Analysis of Hsp70 effects on HIV-1 nuclear import and replication in macrophages. (A) Macrophages (1 × 10⁶ cells) were subjected to mild heat shock (47°C, one hour) and then cultured for indicated time. Intracellular Hsp70 was measured by ELISA. Results are mean ± SE of triplicate cultures. (B) Macrophages subjected or not to heat shock (HS) were infected in triplicates with the wild-type (wt) or Vpr-deficient (ΔVpr) HIV-1 NLHXADA.³⁶  Viral replication was assessed at the indicated time points by measuring RT activity in the culture supernatants. Results are mean ± SE of triplicates. (C) Triplicate macrophage cultures were infected with the wild-type (wt) or Vpr-deficient (ΔVpr) HIV-1 NLHXADA³⁶  and cultured in the absence or presence of 100 ng/mL recombinant human Hsp70. Viral replication was assessed as in panel B, and results are presented as mean ± SE. (D) At 24 hours after infection of macrophage cultures with Vpr-deficient HIV-1 NLHXADAΔVpr,³⁶  cells were lysed and assayed for 2-LTR– and pol-specific (a control for total HIV-1 DNA) DNA products by PCR.³¹  Results are representative of 2 independent experiments.