Figure 5

IN-CA deficient virus can produce infectious progeny virus. (A) Effect of RAL on the infectivity of WT and D64A viruses. After infection with VSVG-pseudotyped WT (NL-Luc-E(−)) or D64A (NL-Luc-IN-D64A-E(−)) virus in the presence of AZT, RAL or EVG, cells were harvested at 48 hpi and subjected to luciferase assay. Relative luciferase activity compared to a control sample, in which WT virus was infected without any compounds, were plotted. Concentration of AZT was 0, 1, 10 and 100 μM, whereas concentrations of RAL and EVG were 0, 0.1, 1 and 10 μM. Black circles, WT virus; gray circles, D64A virus; ND, not detected. Representative data of two independent experiments was shown. Error bars, s.d. of triplicate assays. (B) Functional evaluation of progeny viruses generated after IN-CA independent infection. MT-4 cells were infected in the presence of RAL with replication-competent WT virus (NL4-3). Then conditioned medium was harvested every 2 d, and infectivity of progeny virus present in the conditioned medium was evaluated using MAGIC5 cells. (C) Similar experiment with (B) was done using D64A (NL-IN-D64A) virus. Representative results of three independent experiments are shown. (D) Secondary virus generated from MDMs infected with virus in the presence of RAL. MDMs were infected with a replication-competent NL4-3 virus with an env gene, which was derived from R5-tropic ADA virus (NL-ADA-R(−)). Then, HIV-1 RNA copy number in the conditioned medium was quantified by RT-qPCR analysis. To evaluate effects of DNA damaging agents, 2.5 μM etoposide or 1.25 μM bleomycin were added from 0–2 dpi. To exclude the possibility that carry-over virions, which were remnant viruses that could not be completely removed after the initial infection, we included control sample, in which a fusion inhibitor enfuvirtide (ENF) was continuously added from 0 dpi to the end of assay.