FIGURE 9.

Exosomes derived from transfected (pNL4-3) or infected (89.6) Jurkat cell culture supernatants contain TAR RNA. A, early phase log growing Jurkat cells (2.5 × 10⁷) were transfected with 20 μg of pNL4-3 using electroporation or infected with 89.6 HIV-1 strain (∼200 ng of p24-positive virus) and maintained for 5 days in exosome-free media. Supernatants from these transfected and infected cells (50 ml each) were further processed for the presence of exosomes. pUC19 (20 μg) served as negative control for electroporation. Western blots were carried out using WCE or with exosomes using antibodies against CD45, Hsp70, p24, and cytochrome c. B, exosomes isolated from transfected and infected Jurkats were analyzed by qRT-PCR for the presence of TAR RNA. Error bars show the standard deviation of three independent measurements. Exosomes isolated from infected Jurkat cell culture supernatants (C) and transfected Jurkat culture supernatants (D) were utilized in infectivity experiments using U937 cells. Briefly, U937 cells were incubated for 24 h with each set of exosomes and then infected with 89.6 strain of HIV-1 (0.5 ng/p24/μl; 100 μl). Supernatants were analyzed 5 days post-infection for RT activity. Data were obtained from three independent replicates of each sample. Error bars show ± S.D.