This system employs a luciferase (GLuc)-based reporter genome (HIV-1inGLuc), which expresses and secretes GLuc only after splicing of an intron (inGLuc), packaging into viral particles and infection of target cells [20], [35]. the indicated time points. The data are SB271046 HCl displayed as the GLuc signal ratio over the signal at t?=?0 hr. These results indicate that 36C48 hr are required for an optimal level of signal. Error bars represent the standard deviation of the combination of 2C3 experiments each done in triplicate. (C) Cell-free and co-culture infections were adjusted to result in 10% infection of target cells. Percent infection was determined based on flow cytometry analysis of HIV-1 Gag expression at 24 hr post-infection. Error bars represent the standard deviation of 10 measurements from 5 experiments. (D) Primary CD4+ T cells were infected by cell-free inoculation or co-culture infection as in panel (A) and the infected population of primary CD4+ T cells was sorted 36 hr after infection in order to determine the actual viral MOI resulting from either mechanism SB271046 HCl of viral transmission. Sorting gates were placed based on an efavirenz-treated control (1 M). The purity of the sorted population is shown. (E) GLuc signals obtained after cell-free or co-culture infection. (F) The viral MOI was determined by measuring HIV-1 integration by and Hanna, respectively [60], [65].(PDF) ppat.1003982.s002.pdf (771K) GUID:?154A23C1-EE4E-4D7E-8AF0-A69520C80934 Figure S3: Most PIs potently inhibit HIV-1NL4-3 cell-to-cell transmission. (A) Experimental outline for testing PIs against HIV-1 cell-to-cell transmission. Briefly, Jurkat-inGLuc cells were inoculated with HIV-1NL4-3, washed, stimulated with PMA, washed and cultured in the presence of increasing concentrations of PIs. One set of cells was incubated for 12 hr at 37C prior to co-culturing with target primary CD4+ T cells. Co-cultures were incubated for 42 hr followed by measuring GLuc. The other set of cells hHR21 was incubated without target cells for 54 hr at 37C. This corresponds to the cell-free virus generated and released by donor cells. The viral supernatant was tittered on target primary CD4+ T cells or TZMbl cells and measured GLuc signal 36 hr later. (B) Inhibition curves for the data shown in Fig. 2B. Error bars represent the standard deviation from the combination of at least two individual experiments each done in triplicate.(PDF) ppat.1003982.s003.pdf (454K) GUID:?7BEB8E59-5C57-48E3-8293-6498679DD759 Figure S4: Treatment with antiretroviral inhibitors does not cause a significant effect on the viability of the primary CD4+ T cells. Viability of cells infected by cell-free HIV-1NL4-3 or co-culture at 36 hr post-infection determined with the CellTiter-Glo kit. The data are displayed as the percent viability compared to DMSO control. Error bars represent the standard deviation for 3 measurements.(PDF) ppat.1003982.s004.pdf (407K) GUID:?8FE33665-9DBC-4156-A345-21B09F2A5036 Figure S5: Most NNRTIs, Ent-Is, and PIs potently inhibit HIV-1TRJO.c cell-to-cell transmission. Tested the effect of selected antiretroviral inhibitors against cell-free and cell-to-cell transmission of the founder virus HIV-1TRJO.c. (A) The percentage of infected target cells was equivalent regardless of the mode of transmission. (B) Inhibition curves for the data shown in Fig. 2C. Cell-free virus signal for samples treated with PIs was measured by titrating virus produced from donor cells on primary CD4+ T SB271046 HCl cells. (C) Viability of cells after co-culture or cell-free infection. Error bars represent the standard deviation from the SB271046 HCl combination of at least two individual experiments each done in triplicate.(PDF) ppat.1003982.s005.pdf (672K) GUID:?EE99D38E-5CF1-4AD3-8634-C1E0064E7385 Figure S6: Most NNRTIs and Ent-Is keep a high instantaneous inhibitory potential (IIP) against HIV-1NL4-3 cell-to-cell transmission. Complete IIP data set for inhibitors presented in Fig. 3A, B.(PDF) ppat.1003982.s006.pdf (528K) GUID:?383C5083-58BD-49A3-B704-6441D92A6809 Figure S7: Most NNRTIs and entry inhibitors keep a high instantaneous inhibitory potential (IIP) against HIV-1TRJO.c cell-to-cell transmission. Complete IIP for the HIV-1TRJO.c data set presented in Fig. 3C.(PDF) ppat.1003982.s007.pdf (317K) GUID:?0BFB372C-4ACC-4AED-916B-A32AC39BC6A9 Figure S8: Combinations of NRTIs are highly effective against HIV-1 cell-to-cell transmission. (A) An experiment as in Fig. 4A, B for HIV-1NL4-3 was performed for the combinations of 3TC with TFV and 3TC with AZT (B) The average IIP for all drug combinations presented in Fig. 4 was compared to the average IIP of single inhibitor treatment. Error bars represent the standard deviation from the combination of at least two individual experiments each done in triplicate.(PDF) ppat.1003982.s008.pdf (606K) GUID:?756F4CE5-BB8D-46B1-BEE1-9C28505FDCAF Figure S9: Drug resistant HIV-1 gains an advantage to spread by cell-to-cell transmission in the presence of drug combinations. (A) An experiment as in Fig. 2 for HIV-1NL4-3 carrying the M184V mutation of reverse transcriptase (black line) compared to wild-type HIV-1NL4-3 (red line) in the presence of increasing concentrations of the 3TC with TFV drug combination. Error bars represent the standard deviation from the combination of 4C5 experiments.(PDF) ppat.1003982.s009.pdf (228K) GUID:?62BAE6EF-953B-4840-A6EB-BB0F16FC8E01 Abstract HIV-1 cell-to-cell.