FIGURE 6.

Hyperthermia enhances HIV infectivity. (A) Comparison of WT, PR-mutant, and CA-mutant infectivity assayed at 37 °C and 39.5 °C was determined by TCID₅₀ values derived from serial half-log dilutions of each virus stock in quadruplicate wells of PHA-activated PBMC (1 × 10⁵ cells/well) and calculated as a reciprocal of the dilution at which 50% of the wells contained detectable (≥50 pg/ml) HIV p24 measured by ELISA 7 days after inoculation. Results are represented as mean fold-increase in HIV infectivity at 39.5 °C ± SEM for three independent titration experiments. (B) Flow cytometry analysis of PBMC cultures incubated at 37 °C and 39.5 °C for 7 days using the Trucount assay for absolute PBMC cell number and amine aqua exclusion dye to determine cell viability. The flow cytometry plots are one of three independent assays, and the mean total cell number/ml ± SEM was 1171 ± 470 for PMBC incubated at 37 °C and 1190 ± 501 for PBMC incubated at 39.5° C. The mean percent cell viability ± SEM was 60 ± 3 for PMBC incubated at 37 °C and 63 ± 2 for PBMC incubated at 39.5 °C. (C) Hyperthermia increases cellular HSP90AB1 levels. Western blot analysis on 1 μg of protein from cell lysate extracted from PBMC incubated at 37 °C and 39.5 °C for 7 days and probed for HSP90AB1 and alpha smooth muscle actin. The mean fold-increase in HSP90AB1 expression ± SEM was 3.6 ± 0.14, and the western blot represents one of three independent assays. (D) Comparison of WT, PR-mutant, and CA-mutant infectivity assayed at 37 °C and 39.5 °C in a single-cycle infection assay. The TZM-bl luciferase reporter cell line was assayed for relative light units (RLU) 48 h after inoculation. The graph represents mean RLU ± SEM (error bars) from three independent inoculation assays. (E) Flow cytometry analysis of TZM-bl cultures incubated at 37 °C and 39.5 °C for 48 h using the Trucount assay for absolute cell number and amine aqua exclusion dye to determine cell viability. The flow cytometry plots are representative of three independent assays, and the mean total cell number/ml ± SEM was 223 ± 33 for TZM-bl cells incubated at 37 °C and 172 ± 43 for TZM-bl cells incubated at 39.5 °C. The mean percent cell viability ± SEM was 97 ± 0.6 for TZM-bl cells incubated at 37° C and 96 ± 0.8 for TZM-bl cells incubated at 39.5 °C. (F) Hyperthermia increases cellular HSP90AB1 levels. Western blot analysis of 1 μg of protein from lysate extracted from TZM-bl cells incubated at 37 °C and 39.5 °C for 48 h and probed for HSP90AB1 and actin. The mean fold-increase in HSP90AB1 expression ± SEM was 2.8 ± 0.21, and the western blot represents one of three independent assays.