Figure 2.

HIV infection reduces the basal OCR in surviving cells. To determine the function of the mitochondria in the population of surviving cells, the OCR (oxygen consumption rate) was determined. Oxygen consumption by complex IV is generally accepted as the main source of oxygen consumption in cells (see the cartoon in I). In these experiments, Complex IV activity can be inhibited by Antimycin A (A), and rotenone (R) can shut down complex I activity. Using both inhibitors will completely shut down all ETC activity. Complex V activity can be shut down using oligomycin (O), which will thereby shut down all ATP generated by OXPHOS. The maximal capabilities of mitochondrial ETC function are analyzed by the use of FCCP, an inducer of inner membrane pore formation that dissipates the chemical gradient between the intermembrane space and the matrix. The Seahorse XFp analyzer takes advantage of a sensitive oxygen meter to measure oxygen consumption rates (OCR) in response to the treatments described above. (A) Determination of basal respiration, ATP production, maximal respiration and spare capacity using oligomycin, FCCP, and Antimycin A plus rotenone, respectively. Early during infection, there was a reduction in the basal OCR before O treatment. OCR determinations at the mid (B) or late stage of infection (C). (D) shows amplification of the OCR curve during late stages shown in C to correct for the surviving cell number. (E) Quantification of the basal OCR (*p = 0.0194, n = 6). (F) Quantification of oligomycin response at early, mid and late stages of infection. Only during the early and late stages, where cell death was minimal, were significant differences observed. Red bars correspond to HIV infected cultures (^&p = 0.0033, ^#p = 0.00009, n = 3). (G) Quantification of coupling efficiency in response to FCCP. (H) Quantification of spare respiratory capacity. (I) A diagram showing the site of action of each inhibitor.