Figure 2
Hsp16 suppresses HIV-1 replication in CD4+ T-lymphocytes and macrophages. (A, B) Effect of Hsp16 on HIV-1 replication in CD4+ T-lymphocytes. (A) Western blot analysis shows level of Hsp16 in HIV-infected CD4+ H9 cells. Lane 1, mock-infected H9 cells; lane 2, HIV-infected H9 cells carrying empty vector pcDNA3.1; lane 3, HIV-infected cells hsp16-expressing plasmid. Control, protein loading control. (B) Suppression of HIV-1 viral replication by Hsp16 requires C-terminal end of Vpr. 3 x 106 to 5 x 106 of hsp16-expressing H9 or CEM-SS cells were either mock infected or infected with 2.0 x 103 TCID50 of HIV-1LAI or IIIB. Equal infection of the cells was further verified by measuring viral RNA levels 24 hr after viral inoculation. Viral replication was determined by p24 antigen levels. (C) Analysis of Hsp16 or HSP27 effects on HIV-1 replication in macrophages. Increasing concentration (1, 5 or 10 μg/ml) of purified recombinant fission yeast Hsp16 or 100 μg/ml of recombinant human HSP27 protein was added to 1 x 106 primary human macrophages infected with HIV-1ADA.. Viral inoculates were equalized according to reverse transcriptase (RT) activity (5 x 105 counts per minute/106cells. Viral replication was monitored 7 and 10 days after infection by measuring reverse transcriptase (RT) activity in culture supernatants. To neutralize the effect of potential contamination with endotoxin [45], 10 μg/ml of Polymyxin B (PMB) was added [45]. Results are mean ± SE of triplicates.