technique to detect infected target cell frequency in primary CD4+ T cell infection. Donors had been labelled with CFSE and disease was assayed by movement cytometry pursuing p24 staining for HIV Gag. Best row can be coculture disease, bottom row can be cell-free disease. Percent of contaminated targets in the populace (bottom correct quadrant) demonstrated in reddish colored, and ideals for additional subpopulations in dark.(TIF) ppat.1005964.s007.tif (1.8M) GUID:?47E97229-C6E9-48AE-AC5C-FA7E031089BE S1 Desk: Markers for infection. (TIF) ppat.1005964.s008.tif (1.4M) GUID:?467BBD6C-C4F2-4060-99A7-EF90771B7D0A S1 Film: Time-lapse microscopy of RevCEM clone infection. Cells had been imaged for GFP, mCherry, and CTFR fluorescence using time-lapse microscopy. Period is hours:mins post-infection, bar can be 20M. Contaminated GFP+, mCherry+ focus on cells show up as yellowish, CTFR+ donor cells as blue. ATV was added after clean and prior to the start of film to bracket disease to a 2-hour period window. Therefore few fresh transmissions of practical pathogen occurred through the film.(MP4) ppat.1005964.s009.mp4 (340K) GUID:?1C582BC0-9D42-4AC5-96C4-47DF5024C59F S2 Film: Time-lapse microscopy of MT4 cell infection by cell-free HIV. Cells had been imaged for YFP and mCherry, fluorescence using time-lapse microscopy. Period is hours:mins post-infection, bar can be 20M. Contaminated YFP+, mCherry+ cells show up as yellowish. ATV was added after clean and prior to the start of film to bracket disease to a 2-hour period home window.(MP4) ppat.1005964.s010.mp4 (310K) GUID:?6538DC21-8175-42B9-BF76-98FCA6DF1003 S1 Script: Global fitted of time-lapse data using Gamma distribution. Python.(PY) ppat.1005964.s011.pcon (9.6K) GUID:?411A48B6-D4BC-4E47-9319-106887F9B4DD S2 Script: Medication sensitivity magic size. Matlab.(M) ppat.1005964.s012.m (3.0K) GUID:?057A281E-5F8E-4E5F-8103-C4728CAbdominal4C82 Data Availability StatementAll relevant data are inside the paper and its own Supporting Information documents. Abstract Cell-to-cell pass on of HIV, a aimed setting of viral transmitting, has been noticed to become more fast than cell-free disease. However, a system for earlier starting point of viral gene manifestation in cell-to-cell pass on once was uncharacterized. Right here we utilized time-lapse microscopy coupled with computerized image evaluation to quantify the timing from the starting point of HIV gene manifestation inside a fluorescent reporter cell range, aswell as solitary cell staining for disease as time passes in major cells. We likened cell-to-cell pass on of HIV to cell-free disease, and limited both types of transmitting.